Australian Croc Farming Research

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Crocodile Farming Research: Hatching to Harvest A report for the Rural Industries Research and Development Corporation by SKJ Peucker, BM Davis and Dr RJ van Barneveld September 2005 RIRDC Publication No 05/152 RIRDC Project No DAQ-287A

Transcript of Australian Croc Farming Research

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Crocodile Farming Research: Hatching to Harvest

A report for the Rural Industries Research and Development Corporation by SKJ Peucker, BM Davis and Dr RJ van Barneveld September 2005 RIRDC Publication No 05/152 RIRDC Project No DAQ-287A

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© 2005 Rural Industries Research and Development Corporation. All rights reserved. ISBN 1 74151 215 8 ISSN 1440-6845 Crocodile Farming Research: Hatching to Harvest Publication No. 05/152 Project No. DAQ-287A The information contained in this publication is intended for general use to assist public knowledge and discussion and to help improve the development of sustainable industries. The information should not be relied upon for the purpose of a particular matter. Specialist and/or appropriate legal advice should be obtained before any action or decision is taken on the basis of any material in this document. The Commonwealth of Australia, Rural Industries Research and Development Corporation, the authors or contributors do not assume liability of any kind whatsoever resulting from any person's use or reliance upon the content of this document. This publication is copyright. However, RIRDC encourages wide dissemination of its research, providing the Corporation is clearly acknowledged. For any other enquiries concerning reproduction, contact the Publications Manager on phone 02 6272 3186. Researcher Contact Details Steve Peucker Department of Primary Industries and Fisheries PO Box 1085 TOWNSVILLE QLD 4810 Phone: 07 4722 2608 Fax: 07 4778 2970 Email: [email protected]

In submitting this report, the researcher has agreed to RIRDC publishing this material in its edited form. RIRDC Contact Details Rural Industries Research and Development Corporation Level 1, AMA House 42 Macquarie Street BARTON ACT 2600 PO Box 4776 KINGSTON ACT 2604 Phone: 02 6272 4819 Fax: 02 6272 5877 Email: [email protected]. Website: http://www.rirdc.gov.au Published in September 2005 Printed on environmentally friendly paper by Canprint

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Foreword The primary objective of this broad based crocodile research and development program is to assist with the profitability and sustainability of commercial crocodile production in Australia. The program addresses industry priorities such as pelleted feed, husbandry practices and industry economics. The development of manufactured feed for crocodiles is the main focus of the research program. Traditionally, crocodiles are fed on diets of red meat, poultry or poultry by-products such as chicken heads or necks. Fresh meat contains large amounts of water, which in itself is a cost to farmers. The feed necessitates the use of large freezers for storage. Transport and handling can be difficult and expensive. Manufactured feed should reduce the feed costs associated with growing crocodiles by improving the supply of nutrients, the efficient of nutrients and reducing the cost of diets. Crocprofit was developed in response to enquiries, by people interested in investigating establishment of or investment in crocodile farming. Crocprofit is an information package developed to assist producers and potential investors. The program is based on the cost-benefit analysis technique. It allows people to evaluate the economics of crocodile farming by using their own input parameters before any establishment or investment occurs. Part of the research program deals with crocodile diseases. Observational studies have been carried out on the bacterial and fungal contamination of farmed Johnstone River crocodile eggs. The report presents some interesting results and forms a framework that should be repeated on the more commercially orientated estuarine crocodile where its value would have a greater impact. The second phase of the electrical stunning equipment project is reported. This aspect deals with the animal welfare issue and effects of using this equipment on crocodiles. In a trial comparing the capture of crocodiles using electrical stunning and the traditional noose and pole method the former proved less stressful to the animals and also to the people doing the capturing. This project was funded from RIRDC Core Funds which are provided by the Australian Government. This report, an addition to RIRDC’s diverse range of over 1500 research publications, forms part of our New Animal Products R&D program, which aims to accelerate the development of viable new animal industries. Most of our publications are available for viewing, downloading or purchasing online through our website: • downloads at www.rirdc.gov.au/fullreports/index.html • purchases at www.rirdc.gov.au/eshop Peter O’Brien Managing Director Rural Industries Research and Development Corporation

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Acknowledgments Organisational support is imperative for the successful delivery of crocodile research and development outcomes. Central to this support is the Queensland Department of Primary Industries and Fisheries (DPI&F) which is responsible for having financed the very fine, environmentally controlled crocodile research facility at Townsville. RIRDC is thanked for its operational financing of the project. Australia’s commercial crocodile industry is thanked for its contribution of animals for research purposes. The Crocodile Nutrition Group (CNG) is thanked for its participation, advice, and encouragement for the manufactured feed research/development program. Several individuals have made a special contribution to the crocodile R&D program and include:

• Dr Peter McInnes from RIRDC

• Rob Jack, Lyndell Morrissy and Bill Johnston from DPI&F

• Ian Graham, Electrical Contractor, Tolga

• Dr Craig Franklin, University of Queensland

• John and Lillian Lever and staff from Koorana Crocodile Farm

• Peter Fisher and David Wilson, Melaleuca Crocodile Farm

• Beth Symonds, Dr David Booth and Dr Leigh Ward, University of Queensland

• Dr Annette Thomas DPI&F

Presenting research outcomes to producers at distant locations such as the Northern Territory is difficult. The R&D group have been assisted in this task, by the Northern Territory’s Department of Primary Industry and Fisheries (NTDPI&F). A special thanks is extended to Vicki Simlesa who assisted with the organisation and delivery of manufactured feed for on-farm trials in the Northern Territory.

The following people are thanked for their contribution to the research report:

• Bob Mayer from DPI&F for statistical analysis • Honor Stephenson for editing and proof reading.

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Contents Foreword ..............................................................................................................................................III Acknowledgments................................................................................................................................ IV Executive Summary ............................................................................................................................VI 1. Introduction ...................................................................................................................................... 1

Crocodile Farming in Australia: 2002-2005........................................................................................ 1 The 2002-2005 Crocodile Research Program ..................................................................................... 1

2. Objectives .......................................................................................................................................... 3 3. Methodology ..................................................................................................................................... 4 4. Nutrition............................................................................................................................................ 5

Background ......................................................................................................................................... 5 Response of growing crocodiles to increasing levels of dietary fat and the inclusion of the dietary additive kaolin ..................................................................................................................................... 6 The influence of dietary Kaolin and Sodium Bentonite on the growth and feed conversion efficiency of growing crocodiles ......................................................................................................... 8 The development of feeding frequency strategies for grower crocodiles............................................ 9 Newly hatched crocodiles and their initiation and response to manufactured feeds......................... 14 Weaning trial to determine the effect of using fresh blood on feed intake of manufactured diet to hatchling Crocodylus porosus. .......................................................................................................... 18 2004 Hatchling weaning trial comparing a control and a rapid weaning strategy. ........................... 21 The influence of lupin inclusion on the digestibility of manufactured diets for Crocodylus porosus24 Measuring body condition in farmed crocodiles using Bioelectrical Impedance Analysis............... 31

5. Disease ............................................................................................................................................. 33 Observations on freshwater crocodile (Crocodylus johnstoni) eggs collected from two farms in northern Queensland.......................................................................................................................... 33

6. Capture............................................................................................................................................ 81 7. Economics ....................................................................................................................................... 83

CrocProfit .......................................................................................................................................... 83 8. Extension ......................................................................................................................................... 84

17th Working Meeting of the IUCN-SSC Crocodile Specialist Group............................................. 84 Crocodile research seminar ............................................................................................................... 84 Introduction to crocodile farming in Queensland seminars............................................................... 86 On-farm visits.................................................................................................................................... 88 Crocodile Research and Development Bulletin – Volume 3............................................................. 88 Crocodile Capers newsletter.............................................................................................................. 89

9. Discussion of results ....................................................................................................................... 90 10. Implications................................................................................................................................... 92 11. Recommendations ........................................................................................................................ 93 Appendix A. ......................................................................................................................................... 95

Crocodile Nutrition Research - Strategic Directions 2002-2005....................................................... 95 Appendix B......................................................................................................................................... 102

Development of manufactured feeds for Crocodylus porosus ........................................................ 102 References .......................................................................................................................................... 111

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Executive Summary Rural Industries Research and Development Corporation (RIRDC) and the Department of Primary Industries and Fisheries (DPI&F) have a contractual agreement designed to assist the development of the Australian crocodile industry. Both organisations share the goal of a profitable and sustainable crocodile industry, which will achieve its objectives through improved technology. Crocodile farming is described as an emerging industry and as such has less experience with commercial intensive livestock principles than the more established industries such as pigs and poultry. Despite this comparative lack of intensive livestock skills, the crocodile industry is making rapid progress in closing the gap. Crocodile farming is moving from extensive outdoor practices, modelled on wild habitat observations which are much influenced by climatic conditions, to intensive housing with environmentally controlled housing being used for hatchlings and in some cases for grower animals on commercial farms. Several producers are going a step further and creating individual pens for grower animals to prevent fighting and subsequent skin damage, thus placing a more valuable product on the market. Such practices lead to higher returns on investment. This report covers a range of crocodile R & D topics including nutrition, disease, genetics, animal capture, economics and extension practices. The report is intended to provide the reader with a current picture of research findings and recommendations. Some topics are presented in more detail than others, which reflects the state of the R & D progress for that particular topic. Reports on environmental, genetics and housing are mentioned only briefly. Conversely, the research covering manufactured feed for hatchling and grower crocodiles is advanced and is provided in more detail in this report. Twenty years ago little scientific investigation had been undertaken into commercial crocodile production. Virtually no progress had been made on feeding manufactured diets despite the attention of the international research community. Considerable effort had been made in Zimbabwe and South Africa with little to show for those efforts. No one had identified the barrier(s) to the successful initiation of pelleted feeding. Further, no on-farm feed manufacturing equipment had been developed. Neither was there any technology available to show clients how to successfully manufacture pellets. Significant progress on these issues has been achieved by the DPI&F team at Townsville and includes the development of:

• equipment capable of manufacturing pellets on farms at acceptable cost • successful cold press pellet manufacture techniques • better understanding of barriers to acceptance although universal acceptance of manufactured

diets by crocodiles has not been fully achieved • cooperation with client producers who are trialling manufactured feed and are providing

feedback on acceptance and performance is in progress • research on the crocodile nutritional requirements which are not well understood at all at this

juncture. Pelleted feeding is a high industry priority and one that is being pursued rigorously by the research team. As part of the nutrition program a Crocodile Nutrition Group (CNG) has been formed and meets on an annual basis to discuss research findings, compare progress with objectives and plan future directions. A Crocodile Nutrition Research Strategic Directions (2002-2005) document has been compiled which identifies and prioritises areas for research (see Appendix A). Researchers continue to address the issue of weaning hatchlings onto pellets. Following a series of free choice feeding exercises in 2001 recent work has focused on using a range of feed additives or

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attractants to diets. Attractants such as liver digest, prawn digest, chicken head and kangaroo digest, along with additives like liver or roast beef powder have been tried. These have proven to be unsuccessful in initiating feeding in young hatchlings. One finding that appears to be important in the research program is that acceptability of manufactured diets is related to the size of the animal rather than age. Work on developing feeding strategies to grower crocodiles is also continuing. Growth rates of 16 grams/crocodile/day are being achieved. So far researchers have established that it is possible to manufacture on-farm, cold pressed, pelleted feed that crocodiles will eat. In addition, manufactured feed offers a 2.4:1 ratio over offal diets on a dry matter basis. This advantage translates into ingredient, transport and storage savings for producers. Work on diet formulation continues. Results from trials using lupins at different inclusion levels are looking promising. The use of lupins in the diets did not affect intake and had no negative effects on crude protein or gross energy digestibility. The use of ingredients such as lupins offers the opportunity to look at a wider ingredient base and may lead to savings by using cheaper plant-based ingredients. Other areas of research are reported below in less detail. The genetics component of the program did not eventuate for a number of reasons. One reason in particular was the commencement of RIRDC project number US-109A with the title A genetic improvement program for farmed saltwater crocodiles. This was an extensive project and it was felt that our project would add little to the knowledge pool in the presence of this program. However, our program did cooperate with the research people who conducted this work. Stunning equipment to capture crocodiles has been developed that was unique to Australia. This equipment has revolutionised the way in which animals are captured for relocation, measurement, examination and harvesting. Stunning equipment has attracted considerable international as well as national interest because more animals can be handled in a day’s work with less risk and injury to animals and staff alike. The equipment is now used in South Africa, Zimbabwe, Spain, Papua New Guinea and the USA. These countries collectively produce 70% of the world’s crocodilian skins. Further, stunning as opposed to the traditional method, causes much less stress and fatigue for handlers. However there was some concern, expressed by Animal Ethics Committees, that stunning could be causing more stress to crocodiles. Consequently Departmental researchers collaborated with crocodile physiologists from the University of Queensland to examine stress levels in crocodiles captured by electrical stunning equipment and the traditional method of using a noose and pole. With the noose and pole method animals do a body roll, struggle and thrash around compared to the quiet and minimal disturbance using the electrical stunning. Blood samples were taken and analysed for stress level indicators. Results demonstrated that capture by electrical stunning is less stressful for the animal. This finding supports the observation of researchers and producers that animals recover more rapidly from capture trauma and commence feeding sooner if they are stunned compared with being caught by noose and pole. CrocProfit is a spreadsheet, forecasting tool for established farmers and potential investors in the crocodile industry. For example it will allow established farmers to estimate the impact of changes in skin price or to determine the effects of shifts in expenditure/income due to moving to manufactured feed or the effects an increase in mortality might have on their operation. The CD also contains reference material and a comprehensive list of contacts for related State Government departments and industry associations. This information package is now available from the DPI&F bookshop and will help considerably to eliminate the confusion and unwise investment decisions which occurred in other emerging industries such as ostrich farming. Crocodile producers nationally are cooperating with the research team. Seminars, demonstrations, on-farm trials, publications and workshops have been employed to keep producers informed of research outcomes. Industry seminars are held annually in Queensland and the Northern Territory.

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An example of this cooperation between researchers and industry is the Crocodile Research Seminar that was held in November 2003 in Cairns. Funded by RIRDC the seminar, for the first time, brought together all RIRDC funded crocodile project researchers under one roof to report their findings. Producers made their thoughts known and provided feedback in an open and constructive way throughout the day’s proceedings. The crocodile R&D program has produced successful outcomes in the areas of:

• nutrition • stunning and other aspects of husbandry • disease management • management • application of R&D results through a rigorous extension program.

Detailed information on outcomes from the R&D program can be found in DPI&F Crocodile Research and Development Bulletin, Volume 3 and RIRDC publications.

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1. Introduction Crocodile Farming in Australia: 2002-2005 The Australian crocodile industry is a relatively new industry, with commercial production commencing in the early 1980’s. Currently established farms are under-going a period of expansion in terms of increasing animal numbers and farm infrastructure (single pen accommodation) to meet an increasing demand for skin and meat products. The advent of single pen accommodation has seen an increase in the quality of skins produced by the Australian industry. In recent times there has been interest from potential overseas investors wishing to establish themselves in the Australian industry or as a way of guaranteeing skin supply for their tanning operations in Europe. The biggest obstacle facing the industry is the availability of animals even in the Northern Territory where ranching is allowed. In Queensland discussions are underway between industry and government agencies on the possibility of carrying out a trial egg-harvesting program. If the trial proceeds and it proves to be economically viable then this will be a boost to the Queensland industry and may encourage new people into the industry. The 2002-2005 Crocodile Research Program The title of this report is Crocodile Farming Research: Hatching to Harvest. This report focuses on three areas of research: nutrition, disease and capturing crocodiles. Two other subjects addressed in this report deal with extension/communication and economics. In recent times there has been interest from clients, investors and indigenous communities contemplating crocodile farming. Crocprofit has been developed to meet this need. To this end the following research and information presented in this report are set out in the table below. Some of the research programs objectives were a continuation from a previous RIRDC project (DAQ-247A) and have already been reported and no additional research has been carried out to date. One objective, the genetics component of this program has since been subject of another RIRDC funded project initiative (US-109A) and it was felt that there would be unnecessary duplication if this objective was met and no additional benefits could be made. Based on the research carried out to date and discussion emanating from the Crocodile Nutrition Group (CNG) nutrition consultant, Dr Robert van Barneveld, has prepared a document outlining the nutrition program’s future direction titled Strategic Direction Document 2002-2005 (Appendix A). This action plan builds on existing research, prioritises the key nutritional bottlenecks to crocodile production by relating research areas to the primary nutritional drivers of profitability (relevant to any intensive animal production systems) – feed costs and feed conversion efficiency. Not only does the document, provide clear justification for the respective research areas but also provides a concise direction for the research program.

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Nutrition Crocodile Nutrition Research Strategic Directions 2002-2005

• Response of growing crocodiles to increasing levels of dietary fat levels and the dietary additive Kaolin

• The influence of dietary additives Kaolin and Sodium Bentonite on the growth and feed conversion efficiency of growing crocodiles

• Development of feeding frequency strategies for grower crocodiles • 2004 Hatchling feed initiation trial and observations • Weaning Trial to determine the effect of using fresh blood on feed intake of

manufactured diet to hatchling Crocodylus porosus • Weaning trial comparing a control and a rapid weaning strategy • Influence of Lupin inclusion on the digestibility of manufactured diets for

Crocodylus porosus. • Measuring body condition in farmed crocodiles using Bioelectrical Impedance

Analysis.

Disease • Observations on bacterial and fungal contamination of farmed freshwater crocodile eggs.

Capture

• Further refinements to electrical stunning equipment to capture crocodiles • Comparing the effects of electrical stunning equipment and noose and pole

method of capture in relation to animal welfare issues.

Economics • CrocProfit – a complete information package for crocodile farmers and potential investors.

Extension • 17th Working Meeting of the ICUN-SSC Crocodile Specialist Group (CSG)

• Crocodile Research Seminar • Introduction to crocodile farming in Queensland • On-farm visits • Crocodile Research and Development Bulletin • Crocodile Capers Newsletter

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2. Objectives 1. To further develop improved, economic, nutritionally balanced feed pellets for both hatchling

and grower sized crocodiles, thereby directly reducing farm costs associated with transport, storage, food preparation, manual addition of vitamin and mineral supplements to diets currently involving fresh meat and reducing the risk of animal disease.

2. To continue research into environmental rearing factors which produces outcomes which meet

DPI&F’s crocodile environmental obligations and is beneficial to industry. Such technology will deliver improved growth rates (with a resulting shorter turn-off time on farms), reduced disease and mortality rates and increased proportions of first grade skins produced.

3. To develop housing standards which are commercially beneficial to industry. 4. To implement the genetic program BLUP to improve the breeding performance of farmed

crocodiles. 5. To encourage more farmers (in all states and the Northern Territory) to use DPI&F’s standard,

computer database recording scheme (CROCTEL) and a spreadsheet which acts as a decision making tool for farmers and investors (CrocProfit). To undertake an annual centralised processing and statistical analysis of all farms’ recorded information (maintaining farm anonymity) and then to discuss each farm’s results personally with that farm’s management. To prepare and distribute an annual report on farm statistics to farmers, researchers and government agencies. To present and discuss overall trends at annual industry seminars and to use the results as a basis for R&D planning at Industry Advisory Group meetings.

6. To ensure that research results are accurately and quickly extended to crocodile farms across

Australia by

• conducting annual industry seminars at Cairns and Darwin and sponsoring key farmers and researchers from outside these areas to participate

• publishing research results in ‘Crocodile Research Bulletins’ and in Research Updates

• producing jointly with the NTDPI&F a biennial crocodile industry newsletter called Crocodile Capers.

7. To sponsor half-yearly meetings with the Crocodile Industry Advisory Group to

• review current crocodile R&D undertaken by DPI&F

• plan effective future R&D

so that the individual activities planned under the previous six general activity areas most effectively and efficiently address industry needs.

8. To enhance our working relationship with industry and fellow researchers.

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3. Methodology General research procedures for research experiments Annual cohorts of animals were provided by industry for this work with

• approximately 500 newly hatched animals from a number of large nests, each identified individually by web tags and numbers matched with clutch

• approximately 400 of these subsequently moved into the grower shed at one-year of age. Experiments with discrete-type treatments (eg various attractants) were designed with replicated pen/tank units so that valid statistical analyses could be carried out on ensuing data. Experiments involving a single factor with different levels (eg water temperature) may have unreplicated pen treatments if the main objective is to estimate an overall response pattern. In either situation, close attention was paid to statistical design and setting up of experiments, so that groups of animals initialled assigned to pens were as uniform as possible. Fellow researchers have often published non-conclusive research results, with the explanation the crocodiles exhibit more variation in response to applied treatments than most other animals, both between and within clutches. From experience built up since 1993 the following are regarded as key issues when designing an experiment on hatchling crocodiles to one year of age:

• allow for a ‘settling-down’ period of two weeks between successive experiments (when all animals are subjected to the same conditions)

• grade the animals into two or three size classes as required • allocate animals to tank/size groups using the same mix of clutches (as much as possible,

given the actual numbers in each clutch) using an initial randomisation within these constraints

• fine-tune the allocation (swapping within clutches) so that, for each size-class, tanks contain animals with the same average (fasted) liveweight and also the same range in liveweight

• use a separate ‘give and take’ tank in which to rear runts and any animals which become sick or diseased.

In terms of carrying out the experiment, the following base guidelines are used for hatchling animal and grower crocodiles:

• fast the animals for at least 48 hours prior to measuring • measure animals only at the start and at the end of an experiment (usually treatment

differences are statistically significant by 8-10 weeks) • weigh food offered and remnants of food uneaten at each feed (this gives an indication of

growth rate in each tank during the experiment) • feed animals daily from Monday to Friday then fast them over the weekend to assist in

complete digestion of food in their system • record belly scale patterns at an early age (either by photographs or by photocopies) • record liveweight, total length, snout-vent length and cranial skull length at each measuring. • Some variation to trial length and feeding regime is used for grower crocodiles.

Analysis and publication of research results Mr Bob Mayer, who is the research group’s biometrician, has responsibilities for data analysis. Dr Robert van Barneveld, a nutrition consultant, assists in interpreting the statistical findings. The Crocodile Nutrition Group provides advice and feedback. Individual members of the DPI&F team have cooperated in writing up the results for the different styles of report (Research Bulletin, Research Update, Crocodile Capers, papers for scientific journals, papers/posters for conferences).

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4. Nutrition Background The Australian crocodile industry views the development of manufactured feed as its highest priority. To date research into manufactured feed for crocodiles have proven to be complex, and at times frustrating. Feed initiation and feed intake are areas that have proven to be difficult with young hatchlings. However, progress with the acceptance of pelleted feed for grower crocodiles is much more encouraging. Good progress has also been made in the areas of diet specifications, development of feeding strategies and pellet manufacture. On-farm trials are occurring in Queensland and the Northern Territory and for the first time animals have been taken through to harvesting size with a trial at the Johnstone River Crocodile Farm at Innisfail, north Queensland. Details of these trials will be the subject of a final report in November 2005 (Project No.DAQ-300A). Despite some intensive trial work using feed additives and attractants in diets we have been unsuccessful in gaining rapid acceptance of manufactured diets by young hatchlings. The exact reason for this remains unclear. It may be associated with pellet texture, taste or odour or it may in fact be simply instinctive in origin. It would appear that the development of weaning strategies that gradually wean hatchlings from meat diets to pellets will be needed if pellets are to be successfully fed to farmed crocodiles. To this end, a series of weaning strategy trials have been carried out and details are contained in this report. Results have been mixed, unsuccessful with young hatchlings but good acceptance with crocodiles greater than 800 grams in body weight. From these results, it appears that pellet acceptance appears to be linked to the size/weight of animals rather than age. If this is so, then it maybe a case of feeding hatchlings on traditional meat diets until they reach 800 grams and then introduce them to manufactured feed. Trials in 2005 will identify a target weight that young crocodiles will readily accept manufactured feed. Once manufactured feed is accepted crocodile growth is progressive and sustainable with an average weight gain of 16grams/crocodile/day being achieved. This is in the mid range of the figures presented in the RIRDC publication Benchmarks for New Animal Products (Project No. WHP-2A). In recent years research animals have been fed on manufactured feed for periods of up to two and half years at the Department’s research facilities before being returned to producers. These animals have been well grown and their performance acceptable to commercial producers. While feed initiation has proved difficult, good progress has been made on diet composition (see table below). Initially diet formulation was based on suggested dietary allowances for crocodiles described by Staton and Vernon in Intensive Tropical Animal Production Proceedings (1991) and from the outcomes of a crocodile nutrition workshop held in 1998 at Townsville. The table below details what was considered a desirable pellet in 1998 and what the standard diet is in 2004. Research conducted at the DPI&F facilities has also formed the basis for the reduction in dietary fat content, the need for fresh meat product, manufacturing capacity and physical diet requirements and the overall diet costs.

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1998 desirable pellets 2004 pellets Dry matter content 60% Crude protein content 35-45% Crude fat 10-16% Fresh meat product 20-50% Binders nil Cost $1-1.50 /kg

Dry Matter content 63% Crude protein content 42% ave Crude fat 5% Fresh meat product 0% Binders nil Cost $1-00 -$1.60/kg Mycotoxin binder (fusariotoxins) Shell grit Acidifiers Additional vitamin D Iron supplementation

Response of growing crocodiles to increasing levels of dietary fat and the inclusion of the dietary additive kaolin A trial was carried out to compare the effects of three different dietary fat levels and kaolin on crocodile growth in May 2002. Fat content was chosen, as it would most likely induce changes in growth response. Importantly fat levels will have influence on the shape of the animal which is an important consideration for skin buyers. Skin buyers prefer animals with a narrow rectangle shaped body rather than a short squared shaped animal that is often grown on a diet of chicken heads. Kaolin also known as Chinese clay, is fine fluffy white clay. Kaolin was added to the diet to see if it could slow the rate of digesta transit time in crocodiles as it was felt that the nutrients in the feed were passing too rapidly through the gut of the crocodile and that maximum benefit was not being obtained. Kaolin has been shown to positively affect the nutrient utilisation by influencing the digesta transit time and digestive capacity in the abalone and poultry industries. Trial design In May 2002, 390 animals aged twelve months were housed in the grower research facility. Animals were allocated on size and clutch. Thirty-five animals were in each small/medium (S/M) group and thirty animals were in each medium/large (M/L) group. The trial was conducted over a twenty-week period. This group of crocodiles had already been successfully weaned onto pellets. Crocodiles were fed diets that were formulated to contain 5, 10 and 15% crude fat with crude protein at 42% on a dry matter basis. Chicken tallow was used as the dietary fat source. Diets were formulated on a least-cost basis. Dietary moisture content had to be reduced to accommodate the increased levels of dietary fat. All diets were based on the same ingredient set, however inclusion levels obviously varied between diets. Discussion Animals in both size groups, fed the diet containing 5% performed slightly better in terms of total weight gain (Figure 4.1) daily weight gain (Figure 4.2) and total weight and length gained (Figure 4.33) than the 10% dietary fat group and significantly better than animals fed the 15% fat diet. Feed Conversion Efficiencies (FCE) for the treatments are presented in Figure 4.4 below. The M/L group fed a diet containing 10% fat had the best FCE for that size group while in the S/M group animals fed 5% dietary fat performed best. Results from this trial have allowed for the formulation of standard grower crocodile diets without the need for adding an additional fat source. This reduces the number of ingredients used, storage required and the overall cost of diets. It was difficult to determine if there was any benefit in using of kaolin as a feed additive in this trial. The kaolin content of the 5% fat diet was higher than in the 10 and 15% fat diets and therefore we are uncertain whether the growth response exhibited by the crocodiles was in response to dietary fat or dietary kaolin level. Subsequently another trial was carried out using 5% dietary fat levels to investigate if the kaolin had any effect.

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Figure 4.3 Total length gain of animals fed 3 different levels of dietary fat

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Figure 4.4 Feed Conversion Efficiency of animals fed 3 different levels of dietary fat. The influence of dietary Kaolin and Sodium Bentonite on the growth and feed conversion efficiency of growing crocodiles This trial was a continuation of the work mentioned previously to determine the value, if any, of adding kaolin to the diet. Three treatments were used, kaolin, sodium bentonite and water. Kaolin has been described previously. Sodium bentonite is similar in appearance to kaolin. It is a very fine cream-coloured powder and is used as a binder in pellet manufacture. The trial commenced in December 2002 and finished in March 2003 and involved 366 animals aged 19 months old. Diets contained 42% crude protein and 5% crude fat. Animals were allocated on size and clutch with thirty-five in each small/medium group and twenty-six in each medium/large group. Figures 4.5 and 4.6 below show that neither kaolin nor sodium bentonite had any benefit in animal growth or FCE.

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Figure 4.5 Weight gain of animals fed three different additives

2.52.72.93.13.33.53.73.94.14.3

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0.00.51.01.52.02.53.03.54.04.5

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Figure 4.6 Feed Conversion Efficiency of animals fed three different additives The development of feeding frequency strategies for grower crocodiles In 2003/2004 the research program developed in two distinct areas. For hatchlings the research has focused on promoting the initiation of feeding. For juvenile animals that have been successfully weaned onto manufactured feed, part of the research program has centred on developing feeding strategies that will optimise the growth of animals fed manufactured diets. Two experiments are reported in this paper. These two experiments were carried out using different feeding regimes and involved housing animals in groups or individually in cages. The development of feeding strategies directed at establishing optimum feeding regimes for crocodiles at various production ages is an important component of the research program. Feed intake can be influenced by many factors. The smell, texture and taste of the food offered, the capacity of the animal to utilise the nutrients in the diet, environmental factors and the social behaviour of the animals are important considerations if the nutrition for the animal is to be optimised. Other factors to consider from a producer’s point of view will be to grow the animals out in the shortest possible time leading to a reduction in production costs through savings in labour and feed. Current feeding strategies on commercial farms vary. Some farms feed every second day while others feed three times per week. Feeding regimes used on farms may result in overfeeding with an increase in feed wastage which will add to the production costs. Conversely, “over feeding” may be necessary under commercial conditions to ensure all animals have ready access to feed and reach their commercial potential in the shortest possible time. The aim of these two experiments was to determine the optimal feeding regime for crocodiles fed on manufactured feed. Experiment 1 Design Group Housed Animals (Rooms) Experiment 1 was conducted in environmentally controlled grower research facilities over a 115 day period from the 24th March to the 16th July 2003. Animals were 22 months old at the commencement of the experiment. They were divided into two size groups: small/medium (S/M) and medium/large (M/L). Stocking densities were 48 animals per pen in the S/M group and 37 animals per pen in the M/L group representing densities of 2.6 animals /sq metre and 2 animals/sq metre respectively. Average initial weight and length for the S/M group was 3674 grams and

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1091mm and for the M/L group 5951 grams and 1235mm. The feeding strategies used in this first experiment were 2, 3, 5 or 7 days per week. A standard manufactured diet was used in the experiment which contained 42.5% crude protein and 6% crude fat. This experiment was not replicated in design. Individually Caged Animals Sixteen animals, four animals from each of four clutches, were housed in individual cages. Animals averaged 1223mm in length and 6364 grams in weight. The feed strategies were 2, 3, 5 or 7 days per week. Cages measured 520 cm in length, 60 cm in width and 40 cm in height. They were manufactured from 50mm wide security screen mesh. A canvas blind was used as a divider between cages. The blinds were used for three purposes: • To minimise any dominance effect which might adversely impact on feed intake • To reduce the possible effect on animals in neighbouring cages by animals releasing

pheromones from the chin glands (personal comm. C. Manolis) • To serve as a barrier to prevent leftover pellets drifting into neighbouring cages thus ensuring

accurate measurements of animal intakes and feed residues. Experiment 2 Design Group Housed Animals Experiment 2 followed directly on from Experiment 1 and ran for a period of 67 days from the 17th July to the 22nd September 2003. Animals were 26 months old at the commencement of the experiment. They were divided into two size groups with the same animal numbers and stocking densities as Experiment 1. The experiment was not replicated in its design. Average initial weight and length were 5316 grams and 1204mm for the S/M group and 8458 grams and 1405mm for the M/L group. The feeding strategies employed in this experiment differed slightly from Experiment 1. Group housed animals were fed 2, 3, 4, or 5 days per week on our standard manufactured diet which contained 42.5% crude protein and 6% crude fat. Individually Caged Animals Fifteen animals, three from each of five clutches, were used in Experiment 2 and animals were fed 2, 3 or 5 days per week. Average initial weight for caged animals was 6364 grams and average length was1313 mm. Results Experiment 1 No significant differences were demonstrated for results for animals housed in cages and those housed in groups. Clutch, animal size and stocking densities had a greater influence on results. In individually housed animals those fed 3 times per week performed better in terms of food conversion efficiency (FCE) than the other treatments. In terms of liveweight gain animals fed 7 days per week had an average weight increase of 28.3% while those fed 2, 3 and 5 feeds per week had increases of 22.9, 26.6 and 25.8% respectively. In the S/M group, animals fed 7 days per week out-performed the other treatment groups in weight gain (55.9%) and body length (11.7%). This compared to 50.8, 57.7 and 44.8% for weight gain and 10.7, 10.8 and 9.8% in body length gain for 2, 3 and 5 day feeding groups. Seven days per week feeding had a slightly poorer FCE than groups fed 2 and 3 days per week (see graph below). In M/L group animals fed 7 days per week were heavier and longer than animals fed 2, 3 and 5 days per week. With regard to FCE, animals fed 7 days a week were again marginally better than 5 days per week with 3 and 2 day feedings performing less efficiently.

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Figure 4.7 Experiment 1 - Feed Conversion Efficiencies for Caged and Room Housed Animals Figure 4.8 Experiment 1 – Average Initial and Final Weights – Caged and Room Housed Animals Figure 4.9 Experiment 1 – Average Initial and Final Lengths – Room Housed Animals

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Results Experiment 2 For the animals housed individually in cages there were no statistical differences in results between the three feeding treatments or between the clutches. All animals gained weight and length and there were no adverse affects on the animals by housing them individually. There were consistent patterns in the percentage increase in values for total length and weight gain, food eaten and FCEs with the number of feeds the animals received each week. Animals on the 5 days a week feeding performed slightly better than those animals fed 4 days which performed slightly better than those fed 3 days. Total body length percentage increases were 5.1, 5.4 and 6.1 % and bodyweight increases were 21.6, 25.0 and 26.2% and FCEs were 1.71, 1.76 and 1.66 for the 3, 4 and 5 feeds per week respectively. Animals housed in rooms fed 4 and 5 times per week did better than the 2 and 3 days per week. Room 3 which house a M/L group fed twice per week experienced one animal dominating over the rest of the animals which led to poorer growth and FCE results. The results from this room are included. As the number of feeds per week increased so did the weight and body length in the M/L group. Weight gains were 0.6% (room with dominance problem), 8.5, 10.1 and 11.6% and total body length increases were 2.5, 3.6, 4.1 and 4.1% The S/M group ate less than their larger siblings but had better bodyweight gains. In the S/M group, animals fed 4 times per week performed better in terms of growth rate than the other groups but had a slightly higher FCE than animals fed 2 or 5 days per week but lower FCE than the 3 day/week feed group. Percentage increases in bodyweight ranged from 25.2% for the animals fed 4 days/week to 17.5% for those fed three days /week. Animals fed 2 and 5 days a week had increases of 19.5 and 21.7% respectively. Figure 4.10 Experiment 2 – Feed Conversion Efficiencies – Caged and Room Housed Animals Figure 4.11 Experiment 2 – Average Initial and Final Weights – Caged and Room Housed Animals Figure 4.12 Experiment 2 – Average Initial and Final Lengths –Caged and Room Housed Animals

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Discussion The feeding strategies employed by farms will have a direct influence on cost of production. Identifying the optimal feeding strategy for animals fed manufactured feed that minimises feeding costs and reduces labour inputs and allows for growth at acceptable levels will improve farm profitability. The aim of these experiments was to examine the performance of juvenile crocodiles fed manufactured feed employing different feeding strategies. Crocodiles are efficient converters of feed into body tissue. This is particularly so in wild Crocodylus porosus (C. porosus) which have reported conversion efficiencies of about 82% (Webb et al. 1991). The percentage for farmed animals is considerably lower. Published values for farmed raised animals vary with age. Garnett et al (1986) reported 17, 34 and 37% for juvenile farmed crocodiles fed diets of fish, beef or pork respectively. Two separate experiments by Webb et al. (1983) using six and eleven and half month old Crocodylus Johnstoni (C. Johnstoni) found no significant difference in growth rate between animals fed daily and those fed five days per week. Mayer (1995B) found that seven month old C. porosus fed a mixture of chicken heads, beef and kangaroo meat produced growth rates of 7-21% with the small group of animals having the poorest FCE. Mayer et al. (1997) during a ‘rearing density’ experiment using ten month old C. porosus fed a mixture of pork and chicken heads produced growth rates of between 23 and 31%. In the wild C. porosus are opportunistic feeders and the authors (Webb et al 1991) hypothesised “that the physiological mechanisms associated with digestion and assimilation may not function as efficiently when the stomach is repeatedly filled to capacity as occurs in farmed raised animals”. These experiments present the growth rates of C. porosus fed solely on manufactured feed. No cost analysis was carried out. The key nutritional drivers to profitability for any intensively farmed animal enterprise are described by van Barneveld (2003) in Appendix A. Here the author lists the main areas that make up the nutritional component of the costs of production. One of the drivers reported by van Barneveld is in the area of FCE. FCE is a measurement of the amount of food that is eaten to the weight gained by the animal. In these experiments the key component to establishing the optimum feeding strategy is to examine the FCE of animals fed different feeding strategies. The lower the FCE the better the result. The FCEs for the animals housed in cages and rooms are shown in Figures 4.7 and 4.10 for both experiments. FCEs for animals housed in rooms were poorer for all groups than for animals housed in cages. For caged animals the FCE ranged from 1.66:1 to 2.2:1. For animals housed in rooms the FCEs were poorer ranging from 2.3:1 to 4.9:1. The FCE of 30.22 and poor overall weight gain for the M/L group fed twice weekly should be discounted as there was a high incidence of dominance in this group. This only serves to highlight the problem of social interaction and represents one of the problems encountered when working with this age group of crocodiles. The result for animals

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housed in cages is not unexpected. The use of individual cages from a researcher’s point of view reduces the social and other influences that can affect feed intake. Across both experiments there was little difference in growth and feeding efficiency between 2, 3, 4, 5 and 7 feeds per week. In the M/L group there was a trend showing an increase in weight with the increase in the times that animals were fed per week which is not unexpected. There was little difference in total length gain between animals fed 3, 4 and 5 days per week. From the results reported here a feeding frequency of 3 days per week for the M/L group would appear to strike a balance between FCE, growth rate and labour required to feed out and any extra cleaning that would be required from additional feedings. For the S/M group there was little difference in the different treatments. In terms of FCEs in Experiment 1 those fed twice per week performed slightly better than the other treatments while in Experiment 2 animals fed 2 and 5 days per week did better at converting feed to weight than those fed 3 and 4 days per week while those fed 3 and 4 days per week had the biggest gains in bodyweight and length. Group size played a part and smaller groups generally had a better FCE over all treatments than larger groups and this trend was found in both experiments. Newly hatched crocodiles and their initiation and response to manufactured feeds Feed initiation of hatchlings is a critical step in the commercial introduction of manufactured crocodile diets. This is the case whether it is in trying to get very young hatchlings to accept manufactured feed or through a process of weaning older juvenile animals over time period to gain acceptance. Getting animals settled and adapted to a routine feeding regime is vital to ensure the long-term survival and acceptable growth standards for hatchlings. Past research efforts have focused on pellet texture and odour and recently taste. Taste of the pellets have been manipulated through various attractants such as those tried in this experiment. While these three characteristics have been recognised as important in gaining acceptance of manufactured feed for crocodiles (Jansen-Van Vuuren 1995) in our research program there seems little recognition of pellets as a source of feed by C. porosus hatchlings for what ever reason. The literature details little information on feed recognition in crocodilians and in particular what actually triggers feed initiation in C.porosus hatchlings. Even when hatchlings are fed on traditional diets of meat and chicken heads here is often a percentage of animals that do not commence eating, resulting in ‘runts”. Runts are animals which fail to thrive and eventually died or display retarded growth. Various authors, Manolis et al. (1989) and Garnett & Murray (1986) found that young hatchlings exhibited clutch specific preferences for certain feeds. Manolis (1989) suggests that hatchlings crocodiles maybe genetically pre-programmed to recognise certain smells, tastes and movements in their environment as representing food. Considerable problems have been experienced in getting hatchlings to initiate feeding on manufactured diets. The objective of getting young hatchlings to accept manufactured pellets (containing no fresh meat) is considerable. To this end a series of trials and observations were carried out with the 2004 group of hatchlings using numerous feed additives and attractants to:

• examine the use of attractants to initiate feeding • increase the intensity of the feeding • increase the acceptability of manufactured diets to hatchlings.

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Trial Design Two hundred and one hatchlings, from five clutches, were allotted to six tanks housed in three rooms of the hatchling facility. Hatchlings were individually identified by applying two consecutive numbered stainless steel tags to the web of each hind foot. Hatchlings were weighed and measured upon arrival. The average weight of the hatchlings was 73 grams with an average length of 310 millimetres. The hatchlings were aged 8-10 days at the beginning of the trial, which commenced on 3rd April and concluded on the 5th May. Hatchlings were fed a total of 27 times over the 33-day trial period. Animals were weighed again on the 27th April.(Graph 4.13). Animals were fed using small 24hour automatic belt fish feeders and/or pellets placed white plastic feed trays. Animals were observed at feeding time for their reaction to the different attractants via close circuit monitors and feed intakes were recorded. Trial diets and attractants used Ten kilogram batches of pellets were manufactured using either the standard hatchling and grower diets with the addition of an attractant. The following diet and attractant combinations were used:

• Grower diet and 1litre chicken head digest • Grower diet and 1litre beef liver digest • Hatchling diet with 50% kangaroo meat • Grower diet with 3 grams beef liver powder • Hatchling diet/no oil/ and 25% kangaroo meat • Hatchling diet/no oil and prawn digest • Hatchling diet pellets, coated in fresh sheep blood • Hatchling diet and hen egg yolk • Hatchling diet with no attractant • Hatchling diet and 500mls beef liver digest • Grower diet with 1litre of prawn digest • Grower diet with kangaroo digest.

Digest preparation process Five kilograms of raw material (ie prawns, chicken heads, kangaroo and liver) were minced through a five ml die and added to a 20L plastic drum. 1.25L of water was added and thoroughly mixed together using a hand drill and stirring rod for 10 minutes

• 625 mls of phosphoric acid was then added, and mix for a further five minutes • the pH of the mix was then measured using lab sticks and adjusted to 2.0 pH by either

adding more phosphoric acid or concentrated sodium hydroxide • once adjusted the mixture was mix again for another five minutes • the drum was then sealed and placed in a warm place for four weeks prior to use.

Discussion At no stage during the trial or from observations made did we observe any mass intensity of feeding or acceptance of manufactured feed using any of the various attractants. In the groups fed pellets containing the attractants, liver digest, chicken head digest and pellets containing 25 and 50% kangaroo meat some six to eight animals could be seen eating pellets from the feed trays or off the floor of the tank. Graph 4.14 and 4.15 show a small number of animals that increased weight. Animals lost body weight, but increase in body length and head length over the 25 day period. This is not unusual occurrence and has been report previously by (Whitehead 1990). Some increased feed response was gained using fresh blood as an attractant (see graph 4.16 and 4.17). This work was repeated and has been reported in a separate article in this document. The

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various attractants tested did not stimulate feed intake to a level that would be considered viable by industry. The loss of body weight does not support the use of attractants as a stand alone method of initiating feeding in young hatchlings. A staged reduction of dietary meat content and animal size appear to be the most predictable parameters for getting young crocodiles to accept manufactured diets. Graph 4.13 Comparison of initial weight at 2/4/04 and at 27/4/04 Graph 4.14 Crocodile weight comparisons – 2/4/04-27/4/04 for Tank 1B (fed grower diet and attractants Liver Digest, GD and Fresh Blood, GD and Liver Powder) Graph 4.15 Crocodile weight comparisons – 2/4/04-27/4/04 for Tank 3A (Hatchling Diet – No Oil/25% Meat, HD, Fresh Blood, HD – Egg Yolk)

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Graph 4.16 Response to various attractants for hatchlings in Room 1 Tank B Graph 4.17 Response to various attractants for hatchlings in Room 3 Tank A

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Belt feeders Belt feeders were used to deliver pellets to hatchlings and to create movement and to try and stimulating a feeding response. Two types of belt feeders were used during the trial and observations. Electric belt feeders that operated of 12 volts and belt feeders that operated off a timing mechanism that was engaged when the feeding belt was extended out along the feeder. Both types of feeders operated for 12hour periods. Some of these were borrowed and some were purchased for the trial. Two feeders per tank were used. The feeders were supported over the tanks by wooden slats and placed parallel to the hide-boards in each tank. Pellets would drop directly down and in front of hatchlings who were under the hideboard. Advantages of using belt feeders • feeders did create a movement of pellets by dropping pellets to the floor of the tank • the movement of the pellets did attract the attention of a small number of hatchlings • in some tanks, 6-8 hatchlings would gather around the feeder and appeared to be waiting for the

pellets to fall (particularly liver digest flavoured pellets) • feeders allowed the opportunity to offer feed over a 12 hour period • easy to use • no operating problems were encountered with the electric feeders. Disadvantages of using belt feeders • belt feeders were very expensive, $367 each • were a bit slow on occasions, when we wanted to do observations, If pellets were placed to

far back on the belt (20mm from front of belt) you would have to wait up to 20-30 before they fell. This made the observation period long and impractical at times

• some of the borrowed feeders were old and stopped during feeding. If this happened early in the feeding period a lot of food would be left on the feeder. Animals therefore did not have access to food for that period

• some animals laid waiting for pellets to fall, this may have kept some shy feeders from accessing the pellets

• pellets coated in blood, once dried, some would stick to the belt and did not drop into the tanks

• feeder belts needed to be cleaned weekly. This was simply done by using water and a small amount of detergent.

Weaning trial to determine the effect of using fresh blood on feed intake of manufactured diet to hatchling Crocodylus porosus. Getting newly hatched crocodiles to take manufactured pelleted as their sole source of food has proved very difficult. It is our experience that only a small percentage newly hatched crocodiles will commence consuming pellets when they are first introduced but they soon loose interest. They loose interest to the extent that they stop eating pellets altogether and body condition declines. In an attempt to overcome this problem additives or stimulants were added to the diet. Additives took two forms namely a manufactured product and what might be described as a natural product in the form of "digests" were tried. Manufactured additives were presented in a powder form and had chicken, beef and prawn flavours. They were added to the diet in varying amounts but none of the flavours or different amounts encouraged hatchlings to take pellets as their sole source of food. This report covers the use of fresh sheep’s blood as a natural stimulant.

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Trial design Two treatment diets and a control diet were used in this trial. The treatment diets consisted one, plain pellets using our standard hatchling diet. The other treatment consisted of the same diet but with the addition of fresh blood. The blood was sprayed onto the pellets and applied at the rate of 5 or 10mls per feed. The blood was allowed to semi dry before the pellets were fed out. The blood was preserved via the anticoagulant, Alsevers* solution. The control diet consisted of 70% kangaroo meat and 30% chicken heads and 1% vitamin and mineral supplement. Two hatchling tanks were partitioned into five smaller compartments each, measuring 600 mm wide by 1300 mm long. Painted 5mm ply board was used as partitions within the tanks. Pens 1-5 were in one tank and pens 6-10 were in the second tank. Ten animals each, from three clutches were selected based on bodyweight. Hatchlings were four and half months old at the commencement of the trial. Average weight and length for the animals chosen from the clutches were 217.8g and 448.9mm, 193.5g and 423.8mm and 190.4 g and 421.9mm respectively. Hatchlings were housed in groups of three, consisting of one hatchling from each clutch. A settling in period 2 weeks was allowed and in this period all hatchlings were feed standard meat diet. Feed was placed onto small plastic white trays. Trial commenced on 13th September and concluded on 29th October 2004 a total of 47 days including a pre trial settling period. Three pens each were allocated as controls and pellet treatments, with four pens receiving the blood-coated pellets. Allocation of treatments to pens was as follows: • control 1,5 and 9 • pellet (plain) 4,7 and 10 • blood (coated pellets) 2,3,6 and 8

Hatchlings were fed three days per week, with a feeding period of four hours. Lights were turned off at feeding time. Feeding responses were based on observations via the use of closed circuit monitors and feed intake were measured and recorded. Weaning Strategy Treatment Week 1 Week 2 Week

3 Week 4

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100% meat

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70% meat & 30% pellets

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100% pellets

Table 4.19 detailing the weaning strategies used during trial *Recipe for Alsevers solution: combine 1000mls filtered water and • 20.5 g glucose • 4.2 g sodium chloride (NaCl) • 8g Sodium citrate-trisodium salt • 0.55g Citric acid Stir until contents dissolve and filter through 2µm filter paper. Add 50 mls of alsevers with 250 mls blood.

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Results

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1 209.8 410.6 Control 2 179.7 228.3 Pellets coated with Blood 160 261.6 Pellets coated with Blood

4 193.6 227 Pellets 5 165.1 272 Control 6 249.2 340 Pellets coated with Blood 7 191 255.6 Pellets 8 162.8 243.3 Pellets coated with Blood 9 250.2 404 Control

10 232.4 201 Pellets Table 4.20 detailing average initial and final weights for each

2004 Hatchlings - Weaning Trial Blood Attractability - Average Weight between Pens

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Key C – Control P&B - Pellets coated with Blood P – Pellets Graph 4.21 Showing average weight gain for control and treatment groups Mortalities Three animals died during the trial, all deaths occurred within a space of three days during the fifth week of the trial. Hatchlings were from three different pens, 6, 7 and 10 and treatments. Two of the animals were in good condition, both had gained weight during the trial period, scutes were up and no bite marks were observed. The third hatchling from pen 10 was in poorer condition than the other two, had several bite marks on top of the hind legs and had lost weight. Two animals were sent to Oonoonba Veterinary Laboratory. Post mortem results indicated the cause of death in two of the three animals was Salmonella infection. The third animal died on a weekend and was not submitted for autopsy due to decomposition.

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Discussion Hatchlings in the control groups out performed all treatment groups in terms of average weight and bodylength gain. Average gain for the control groups were 171.6 g in weight and 81.3mm, in length compared to 80g and 62.8 mm and 67g and 50.2 mm for blood/pellet and pellet groups respectively. Control groups showed more consistency in feed intake and response at feeding time and all control group hatchlings gained weight and grew in length compared to hatchlings in all treatment groups (graph 4.21). Hatchlings in the control groups were observed to approach the feeding trays and commence eating first on all but one of the feeding days. The difference in the time it took hatchlings to approach feed trays and commence feeding compared to the control groups varied between one and thirty minutes. On the last feeding day before the trial was terminated it was observed that pens 6 and 7 had not eaten during a 30 minute period and only one animal in pen 2 appeared to be eating during this time. Control fed hatchlings in pen 1 and 9 approached the feed trays and commenced eating before all other pens. One animal in pen 1 and one in 9 were considerably bigger than all the other hatchlings, these two hatchlings gained 312.6 g and 313.8g respectively during the trial period. These two hatchlings accounted for the high average weight gain for these two pens. It was visually noticeable that the controls were growing faster than hatchlings from either of the treatment groups. The difference in size could be in part attributed to the two-week period when the treatment groups were fed 100% pellets. During this period there was dramatic decline in feed intake when the diet changes from 50-50% meat/pellets to 100% pellets. Control animals are not subjected to diet changes to which they would have had to adjust to. Previous observations showed that diet changes leads to checks in growth rates like those experienced here. Results from the two treatment groups varied. The results show that hatchlings in the blood groups did perform better than plain pellet fed animals. Four hatchlings out nine fed plain pellets lost weight while all nine hatchlings fed pellets and blood gained weight. On this result it would appear using blood did have some effect on feed intake and growth response. Hatchlings in pen 10 (pellets) showed little interest in eating pellets, and were not observed eating very often during the trial. Accordingly all three animals in this group lost weight. Hatchlings in the blood coated pellets pen 3 and 8 approached and ate pellets shortly after the controls commenced and always (except one feeding) before hatchlings fed plain pellets 4,7 and 10 commenced eating. The trial was terminated before its due date due to a number of reasons. Firstly there was a lack of response to feeding from pens 6, 7 at the 100% feeding rate, the poor condition of animals in pen 10. The research group has extensively investigated the use of attractants and determined that they offer no value in developing weaning strategies. The results of this trial suggest that the use of blood has minimal effect on feed intake in young hatchlings. This result supports observations made over the past couple of years and reinforces the research groups views that acceptance of manufactured feed is related to some other variables such as the size of the animal. Work during 2005 will focus on establishing a recommended starting weight for animals to be fed manufactured feed. 2004 Hatchling weaning trial comparing a control and a rapid weaning strategy. A series of investigations over recent years has shown that the use attractants or feed additives to increase acceptance of manufactured feed will not work on young hatchlings. Attractants whether in powder or liquid form such as the many tried in the above-mentioned papers have failed to initiate and stimulate the consumption of manufactured feed. Perhaps this resistance has its foundations in the possibility that hatchlings are in some way imprinted on the diet their mothers ate and the imprint message is transmitted to the developing hatchling during its life as an embryo.

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Whatever the reason, it is our experience that hatchling C.porosus will only feed initially on wet meat diets. The first occasion the research group got young crocodiles to eat manufactured feed was in some ways purely by chance. A group of eight month old crocodiles who had never before had access to pellets ate them with vigour. These animals remained on pellets until they were returned to farms some two years later. It was never clear at the time if this response was due to age or size or a combination of these two variables. A further complication was the possible impact of clutch effects. With variations in ages within this 2004 hatchling group, the opportunity presented itself to firstly to look the relationship between acceptance of manufactured feed and age/size of the animals and secondly to examine the success of the weaning strategies used in the trial. Trial Design 518 hatchlings were allocated into two groups based on age, liveweight and clutch. Two weaning strategies were used. A ‘control’ weaning strategy, which weaned hatchlings from 100% meat diet to 100% pellets over seven weeks which included a period of only two weeks on 100% pellets compared to a ‘rapid’ weaning strategy over the same time period but with a quicker increase in the percentage of pellets fed and a period of four weeks on 100% pellets. Hatchlings remained in the same groups as per the attractabilty trial (reported above). Initial body weight (table 4.22 & 4.23**) were based on the hatching weight. It was determined in this instance that another weigh and measure at this point would only add additional stress to the animals. During the attractability trial only a small number had increased weight during this trial. The younger group also had less time exposure to the attractability trial and less effect on hatching liveweight. Animals allocated to tanks in rooms 1-3 (clutches 1-9) were older animals and were 53-61 days old (average 57 days). Animals allocated in rooms 4-6 (clutches 10-20) were younger with an age range of 13-41 days old (average 36 days) at the commencement of the trial. Two late clutches arrived two days after the commencement of the trial and were divided equally between tanks in rooms 4-6. A two-week pre-trial period was allowed where all hatchlings were fed standard meat diet with 1% vitamin and mineral supplement. The trial was carried out over 47 day period from 24th May to 9th July 2004. Hatchlings were fed five times a week, Monday to Friday during the course of the trial. Rapid weaning strategy Control weaning strategy Week 1 diet of 90% meat & 10%

pellets Week 1 diet of 90% meat & 10%

pellets Week 2 diet of 70% meat & 30%

pellets Week 2 diet of 80% meat & 20%

pellets Week 3 diet of 50% meat & 50%

pellets Week 3 diet of 70% meat & 30%

pellets Week 4 diet of 100% pellets Week 4 diet of 60% meat & 40%

pellets Week 5 diet of 100% pellets Week 5 diet of 50% meat & 50%

pellets Week 6 diet of 100 % pellets Week 6 diet of 100 % pellets Week 7 diet of 100 % pellets Week 7 diet of 100% pellets

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Results Rooms 1-3 Individual clutch responses to treatments

No. animals* Initial weight** Final Weight 12/7 Weight gain % weight gain Clutch Control Rapid Control Rapid Control Rapid Control Rapid Control Rapid

1 16 16 65 64 176 154 111 90 165 132 2 21 20 73 75 148 145 75 70 102 90 3 13 14 68 76 173 221 105 145 153 185 4 20 20 66 67 162 121 96 54 140 76 5 17 16 67 68 170 155 103 86 154 122 6 4 4 52 54 145 103 93 49 189 85 7 2 1 52 51 85 47 33 -4 64 -8 8 15 13 65 64 113 117 48 53 74 83

1-5 87 86 68 70 166 159 98 89 143 121 6-8 21 18 56 56 114 89 58 33 109 53

Table 4.22 detailing individual clutch responses to treatments * Animals which survived to the end of the trial ** 27/4 for clutches 1-5 and 5/4 for clutches 6-8

Rooms 4-6 Individual clutch responses to treatments

No. animals* Initial weight** Final Weight 12/7 Weight gain % weight gain

Clutch Cont. Rapid Date Cont. Rapid Cont. Rapid Cont Rapid Cont.

Rapid

9 8 8 13/4 74 74 121 97 48 22 64 31 10 12 12 8/4 71 72 188 170 117 99 166 137 11 5 3 23/4 68 67 77 53 9 -14 13 -21 12 8 7 14/5 75 77 86 67 11 -10 13 -14 13 11 11 14/5 61 63 41 43 -21 -20 -33 -31 14 14 14 29/4 77 75 108 100 32 24 41 31 15 10 10 29/4 63 64 46 45 -18 -18 -28 -29 16 10 11 29/4 75 75 77 65 2 -10 3 -13 17 21 21 29/4 72 72 101 69 29 -3 40 -4 18 11 11 6/5 63 64 67 64 4 0 6 0 19 12 10 6/5 81 82 68 64 -13 -19 -16 -23 20 13 14 6/5 76 77 65 57 -11 -19 -15 -25

Table 4.23 detailing individual clutch responses to treatments • * Animals which survived to the end of the trial • ** On arrival at Oonoonba, when they were tagged

Summary of results based on age Age group Treatme

nt Ave Initial wt.

Ave Final wt.

% weight gain

% mortality

Food eaten/surviving hatchling

FCR

Older Control 71.4 154.8 117 12.0 274 3.3 Older Rapid 71.3 144.9 103 10.4 211 3.0

Younger Control 71.8 89.1 24 7.1 149 9.4 Younger Rapid 71.8 76.3 6 6.6 84 22.6

Significant factor (s)

(none) Age (**) Age (**) (none) Age (**) Diet (*) Age (**)

Table 4.24 details age response to weaning strategies Results Results show that the older group of hatchlings, clutches 1-8 outperformed the younger group clutches 9-20 in terms of percentage weight gain in both control and rapid weaning treatments. Results from the older group show 101/108 (93%) and 98/103 (95%) animals put on weight in the control and rapid treatment groups while only 70/131 (53%) and 45/128 (35%) for the younger aged animals. A total 171/239 (71%) of all animals in the control groups gained weight compared to 143/231 hatchlings (62%) of the hatchlings in the rapid weaning group.

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Mortalities Nine percent or forty-eight animals died during the trial. This represents a much higher percentage than normally occurs during our trials. The mortality rate between the two treatments was evenly spread with twenty-five ‘control’ hatchlings and twenty-three ‘treatment’ hatchlings dying. Twenty-nine of the forty-eight deaths were from the older group, this high number could be attributed to a possible “carry over” effect of minimal feed intake of hatchlings during the initial attractability trial. Older hatchlings spent more time in this previous trial than the younger group and growth and their well-being may have been more compromised resulting in a higher number of deaths. Discussion Individual clutch response: These are reported in tables 4.22 and 4.23. Results were reported this way as hatchlings were not weighed directly before the commencement of this trial. For example the older group hatching weight was used as the starting weigh.** Table 4.22 shows that the older aged clutches in rooms 1-3 all gained weight and out performed the younger hatchling group in rooms 4-6. Overall hatchlings in the control groups (71%) did better than those in the treatment groups (62%). Only two ‘rapid’ weaning groups from clutches three and eight put on more weight than their siblings in the control groups indicating that acceptance of the manufactured feed was reduced. In fact their results were disappointing in both the control and rapid groups except for clutches 9, 10, 14 and 17. Clutches 9 and 10 were the oldest out of the younger group and did perform better than any of the clutches in this group. In hindsight this clutch should have gone into the older group to make the age difference larger. Clutch 14 with percentage weight gains of 41% and 31% and clutch 17 at 40% for control performed the best of the younger group. The 4% loss in weight from clutch 17 using the ‘rapid’ weaned group highlights the variability between animals from the same clutch and further indicates that that rapid weaning is not a suitable feeding strategy for young hatchlings. Rapid weaning strategy did not offer any advantage over the control initiation. The control strategy group consumed more feed per surviving hatchling than the rapid weaned group. There appears to be a positive link between size of the animal and feeding response, this is reinforced by the example mention above. Studies in 2005 will aim to identify the target weight at which animals will make a successful transition a meat diet to manufactured feed. The influence of lupin inclusion on the digestibility of manufactured diets for Crocodylus porosus One of the key areas in relation to the cost of developing manufactured feed for crocodiles is the variety and availability of ingredients that can be used in the diet. Included in this, will be the maximum amount of a particular ingredient that can be used in the diet and the capacity of that ingredient to supply protein and energy for metabolism. Currently only animal-based protein sources such as meat meal, feather meal and poultry offal meal are used in the diets. These animal based-protein meals can be expensive compared with other non-animal protein sources. Some research has been carried out on alternative protein sources for feed in the alligator industry. Researchers have examined the use of vegetable protein in diets for alligators. Coulson and Hernandez (1983) reported that alligators are unable to digest vegetable proteins. Kercheval and Little (1990) reported better growth rate of alligators fed a combination of plant and animal protein or just animal protein over two other formulations based on plant protein and plant protein plus 500mg of taurine/kg. Stanton et al (1986) also identified numerous advantages in using a vegetable based source of protein in alligator diets.

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Lupins have shown significant potential as vegetable protein alternatives to animal proteins in livestock diets (van Barneveld, 1999). They have been used with some success in aquafeeds and Australia now exports in the vicinity of 30,000 tonnes per annum for this purpose. While they have the capacity to supply high levels of digestible protein (with the exception of sulphur based amino acids such as methionine and cystine), lupins also supply significant quantities of soluble and insoluble non-starch polysaccharides. In poultry, a consequence of this is an increase in digesta viscosity and a potential drop in apparent metabolisable energy if lupin inclusion is too high. While the same may be possible in crocodiles given the short digestive tract, the reverse may also be true. In the absence of a crop and gizzard, crocodiles do not have the same capacity as poultry to predigest food and hence may rely more on gut retention time and the subsequent action of digestive enzymes. With this in mind, inclusion of lupins in manufactured diets for crocodiles may not only improve the stability of the pellet (through the soluble NSP’s), but may also increase digesta transit time, and hence, the length of time the crocodile can extract nutrients from manufactured pellets. In two experiments we examine the effect of using Lupins (Lupinus angustifolius) as a source of vegetable protein and its effect on growth rate and digestibility. Experiment Details Housing Two experiments were conducted to examine the effects of using lupins as part of the diet in manufactured feed. Sixteen animals were used in each experiment. Animals were placed in individual cages made from 50mm security mesh screen and measuring 1500 millimetres (mm) long by 590mm wide by 380mm high. The cages were housed in the Department’s environmentally controlled grower research facility at a rate of four cages per room. Temperatures in the rooms were held at approximately 32º C for air and water. Rooms in the grower facility measure six metres long by three metres wide. Two small white plastic feed trays were fixed to the floor of the cages. Canvass partitions were placed between each cage to act as dividers between the cages. These dividers allowed accurate recordings of residual feed and collection faecal samples from each animal. No cross contamination of feed residues or faecal samples occurred. The dividers also reduced the eye contact between animals. The cages were placed in the rooms so that animals could get in and out of the water at will. Experimental Diets A control and three treatment diets were used in both experiments (Table 1). The control diet contained no lupins and the other three diets contained lupins at inclusion levels of 5, 10 and 15 %, respectively. All four diets had a crude protein level of 52.5%, crude fat level of 6.5% and a dry matter of 74%

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Table 1. Composition (%) of experimental diets. Diet

Ingredient 1 2 3 4 Water 20.000 19.900 19.700 19.500 Wheat gluten 15.000 15.000 15.000 15.000 Lupin meal (dehulled) 0.000 5.000 10.000 15.000 Hydrolysed feather meal 15.900 17.100 17.700 16.900 Meat and bone meal 20.000 15.000 7.500 2.500 Poultry offal meal 19.438 20.429 23.932 24.138 Fishmeal 5.900 3.800 2.200 3.000 Lecithin 0.010 0.010 0.010 0.010 Shellgrit 1.000 1.000 1.000 1.000 Choline chloride 60% 0.100 0.000 0.100 0.100 Lysine HCl 0.052 0.161 0.258 0.252 Mycosorb 0.050 0.050 0.050 0.050 Organic acid 0.050 0.050 0.050 0.050 Vitamins and minerals 0.500 0.500 0.500 0.500 Celite 2.000 2.000 2.000 2.000 Experiment No 1 Design Two animals from eight clutches were selected, based on weight and length. The weight ranged from 1470 to 3400 grams and length between 805 to 1050mm. The eight clutches were allocated as two replicates of a randomised incomplete block design. Animals were seventeen months old at the commencement of the experiment. Each diet was allocated to a cage in each of the four rooms according to a Latin square design so that each treatment occurred in each of the cage positions within the rooms. The two animals from each clutch had differing diets. This experiment was conducted over 48 day period from 18th June to 4th August 2004. Animals were fed three times per week on Mondays, Wednesdays and Fridays. Digestibility of diets was determined by collecting spot samples of faeces during the course of the experiment. The faecal samples were collected, bulked and frozen, prior to mixing and subsampling for chemical analysis. Celite was included as an acid-insoluble ash marker in the diets so that total faeces collection was not necessary. Experiment No 2 Design In this experiment only two clutches were used with eight animals from each. Animals had a weight and body length range from 2560 to 3190 grams and 973mm to 1055mm. Again the clutches were allocated as a randomised incomplete block design with two animals each per clutch per room. Animals were approximately nineteen months old at the commencement of the experiment. This experiment was carried out over 65 days from the 8th September to 11th November 2004. The feeding regime was the same as for Experiment 1. Digestibility of diets was determined by collecting spot samples of faeces during the course of the experiment. The faecal samples were collected, bulked and frozen, prior to mixing and subsampling for chemical analysis. Celite was included as an acid-insoluble ash marker in the diets so that total faeces collection was not necessary.

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Results Experiment No 1 Results from this experiment suggest that clutch effect had a significant effect on the results. A pair of animals from one clutch both lost weight. Four animals “sulked” when removed from their group situation and placed in cages. Three animals subsequently lost weight and the fourth animal remained the same weight throughout the experiment period. All animals gained in length. “Sulking” did not appear to be related to the particular treatment each animal was fed. One animal was on the 5% lupin diet, two animals were on the 10% lupin diet and one animal on the 15% lupin diet. There was a reduced weight gain with an increase in the percentage of lupin included in the diet. It should also be noted that no animals on the control diet “sulked”. Estimated weight gain means for the eight clutches (allowing for different diets) ranged from –7% to +26%. Results for total length gain, food eaten and food conversion ratio (FCR) were made without the three animals that lost weight. For percentage total length increase, there was a decreasing trend but it was not significant. All sixteen animals grew in length, even those that lost weight. For food eaten there was a decreasing trend with an increase in lupin level but this was not significant. For FCR there was a decreasing trend as the percentage of lupins increased but due to the small numbers involved this result is questionable. Feed intake was not affected by increasing the percentage of lupin in the diet but there appears some effect on FCR and hence weight gain. No significant difference existed between the digestible crude protein and digestible energy content of the diets, regardless of lupin inclusion level (Table 2), however, limited quantities of faeces were available from crocodiles fed diets containing 10% and 15% lupins, respectively, so some caution is required when interpreting these results.

Table 2. Experiment 1: Crude protein and gross energy digestibility of diets containing 0, 5, 10 or 15% dehulled lupins (L.angustifolius).

Lupin Inclusion % 0 5 10 15

Crude protein 20.3

1 20.21 17.91 17.80

Gross energy 14.01

14.07 11.70 12.48

Despite the fact that there are no differences between diets in terms of crude protein and gross energy digestibility, it is important to note that only 18-20% of the crude protein and 12-14% of the gross energy from manufactured diets is being digested by the crocodiles. If efficiency is to optimised through the use of manufactured diets, then significant improvement is required in the proportion of nutrients digested. This level of digestion will also have an influence on nutrient levels in effluent outflows. Experiment No 2 The second experiment was carried out in part due to four animals that “sulked” in the first experiment and some additional analyses were carried out in this experiment. Results from this experiment are set out in Table 3. In this experiment there was no significant difference between the control and the three treatments. All animals gained weight and grew in length. The difference between clutch again highlights the clutch variation even when attempts to reduce this were made by using only two clutches as opposed to the eight used in Experiment 1. The 15% treatment produced the best result (clutch 1) in all of the six parameters measured while the same treatment, for animals from clutch 2 were at the lower end of performance. The results between the other treatments and control were not significantly different. Lupin content in the diet did not have an influence on feed intake or FCR.

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Table 3. Experiment 2. Growth responses of crocodiles fed diets containing 0, 5, 10 or 15% dehulled lupin meal.

Lupin

content % (L)

Clutch (C)

Incr. in total length (mm)

Incr. in head length (mm)

Incr. in tail circum (cm)

Incr. in body weight (g)

Food eaten (g/animal)

F.C.R.

0 1 91 ab 8.5 a 1.5 bcd 500 bc 940 b 1.91 bc 0 2 41 b 9.0 a 1.5 bcd 630 bc 1131 b 1.80 bc 5 1 39 b 7.5 a 0.7 d 365 c 1017 b 2.76 a 5 2 51 b 3.5 a 1.2 cd 470 bc 1148 b 2.45 ab

10 1 67 ab 9.0 a 2.2 ab 700 bc 1433 b 2.11 abc 10 2 55 b 9.5 a 2.0 bc 795 b 1379 b 1.74 c 15 1 121 a 16.5 a 3.0 a 1235 a 2002 a 1.63 c 15 2 38 b 6.0 a 1.7 bc 560 bc 1065 b 2.05 bc

Sig. effects C (*) L(**), L.C(*)

L(*), L.C(*) L(*), L.C(*) L(*)

Figure 1. Experiment 2: Crude protein and energy digestibility of diets fed to crocodiles containing 0, 5, 10

or 15% dehulled lupin meal. The digestibility data generated in experiment 2 demonstrates that lupin inclusion level has little or no influence on the protein or energy digestibility of the diet (Figure 1). The data suggests that lupin inclusion is actually beneficial, however, the poor relationship between the growth and FCR data and the digestibility data for the control diet suggests that the control diet data may be erroneous and requires further investigation. Important points to note from this data are that crocodiles have a high capacity to digest protein and energy from manufactured diets (relevant to production efficiency and potential environmental impacts of crocodile production), that potential exists to include alternative protein sources in crocodile diets and crocodiles have the capacity to digest diets containing significant proportions of carbohydrate.

-40

-20

0

20

40

60

80

100

Control 5% Lupin 10% Lupin 15% LupinTreatment

Dig

estib

ility

(%)

Digestible crude protein Digestible energy

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Combined Results from experiments 1 & 2: Table 4.30

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When the results from both Experiments are combined, they show no significant response to the use of lupins in the diets and no overall pattern for weight and length gain. The best and worst results were obtained from the 15% lupin diet in terms of weight and length gain. There was large variability in the amount of feed eaten and FCR however indicating a clutch response rather than a treatment response. Discussion This experiment demonstrated:

1. That clutch effects are still significant, even when crocodiles are individually housed, and experimental design must accommodate these influences.

2. The number of replicates required per treatment must be increased if significant differences are to be measured in nutrition experiments.

3. Crocodiles have the capacity to digest significant proportions of protein and energy from manufactured diets, even when the diets contain moderately high levels of carbohydrate.

4. Inclusion of lupins in diets did not reduce growth rates significantly nor did it have any negative effects on the digestibility of protein or energy.

5. At this stage of development, it appears that inclusion of vegetable proteins, such as lupins, in crocodile diets has no effect on intake. However, until the overall intake of manufactured diets can be improved, it cannot be concluded that vegetable proteins represent a real alternative to animal proteins in crocodile diets.

Conclusion At this stage of manufactured diet development, and based on the results of the current experiment, there appears to be some scope to include vegetable proteins at the expense of animal proteins to reduce diet costs. Notes Coulson, R.A. and Hernandez, T. 1983. Alligator metabolism, studies of chemical reactions in vivo. Pergamon Press, New York. Kercheval, D.R. and Little, P.L. 1990. Comparative growth rates of young Alligators utilizing rations of plant and/or animal origin. Volume 1 Proceeding of the 10th Working meeting of the Crocodile Specialist Group of the Species Survival Commission of the IUCN – The World Conservation Union. Gainesville, Florida. USA23rd-27th April 1990 Staton, M.A., Brisbin, I.L., and Pesti, G.M. 1986. Feed formulation for alligators: An overview and initial studies. Proceeding of the 8th Working meeting of the Crocodile Specialist Group of the Species Survival Commission of the IUCN. Quito Ecuador. 13th-18th October 1986 van Barneveld, R.J. (1999). Understanding the nutritional value of lupins to improve livestock production efficiency. Nutrition Research Reviews. 12: 1-30.

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Measuring body condition in farmed crocodiles using Bioelectrical Impedance Analysis At a meeting of the CNG in 2001 the question was raised as to which was the best method of determining body condition in crocodiles. Measuring body condition would be a useful parameter as we develop diets for growing crocodiles. Measuring the tail circumference is one method that could be used. This method can be subjective; it also needs consistency in point of measurement. The idea of using Bioelectrical Impedance Analysis (BIA) as a tool was raised as a possible tool that could be used. BIA technology has been successfully used in health and nutritional studies in humans for the last 30 years and has been used in predicting saleable meat in live cattle and carcases. The technology is relatively cheap, costing $1870.00. It is user friendly, offering a rapid, accurate, non-invasive method of determining body condition. The unit is small and compact and runs on two AA batteries. The size and portability of the unit makes it ideal for use at our research facilities and for use in on-farm trials. Calibration of BIA unit Having investigated the possibility of using BIA in the research program, the next step was to purchase a unit. It was not possible to buy an off the shelf unit that was ready for use on crocodiles so the unit had to be calibrated. This involved a collaborative effort involving Departmental personnel, researchers from the University of Queensland and industry. Twenty male animals were used to determine the prediction model for Total Body Water (TBW). Five individuals in the following weight classes 0-2 kilograms, 2.1-10 kilograms, 10.1-15.0 kilograms and 15.1-20 kilograms were chosen. Animals were aged from 12-36 months, with differing body condition. Seven measurements were taken: • Independent measure of TBW using tritium label water • Estimated measure of extracellular water using Sodium bromide • Individual animal length • Individual animal weight • Individual animal sex • Impedance reading from BIA • Carcass analysis. Independent measurement of TBW The independent measure of TBW is measured by isotope dilution using injections of tritiated water at as dose rate of 0.5mL and 0.5ml/kg of 75 mmol Sodium Bromide was injected directly into the occipital sinus behind the head of the crocodile. After the injections the animals were left for periods of up to four hours to allow for the substances to equilibrate through the animal. A 3mg blood sample was taken from each animal using a 23 gauge 1.5-inch needle tip. The blood was sub-divided into a heparinized mirco-haematocrit tubes for vacuum distillation and into plastic microtubes for centrifuging and plasma collection. Analysis was carried out on the blood samples. Crocodile measurements Selected animals were either caught by hand or captured using electrical stunning equipment depending on their size. Animals less than a metre in total length were caught by hand and those over a metre were stunned. Their length, weight, and sex were recorded and their body condition was visually assessed.

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Measurement using BIA BIA measures an animal’s body impedance to an applied electrical current. The body is divided into two compartments, a lean compartment, which contains virtually all the water and conducting electrolytes and a fat compartment, which contains little water and is hence non-conducting. The value of the impedance combined with the animals length and weight produce a reading of the animals TBW. TBW is a good estimate of lean body mass from which a fat free mass and fat mass can be calculated. Animals were placed on their backs on a dry surface. Colour code alligator leads were attached to the animal using four small fine 25gauge needles. Needles were inserted under the scutes of the animal at the wrist and elbow on the front left leg and on the tail scutes numbers seven and eleven below the cloacal opening. Four measurements are taken: impedance, resistance, phase and reactance for each animal. Figure 4.30 shows an animal with the BIA taking measurements Carcass analysis Carcass analysis was carried out on crocodiles as an additional method to verify that TBW is a good predictor of body condition in crocodiles. The skin was removed from the animals. Whole carcasses of the smaller animals were put through a fine mincer and a sample of 100-150 grams taken. Carcasses from the larger crocodiles were cut vertically down the middle and one half of the carcass was put through the mincer and a 100-150 sub sample was taken. Samples were sent to the Department’s Laboratory at Yeerongpilly to determine percent dry mass, ash, nitrogen and lipid levels. Lipid content expressed as a percentage of wet mass ranged from 6.43 to 18.66%. This analysis allows for the Fat Free Mass to be programmed into the unit.

Development of formula to be programmed into BIA A multiple regression analysis was used on the data collected. Combining the independent TBW, the impedance measurements, crocodile’s length and weight and carcass analysis. A prediction formula has been developed and has been programmed into the unit. The unit has been trialled on animals at the research facility and is relatively simple to use. Use in development of manufactured feed for crocodiles Bioelectrical impedance appears to be a useful tool in developing manufactured feed for crocodiles. The technology is well suited for measuring changes in body composition of individuals or groups of animals over time. BIA can be used to monitor animals on different diets to determine how diets affect body composition. BIA will be useful in establishing benchmarks for crocodiles fed on traditional diets such as chicken heads so comparisons can be made with crocodiles on manufactured diets. The equipment has been used in practice runs and is now ready for use in the trials.

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5. Disease Observations on freshwater crocodile (Crocodylus johnstoni) eggs collected from two farms in northern Queensland Introduction The Australian fresh-water crocodile (Crocodylus johnstoni) is not commonly grown in commercial ventures as its skin does not command the high-grade price received by farmers for the skin of the salt-water crocodile (C. porosus). It is reared mainly in farms where there is also a tourist attraction attached to the commercial enterprise. The ‘freshie’ tends to nest in sandy areas near riverbanks or permanent water sites in the warm, humid parts of Australia – particularly the north of Western Australia, Northern Territory and Queensland. A considerable amount of knowledge has been gathered on the nesting biology, physiology and hatchling capabilities of C. johnstoni and its eggs. Little information has been collected about the microbiological contamination of crocodile eggs, including the shell and yolk. The crocodile egg is composed of a hard shell (mainly calcite) and a reasonably thick, hydrated membrane encompassing a spherical yolk mass within an albumin surround. A thin vitelline membrane encloses the yolk. Differentiation of the fertile egg progresses within the oviduct so that at lay, the embryo is at the 10-20 somite stage. The embryo will attach to the shell membrane within 24 hours of being laid and between 2 and 10 days is at its most vulnerable. Any rotation or shearing due to handling at this time can cause the embryo to detach and die. Therefore transport and handling of eggs is a very important part of the incubation process. Infertile eggs (no sign of embryonic development; no opaque band; no build up of sub-embryonic fluid) are used to approximate the condition that exists in the oviduct before significant embryonic development occurs. The proportion of infertile eggs is quite often dependant on female age, being highest for very young and very old females. Wild animals tend to have a higher fertility rate than captive animals and this may have something to do with the natural diet and movement of the wild crocodiles. Some 217 fresh-water crocodiles eggs (gathered from 19 clutches) were collected from two farms in northern Queensland during one summer season. Although researchers tried to obtain eggs from wild freshwater crocodiles in Queensland, none were forthcoming so any results had to be compared with previous reports of data collected from wild fresh-water crocodiles in the Northern Territory. Results amassed included hatchability; fertility; egg weight, length and width; hatchling weight and length; shell and membrane thickness; yolk and albumen content; bacterial and fungal contamination; water loss.

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Fertility and hatchability Eggs were received from the farms within one week of lay and placed into converted chicken egg incubators that allowed for the placement of approximately 30 eggs over three trays. The eggs were not cleaned or washed before placement but were nested on clean, dry vermiculite at the start of the incubation period. An air stone was used in a tray of water at the bottom of each incubator to maintain humidity over 95%. Water was added to the trays when required. The humidity of most of the incubators (five in all were used) maintained at >98%, however the levels varied from incubator to incubator depending upon the quality of the door seal. The heating lamp was arranged at the top of the incubator and the temperature was maintained at 32oC. However, this arrangement led to decreased moisture levels in the top trays and greater moisture levels in the bottom trays. This was particularly noticed as the incubation progressed and the vermiculite in the lower trays by the end of the trial was soaking wet. No attempt was made to change or dry out the vermiculite, rather allowing the incubation to take its course despite the moisture gradation. The room was air-conditioned during the incubation period. For the purposes of this work, fertile eggs were deduced by the presence of definite banding not by microscopic determination of embryonic growth. Where a band was present but did not progress, this has been classified as ‘Band Not Progress’ (BNP) unless an embryonic form was visible to the naked eye. Then the classification was as follows. Early stage embryonic death was classified by the presence of an embryo to 100mm in total length, mid stage embryonic death was classified by the presence of an embryo from 100 to 200mm in total length while late stage embryonic death was classified by the presence of an embryo greater than 200mm in length. The fertility rate for the freshwater crocodile eggs from the two farms (A and B) was similar – 54.3% for 7 clutches from Farm A and 58.8% for 12 clutches from Farm B. Two clutches from Farm A and three clutches from Farm B had a fertility rate less than or equal to 20%. The hatchability rate (based on the number of eggs hatched from all fertile eggs laid) was 59.1% for Farm A and 82.6% for Farm B. Tables 5.1 and 5.2 summarize the results obtained. It will be noted in Table 5.1 that clutch 4 has been split into two clutches and labelled 4a and 4b. This is because there appeared to be two separate sets of eggs of dissimilar weights (Table 5.4) even though they were sent down as one clutch. Only two clutches from Farm A and four clutches from Farm B had a 100% hatch rate. Visible embryonic death was observed in four clutches (ranging from 1 to 6 eggs per clutch) from Farm A and five clutches (ranging from 1 to 5 eggs per clutch) from Farm B. There are a number of factors that result in infertile eggs and embryonic deaths in crocodile eggs. These include young female breeders laying for the first time, the nutrition of the female breeder, conditions of growth before lay, nest components, nest or incubation temperature and humidity values, cracked eggs (through handling or the female breeder walking on them), excess water loss from or water gain in the egg, and bacterial and/or fungal contamination. Transportation of eggs one day after lay (the embryo is attached to the shell membrane) and before 14 days after lay (when the respiratory and excretory functions are sufficiently developed) can also lead to death of embryos. It is possible that some of the eggs where the band did not progress may have had growth terminated during handling and transportation. Most of these factors and the possibilities of their influencing the eggs received in these trials will be discussed in the appropriate sections.

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Table 5.1 Fertility and hatchability data for freshwater crocodile eggs from Farm A

Clutch Number No. of Eggs in

Clutch No. Eggs Fertile

(%) No. Eggs Hatched

(%) No. Fertile Eggs

Hatched (%) Dead-In-Shell 1 11 10 (90.9%) 10 (90.9%) 10 (100%) 0 2 13 10 (76.9%) 2 (15.4%) 2 (20.0%) 6 (2EED/ 3MED/

1LED) 3 11 10 (90.9%) 8 (72.7%) 8 (88.0%) 1 EED 4a 7 2 (28.6%) 2 (15.4%) 2 (100%) 0 4b 6 1 (16.7%) 0 (0%) 0 (0%) 1 EED 5 18 9 (50.0%) 4 (22.2%) 4 (44.4%) 4 (2 EED/ 1 MED/ 1

LED) 6 15 2 (13.3%) 0 (0%) 0 (0%) 0

Total 81 44 (54.3%) 26 (32.1%) 26 (59.1%) 12 (6 EED/ 4 MED/ 2 LED)

EED – Early stage embryonic death MED – Mid stage embryonic death LED – Late stage embryonic death

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Table 5.2 Fertility and hatchability data for freshwater crocodile eggs from Farm B

Clutch Number No. of Eggs in

Clutch No. Eggs Fertile

(%) No. Eggs Hatched

(%) No. Fertile Eggs

Hatched (%) Dead-In-Shell 1 9 9 (100%) 8 (88.9%) 8 (88.9%) 1 LED 2 10 0 (0%) 0 (0%) 0 (0%) 0 3 12 10 (88.3%) 3 (25.0%) 3 (30.0%) 7 (4 MED/ 3 LED) 4 11 10 (90.9%) 9 (81.8%) 9 (90.0%) 1 LED 5 12 1 (8.3%) 0 (0%) 0 (0%) 0 6 12 9 (75.0%) 3 (25.0%) 3 (33.3%) 4 (3 EED/ 1 MED) 7 10 9 (90.0%) 9 (90.0%) 9 (100%) 0 8 5 4 (80.0%) 4 (80.0%) 4 (100%) 0 9 18 4 (22.2%) 2 (11.1%) 2 (50.0%) 0

10 12 11 (91.7%) 11 (91.7%) 11 (100%) 0 11 10 2 (20.0%) 2 (20.0%) 2 (100%) 0 12 15 11 (73.3%) 6 (40.0%) 6 (54.5%) 2 (1 EED/ 1 LED)

Total 136 80 (58.8%) 57 (41.9%) 57 (82.6%) 15 (4 EED/ 5 MED/ 6 LED)

EED – Early stage embryonic death MED – Mid stage embryonic death LED – Late stage embryonic death

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It is unfortunate that no freshwater crocodile eggs were obtained from the wild in Queensland so that data could be compared. All that could be done is compare our data to published data relating to wild C. johnstoni eggs collected in the Northern Territory. The variation in clutch size, fertility and hatchability rates for the two farms as compared with data from wild NT fresh-water crocodiles are shown in Table 5.3. The clutch size is consistent between the three sets of data. The hatchability of fertile eggs was much higher in Farm B eggs than Farm A even though their fertility rate was similar. The hatchability rate of wild eggs is greatly reduced due to loss of eggs by flooding and predation, a problem not encountered in the farm raised eggs. However, farm incubated eggs can be lost to increased water levels in situations where fertile eggs can take up water, swell and crack. This is usually not a great problem as long as the shell membrane stays intact. Table 5.3 Comparison of clutch size, fertility and hatchability rates for Farm A, Farm B, and wild NT freshwater crocodile eggs

Crocodile

Eggs Clutch Size Fertility Hatchability

(all eggs) Hatchability (fertile eggs)

Farm A 11.6 (6-18) 54.3% 32.1% 59.1% Farm B 11.3 (5-18) 58.8% 41.9% 82.6%

Wild NT 13.2 (4-21) 96.0% 30.0% 31.3% Egg dimensions On receipt of the freshwater crocodile eggs from farms A and B, the eggs were measured for length, width, weight and bandwidth. From these dimensions, egg volume and initial egg mass were determined using equations developed by prior researchers into the biology of crocodile eggs. These measurements and calculations were compared with published reports on wild freshwater crocodile eggs examined in the Northern Territory. The average clutch results are shown in Table 5.4 for Farm A and Table 5.5 for Farm B. Table 5.4 Averages for 7 clutches of freshwater crocodile eggs from Farm A and comparison with published data for wild NT freshwater crocodile eggs

Clutch No.

No. of eggs in clutch

Egg length (cm)

Egg width (cm)

Egg volume (cm3)

Initial egg mass (g)

Egg weight

(g)

Total Clutch wt. (g)

1 11 7.17 4.27 68.4 76.4 78.9 867.9 2 13 6.87 4.23 65.0 71.8 73.4 954.2 3 11 7.04 4.19 65.5 72.3 74.9 823.9 4a 7 7.60 4.26 72.0 80.4 81.0 567.0 4b 6 6.85 3.98 56.5 63.6 68.6 411.6 5 18 7.18 4.39 72.7 81.1 82.6 1486.8 6 15 6.83 4.18 62.6 69.8 71.8 1077.0

Average 13.5 (11-18)

7.06 4.23 66.1 73.6 75.9 1031.4

Wild average

13.2 (4-21)

6.64 4.19 68.2 (50-86)

900

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Table 5.5 Averages for 12 clutches of freshwater crocodile eggs from Farm B and comparison with published data for wild NT freshwater crocodile eggs

Clutch No.

No. of eggs in clutch

Egg length (cm)

Egg width (cm)

Egg volume (cm3)

Initial egg mass

(g)

Egg wt.(g)

Total Clutch wt.

(g) 1 9 7.20 4.42 73.6 82.1 80.1 720.9 2 10 6.94 4.32 67.8 75.7 74.0 740 3 12 7.15 4.33 70.1 78.3 79.4 952.8 4 11 6.93 4.28 66.3 73.9 72.7 799.7 5 12 6.55 4.25 61.9 69 69.5 834 6 12 6.84 4.21 62.5 70.7 71.5 858 7 10 6.76 4.22 63.6 71.1 71.6 676 8 5 7.06 4.36 70.4 78.6 79.4 397 9 18 6.88 4.11 60.8 67.9 69.2 1245.6

10 12 7.17 4.14 64.3 71.6 71.8 861.6 11 10 6.79 4.04 57.9 64.7 63.8 638 12 15 7.17 4.21 66.4 74.2 75.3 1129.5

Average 11.3 (5-18) 6.95 4.23 65.4 73.2 73.2 821.1 Wild

average 13.2

(4-21) 6.64 4.19 68.2

(50-86) 900

Initial egg mass in grams was calculated from egg width and length using the equation 11.6L +29.7B – 134, where L equals the length of the egg, B the breadth or width of the egg. This could be used to estimate the original mass when the weight of the egg at date of lay is unknown. As can be seen from the results in both Tables, it equates well to the weight measured at time of receipt. It is often more accurate to compare egg or crocodile weight to egg volume than it is to compare it to either egg width or length. Figures 5.1 and 5.2 show the comparison between egg volume and egg weight for the eggs from the respective Farms. Thus if it is not possible to weigh crocodile eggs on receipt or in the field, it could be estimated from the volume which can be easily determined using the equation KmxLxB2 where L equals the length of the egg, B the breadth or width of the egg and Km is a coefficient factor calculated for specific eggs. For C. johnstoni, Km = 0.584.

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Figure 5.1 Egg Weight vs Egg Volume for Farm A

Figure 5.2 Egg Weight vs Egg Volume for Farm B The average total clutch weight was calculated using the average values for the number of eggs in the clutch times the average weight per egg. For wild freshwater eggs from the Northern Territory, it approximates to 900g. Farm A averaged 1031g and Farm B averaged 821g. Total clutch mass is regarded as an indicator of the size of the female breeder. The breeder contains the entire clutch in the abdomen before laying so the bigger the crocodile, then the bigger the mass (4-5% of body weight) she is able to hold. The average egg weights for eggs from both farms were heavier than those from the wild in the NT. This is no doubt due to the captive females being larger animals than their wild counterparts. Egg width was reasonably consistent at 4.19cm for wild NT eggs and 4.23cm for eggs from both the Farms. The widest egg was 4.56cm for Farm A and 4.57cm for Farm B and the narrowest egg was 3.82cm for Farm A and 3.97cm for Farm B. Mean egg width is also a

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good indicator of female breeder size. The width is a good indication of the oviduct size that is proportional to the body size. The length of the eggs was more varied. Reported data showed an average of 6.64cm for the wild NT eggs while our data showed 7.06cm for Farm A and 6.95cm for Farm B. The longest egg was 7.76cm for Farm A and 7.54cm for Farm B while the shortest eggs were 6.32cm and 6.21cm for Farms A and B respectively. Water loss and crocodile dimensions Crocodile eggs are calcareous and contain a large number of pores of various sizes. This fulfils the basic need to be strong and rigid enough to survive the laying process but also porous enough to allow gaseous exchange for embryonic respiration with only minimal loss of water to the environment. The egg also has to withstand any ingress by bacterial or fungal organisms. Water loss or gain and gaseous exchange between the internal egg and the external environment are very important functions during the growth of the embryo. As incubation proceeds, the ever-growing embryo requires a more efficient gas exchange. Towards the end of incubation, there tends to be enlargement of the pore openings and a weakening of the shell as incubation proceeds. Minerals from the shell are removed and used by the growing embryo. This is mainly with reference to Ca++ but other ions are also utilised. This form of intrinsic degradation has another function in weakening the shell for an easier exit by the hatchling from the egg. The pores go through the shell via a tortuous route ending in contact with the shell membrane that also contains pores. This provides a means of slowing down but not preventing microbial ingress into the egg. The shell membrane (which is quite thick in crocodilian eggs) is the last line of defence against microbial entrance. Normally, water loss is maintained in the range 1-15% for a healthy hatchling to emerge. Losses greater than this will result in massive dehydration and embryonic death. Conditions where there is water gain with accompanying swelling of the egg and associated cracking of the shell can be just as harmful to the growing embryo as too much loss of water. Successful hatching can occur as long as the membrane stays intact. It has been reported that hatchlings of Crocodylus novaguinea can emerge from eggs with a water loss range of –25% to +25%. The diffusion of water and gases through the pores depends on the size and number of pores in the shell, the openness of the pores (age of the egg) and the percentage of water vapour in the atmosphere in the nest or the hatching incubator. The number of eggs placed in an artificial incubator is very important. Generally, there should be 1 kg eggs per 12 litres of free space. Our calculations for the converted chicken incubators allowed the presence of 30 eggs per incubator (10 eggs on each of the 3 trays). Daily opening of the doors to allow the required gaseous exchange, however, also meant an increase in water loss from the incubators. Air stones were used in trays of water in the bottom of the incubators in an attempt to maintain >98% humidity. However, the placement of the heating lamp at the top of the incubator resulted in a gradient of humidity from drier areas in the top trays to moister areas in the bottom trays. In the wild, shells give high conductance to water vapour and because of the rigid nature of the shell, air spaces do not usually develop. In artificial environments, however, especially

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where the control of humidity is below 100% and dehydration is evident, air sacs do occur, mainly between the calcareous shell and the shell membrane. Air spaces were frequently observed with the incubation regime because humidity levels often dropped just below the preferred level in the converted incubators. The exchange of oxygen and carbon dioxide was not investigated throughout this study but the effect of water loss and gain was studied. The average values obtained during the observation study for egg weight, crocodile weight and length (where applicable) and weight loss of the eggs over the 8 week period are shown in Table 5.6 for Farm A and Table 5.7 for Farm B. Weights were measured on a 2 week basis where possible. Comparison with results obtained for wild NT freshwater crocodile eggs are also shown. Table 5.6 Averages for crocodile weight and length compared to % water loss for 7 clutches of freshwater crocodile eggs from Farm A and comparison with wild NT freshwater crocodile egg figures

Clutch No.

No. of eggs in clutch

Egg vol.

(cm3)

Init. egg

mass (g)

Egg wt. (g)

Egg wt. loss (%) (8 wks)

No. of crocs

hatched

Croc. wt. (g)

Croc. wt./ Egg wt. (%)

Croc. length (mm)

1 11 68.4 76.4 78.9 4.3 10 49.3 62.5 ND 2 13 65.0 71.8 73.4 30.7 2 43.4 56.7 ND 3 11 65.5 72.3 74.9 6.1 8 46.5 61.9 ND 4a 7 72.0 80.4 81 4.9 2 ND ND ND 4b 6 56.5 63.6 68.6 4.7 0 NA NA NA 5 18 72.7 81.1 82.6 9.4 4 52.0 63.1 ND 6 15 62.6 69.8 71.8 Infertile/

BNP 0 NA NA NA

Average 13.5 (11-18)

66.1 73.6 75.9 9.85 3.7 47.8 61.1 ND

Wild average

13.2 (4-21)

68.2 (50-86)

42 (33-56)

65 244

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Table 5.7 Averages for crocodile weight and length compared to % water loss for 12 clutches of freshwater crocodile eggs from Farm B and comparison with wild NT freshwater crocodile egg figures

Clutch No.

No. of eggs in clutch

Egg vol.

(cm3)

Initial egg mass (g)

Egg weight

(g)

Egg weight

loss (%) (8 wks)

No. of crocs

hatched

Croc. Wt. (g)

Croc. Wt./ Egg wt. (%)

Croc. length (mm)

1 9 73.6 82.1 80.1 14.7 (t) 8 48.2 60.3 252.3 2 10 67.8 75.7 74.0 All

Infertile 0 NA NA NA

3 12 70.1 78.3 79.4 3.1 (b) 3 55.7 69.3 257.5 4 11 66.3 73.9 72.7 13.9 (t) 9 41.3 57.3 230.5 5 12 61.8 69 69.5 All

infertile 0 NA NA NA

6 12 62.5 70.7 71.5 5.7 (b/t) 3 44.8 61.8 251.0 7 10 63.6 71.1 71.6 4.9 (m) 9 47.3 66.0 256 8 5 70.4 78.6 79.4 1.3

(m/b) 4 53.2 66.7 263.0

9 18 60.8 67.9 69.2 7.7 (b/t) 2 ND ND ND 10 12 64.1 71.6 71.8 4.7 (m) 11 46.3 64.8 255.0 11 10 57.9 64.7 63.8 5.4 (b) 2 45.3 67.4 240.0 12 15 66.4 74.2 75.3 6.5 (t/m) 6 ND ND NA

Average 11.3 (5-18)

65.4 73.2 73.2 6.1 4.75 47.8 64.2 250.7

Wild average

13.2 (4-21)

68.2 (50-86)

42 (33-56)

65 244

As expected, there was a correlation between crocodile weight and laying egg weight (Figures 5.3 and 5.4), and the ratio between the two values (crocodile weight/egg weight), averaged 61.1% for Farm A and 64.2% for Farm B. The average figure for wild NT eggs was 65%. Figures 5.5 and 5.6 show the relationship between crocodile weight and egg volume for the eggs from both Farms.

Figure 5.3 Crocodile Weight vs Egg Weight for Farm A

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Figure 5.4 Crocodile Weight vs Egg Weight for Farm B

Figure 5.5 Crocodile Weight vs Egg Volume for Farm A

Figure 5.6 Crocodile Weight vs Egg Volume for Farm B

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Crocodile length was only measured for Farm B and this showed a positive relationship to crocodile weight (Figure 5.7). It was apparent that the incubators varied in their ability to maintain the humid atmosphere required for the incubation of crocodile eggs. Incubators 4, 5 and 7 maintained the humidity levels better than incubators 1 and 9 and this was possibly dependant on the door seals, the air stones and quality of the heating lamps even though these were of the same brand. As well as this, a humidity gradient existed in each incubator due to the positioning of the heating lamp at the top of the incubators. However, overall water loss was consistent for each clutch irrespective of their positioning in the incubator and the status of the egg.

Figure 5.7 Crocodile Weight vs Crocodile Length for Farm B This consistent water loss or, in some cases, gain for the clutches can be seen in Figure 5.8 for eggs from Farm A and Figure 5.9 for eggs from Farm B where the average egg weight change for the clutches from both farms are displayed. In the situation where water was taken up (Farm B), there was a slight decrease in egg weight before water access occurred. For each clutch, a polynomial, one-degree regression analysis was calculated and the equation listed on the respective graphs.

Figure 5.8 Average Egg Weight Loss for 7 Clutches from Farm A

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Figure 5.9 Average Egg Weight Loss for 10 Clutches from Farm B The water loss from the clutches on Farm A showed a fairly consistent drop over the 8 weeks (Figure 5.8; Table 5.8). However, clutch 2 had a rate at more than four times the other clutches. The average water loss was 20.5% (16.1 to 27.2%) excluding the two cracked eggs that had losses of 51.5% and 62.7%. Even the two hatched eggs had water losses of 16.1% and 17%. The reason for this could have been due to the humidity not being kept high enough during the incubation period but more likely due to a clutch effect that may depend on shell thickness and quality, and/or pore size and number. Shell thickness will be discussed in the next section; however, in this case, the shell thickness of the clutch was far thinner than other clutches from the same farm (Table 5.11), possibly allowing for easier and faster passage of gases and water vapour. Table 5.8 The relationship of average % water loss of freshwater crocodile eggs from Farm A to clutch and status. The range is indicated in brackets.

Clutch Hatch DIS Infertile Infertile/Infected Total

A1 3.5

(2.3 to 5.5) 3.4 3.5

(2.3 to 5.5)

A2 16.6

(16.1 to 17) 20.2

(18 to 21.8) 18.4 26.2

(25.2 to 27.2) 20.5

(16.1 to 27.2)

A3 4.5

(3.2 to 5.9) 3.9 5.9 4.6

(3.2 to 5.9)

A4a 7.4

(6.2 to 8.5) 3.7

(1.3 to 6.1) 4.9

(1.3 to 8.5)

A4b 4 4.8

(2.5 to 9.9) 4.7

(2.5 to 9.9)

A5 8.4

(5.9 to 11.3) 7.1

(6.1 to 8.6) 5.3

(3.7 to 7.6) 6.4

(4.1 to 13.1) 6.8

(3.7 to 13.1)

Total 5.9

(2.3 to 17) 13.1

(6.1 to 21.8) 7.5

(3.4 to 18.4) 8.9

(1.3 to 27.2) 7.9

(1.3 to 27.2) It was observed that the average water loss for hatched eggs from Farm A was 5.9% (with a range of 2.3% to 17%). DIS eggs had an average water loss of 13.1% (6.1% to 21.8%). The average water loss for all clutches was 7.9% (with a range of 1.3% to 27.2%). The six cracked eggs that showed a water loss of 14.4% to 62.7% were not included in the calculations.

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When the eggs from Farm B were received, it was noted which incubators they were placed in as well as the position of the trays. As there were 55 more eggs to incubate, more of the converted chicken incubators were required. The rate of water loss from the clutches on Farm B also showed a fairly consistent drop over the 8 weeks (Table 5.9). However, there was more variation between the clutches from this Farm and this is more likely due to humidity variations in the incubators than to clutch effect. Clutch B3 had 10 of the 12 eggs placed in the bottom tray of incubator 9 and this resulted in 7 DIS eggs (water gains of 1.6% to 14.3%). The remaining two eggs were in the top tray of the same incubator and both hatched with water losses of 6.4% and 7.1%. Table 5.9 The relationship of average % water loss of freshwater crocodile eggs from Farm B to clutch and status. The range is indicated in brackets.

Clutch Hatch DIS Infertile Infertile/Infected Total

B1 9.6

(7.6 to 11.2) 23.2 11.1

(7.6 to 23.2)

B3 1.4

(+9.3 to 7.1) plus 7.1

(+14.3 to +0.5) 6.4 plus 4.2

(+14.3 to 7.1)

B4 9.5

(6.2 to 12.1) 18.9 10.4

(6.2 to 18.9)

B6 6.3

(3.3 to 10.2) 3

(1.6 to 5.1) 6.4

(2.3 to 12.3) 5

(1.6 to 12.3)

B7 3.6

(3 to 4.3) 1.4 3.4

(1.4 to 4.3)

B8 3

(0.3 to 4.4) 0.3 2.5

(0.3 to 4.4)

B9 1.9

(1.5 to 2.2) 1.8

(0.7 to 3.1) 11.8

(1 to 18.5) 7.2

(0.7 to 18.5)

B10 3.5

(1.5 to 5.7) 1.6 3.3

(1.5 to 5.7)

B11 4.5

(4.3 to 4.6) 0.4

(0.2 to 0.5) 0.4

(0.3 to 0.6) 1.4

(0.2 to4.6)

B12 3.9

(2.3 to 4.5) 3.4 4.4 3.5

(2.3 to 5)

Total 5.3

(+9.3 to 12.1) 0.71

(+14.3 to 23.2) 1.2

(0.2 to 3.1) 6.8

(0.3 to 18.5) 4.4

(+14.3 to 23.2) It was observed that the average water loss for hatched eggs from Farm B was 5.3% (with a range of +9.3% to 12.1%). DIS eggs had an average water loss of 0.71% (+14.3% to 23.2%). The average water loss for all clutches was 4.4% (with a range of +14.3% to 23.2%). Thus, with the incubation regime used, the average water loss from the eggs for both farms was similar (5.9% for Farm A and 5.3% for Farm B). Hatched eggs occurred over a range of water gain or loss from +9.3% to 17%. DIS eggs usually occurred when the water gain or loss was too great (+14.3% to 23.2%). Infertile eggs were characterised as either infertile (I) or infertile/infected (I/I), based on the isolation of bacteria and/or fungi from the contents of the eggs when cracked and examined at the end of the trial. Farm B results showed that infertile eggs (1.2%) lost less water than hatched eggs (5.3%) but infertile/infected eggs lost more water than hatched eggs (6.8%)(Table 5.10). This could be due in part to degradation of the membrane and shell of

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the egg by the bacterial and/or fungal contaminants present thus allowing for easier exchange of the gases and water vapour required for microbial growth. As expected, the eggs in the top trays lost more water than those of the middle tray and those in turn lost more water than those of the bottom tray. In the bottom tray, there was often water gain due to the vermiculite bedding absorbing the water in the atmosphere. Table 5.10 shows that this gradient was observed with all status of eggs from hatched through to infertile/infected. Table 5.10 The relationship of average % water loss of freshwater crocodile eggs from Farm B to status and incubator position. The range is indicated in brackets.

Incubator Level Hatch DIS Infertile Infertile/Infected Total

Top 7.8

(2.3 to 12.1) 9.9

(1.6 to 23.2) 3.1 11.95

(4.4 to 18.5) 8.8

(1.6 to 23.2)

Middle 4.1

(2.3 to 9.7) 3.4 1.6 1.4 3.8

(1.4 to 9.7)

Bottom Plus 0.4

(+9.3 to 2.2) Plus 4.7

(+14.3 to 5.1) 0.85

(0.2 to 2.5) 2.3

(0.3 to 9.2) Plus 0.7

(+14.3 to 9.2)

All levels 5.3

(+9.3 to 12.1) 0.71

(+14.3 to 23.2) 1.2

(0.2 to 3.1) 6.8

(0.3 to 18.5) 4.4

(+14.3 to 23.2) This ranged from 8.8% on the top trays to +0.7% in the bottom trays. These observations have reinforced the suggestions that controlled humidity plays a great part in the incubation process of crocodile eggs. Even so, crocodile eggs can survive a wide range of water loss and gain and this is probably related to clutch effect factors that include shell thickness, pore size and numbers, and greater hindrance to contamination. Previous work has indicated that there is a variable but significant increase in egg size with clutch size. This was not evident with our study, however only 19 clutches were examined. It was evident however, that crocodile weight and length were dependant upon the original egg mass rather than to percentage water loss.

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Shell and membrane thickness The crocodile egg has a thick shell and membrane when compared to other eggs, such as bird or turtle eggs. The eggs are calcareous and the thickness varies within the crocodile species. For example, some of the known values (taken at lay) are:- Alligator mississippiensis 0.45-0.65 mm Alligator sinensis (researchers 1) 0.4-0.5 mm Alligator sinensis (researchers 2) 0.3-0.38 mm Crocodylus niloticus 0.45 mm The shell is thicker in the equatorial region than it is at either end. For example, some known values are:- Alligator mississippiensis (at hatch) End – 0.374 mm Equator – 0.413 mm Alligator sinensis (at hatch) End – 0.305 mm Equator – 0.343 mm The thickness also decreases during the incubation process from lay to hatch for both regions, for example, with A. mississippiensis:-

Age of Eggs (Days) Region 0-17 33 44-49 54-63 65-73

End 0.448mm 0.446mm 0.414mm 0.383mm 0.374mm Equator 0.449mm 0.478mm 0.43mm 0.412mm 0.413mm This decrease is due to the shell weakening during the growth phase by intrinsic degradation (Ca++ and other ions removed from the shell by the growing embryo) and to loss of water from the shell. This loss of water is noted as an increase in opacity of the shell or banding which indicates the growth of the embryo and corresponding retraction of the albumen to the poles of the egg during this growth phase. Extrinsic degradation can also occur when nesting material is present, especially in alligator eggs. Both intrinsic and extrinsic degradation is helpful for the hatchling by weakening the shell (especially at the poles) for exit from the egg at hatch. The crocodile egg contains a large number of pores of various sizes that allow gaseous and water exchange for the growing embryo. There are more pores around the equatorial region than at the ends. At lay, the pores have a small surface opening which tend to enlarge as incubation proceeds. The pores do not go straight through to the shell membrane but meander by a tortuous route thus acting as a very useful barrier to bacterial and fungal contaminants. Research has shown that 6% of the pores are open at incubation while some 22-24% of the pores are open at hatch. The rest of the pores tend to be clogged with water and solutes, something to be expected when incubation occurs under conditions of high humidity, especially in the nesting situation in the wild. Porosity, thickness or quality of the shell is not dependant on the size of the crocodile egg but is often related to clutch effect, which is in turn related in part to the age, health and diet of the female crocodile laying the eggs.

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Eggshell conductance refers to the quantity of a given gas that can diffuse in a unit of time through the pores of an eggshell. Conductance is proportional to the area of pores available for diffusion and inversely proportional to the length of the diffusion path (length of the pores). Conductance of respiratory gases (oxygen and carbon dioxide) and water exchange are obviously of great importance for the growth and well being of the embryo/hatchling. Water loss occurs during the incubation phase as mentioned previously. Shells that lose more water also lose more carbon dioxide; and extrinsic degradation by nesting material in wild reared crocodiles will enhance this procedure. However, oxygen exchange does not occur quite as easily. The shell membrane, an interwoven mesh of fibres that also contains pores, is the principal barrier to oxygen exchange due to its relative thickness (0.1 to 0.2 mm depending on the crocodilian species) and state of hydration. There is a range of pore sizes in the eggshells of avian or reptilian species. However, because crocodile nests contain high humidity, there is less need for smaller pores that are essential to minimize water loss. The shell and membrane thicknesses of a reasonable proportion of the freshwater crocodile eggs that were observed were measured during these studies. Tables 5.11 and 5.12 show the average values for eggs from Farm A and B respectively. Because of the significant differences between individual clutches of crocodile eggs, the only valid comparisons that can be made are those between infertile and fertile eggs of the same clutch. However, the overall picture is still interesting and shows similar values for previous work done with numerous crocodile species. Overall for Farm A, hatched eggs had a shell thickness at the equator of 0.339 mm and at the ends of 0.308 mm with a membrane thickness of 0.129 mm. As expected, infertile eggshells were thicker and averaged 0.386 mm at the equator and 0.347 mm at the ends. The ratio of the hatched to infertile values for thickness at the equator and the end of the eggs was 0.88 and 0.89 respectively, indicating a consistent degradation of the shell of growing embryos during the incubation process. For Farm B, hatched eggs had a shell thickness at the equator of 0.377 mm and at the ends of 0.339 mm with a membrane thickness of 0.137 mm. Infertile eggshells were thicker and averaged 0.408 mm at the equator and 0.373 mm at the ends. The ratio of the hatched to infertile values for thickness at the equator and the end of the eggs was 0.92 and 0.91 respectively, indicating a similar rate of degradation of the egg shell as in Farm A. It has been mentioned that the ends of the crocodile egg are thinner than the equatorial regions and this was consistent over both farms with a ratio of 0.91. This covered infertile, DIS and hatched eggs. Research done with ostrich eggs has shown that the larger the egg, the bigger the chick. Also, intermediate size eggs have the best hatching rate. The results from this work on freshwater crocodile eggs have shown a similar pattern. Table 5.13 shows the average values for hatching rates, hatchling weight and shell thickness when the eggs have been sorted into lots of <70g, 70-80g and >80g for both Farm A and Farm B. The best hatching rates for Farm A were in the 70-80g group (61.7% of the eggs) while on Farm B, eggs greater than 70g gave the best hatching rates. The larger hatchlings were from eggs greater than 80g. There was a decrease in shell thickness at both the end and the equatorial region from the smaller to the larger eggs on Farm A. The trend was slightly different on Farm B where the larger eggs maintained a thicker shell in the equatorial region (Table 5.13).

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Table 5.11 Average values of shell and membrane thickness for 6 clutches of freshwater crocodile eggs from Farm A

Croc. Clutch

H/Inf/Con

Croc. Weight

(g)

Shell Thickness

[end (mm)]

Shell Thickness [equator

(mm)]

Membrane Thickness

(mm) Shell Weight

(g) Ratio

End/Equator Ratio H/I

End / Equator A1 (10) H 49.3 0.317 0.353 0.137 NA 0.9 NA NA A1 (1) I NA ND ND ND NA NA A2 (2) H 43.4 0.297 0.285 0.109 NA 1.04 NA NA A2 (6) DIS NA 0.292 0.338 0.12 NA 0.86 A2 (5) I/BNP NA ND ND ND NA NA A3 (8) H 46.5 0.302 0.344 0.129 NA 0.88 NA NA A3 (1) DIS NA ND ND ND NA NA A3 (2) I/BNP NA ND ND ND NA NA A4a (2) H ND 0.27 0.311 0.095 NA 0.87 0.88 0.82 A4a (5) I/BNP NA 0.307 0.38 0.12 NA 0.81 A4b (1) DIS NA ND ND ND NA NA NA NA A4b (5) I/BNP NA ND ND ND NA NA A5 (4) H 52 0.328 0.337 0.137 NA 0.97 0.99 0.94 A5 (4) DIS NA 0.347 0.392 0.147 NA 0.89

A5 (10) I/BNP NA 0.331 0.357 0.14 NA 0.93 A6 (15) I/BNP NA 0.371 0.401 0.118 NA 0.93 NA NA Avg. for Farm A 47.8 0.327 0.362 0.128 NA 0.9 81 eggs

H 0.308 0.339 0.129 NA 0.91 0.89 0.88 DIS 0.314 0.36 0.131 0.87 I/BNP 0.347 0.386 0.126 0.9

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Table 5.12 Average values for shell and membrane thickness for 12 clutches of freshwater crocodile eggs from Farm B

Croc. Clutch

H/Inf/Con

Croc. Weight

(g)

Shell Thickness

[end (mm)]

Shell Thickness [equator

(mm)]

Membrane Thickness

(mm) Shell Weight

(g) Ratio

End/Equator

Ratio H/I End

Ratio H/I Equator

B1 (8) H 48.2 0.311 0.335 0.137 5.34 0.93 NA NA B1 (1) DIS ND ND ND ND ND NA

B2 (10) I 43.4 0.32 0.361 0.154 8.1 0.89 NA NA B3 (3) H 55.7 0.36 0.41 0.143 4.2 0.88 0.89 0.95 B3 (7) DIS NA 0.361 0.38 0.117 4.89 0.95 B3 (2) I NA 0.405 0.43 ND 7.3 0.94 B4 (9) H 41.3 0.3 0.328 0.135 4.42 0.91 NA NA B4 (1) DIS ND 0.26 0.347 ND ND 0.75 B4 (1) I NA ND ND ND ND NA

B5 (12) I/BNP NA 0.322 0.345 0.119 7.7 0.93 NA NA B6 (3) H 44.8 0.38 0.403 0.16 4.58 0.94 0.89 0.89 B6 (4) DIS ND 0.44 0.447 ND ND 0.98 B6 (5) I/BNP NA 0.426 0.453 ND ND 0.94 B7 (9) H 47.3 0.374 0.439 0.143 4.43 0.85 NA NA B7 (1) I NA ND ND ND ND NA B8 (4) H 53.2 0.36 0.405 0.139 5.59 0.89 NA NA B8 (1) I NA ND ND ND ND NA B9 (2) H ND 0.33 0.381 0.157 4.75 0.87 0.82 0.85

B9 (16) I/BNP NA 0.402 0.446 0.136 5.82 0.9 B10 (11) H 46.3 0.323 0.356 0.126 4.54 0.91 0.83 0.84 B10 (1) I NA 0.39 0.423 ND 5.68 0.92 B11 (2) H 45.3 0.377 0.413 0.137 3.64 0.91 1.02 0.99 B11 (8) I NA 0.369 0.418 0.13 5.14 0.88 B12 (6) H ND 0.353 0.392 0.129 5.99 0.9 0.83 0.88

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Croc. Clutch

H/Inf/Con

Croc. Weight

(g)

Shell Thickness

[end (mm)]

Shell Thickness [equator

(mm)]

Membrane Thickness

(mm) Shell Weight

(g) Ratio

End/Equator

Ratio H/I End

Ratio H/I Equator

B12 (2) DIS NA 0.393 0.405 0.155 ND 0.97 B12 (7) I/BNP NA 0.425 0.448 ND 5.73 0.95 Avg. for Farm B 136 eggs 46.8 0.359 0.394 0.135 5.7 0.91 0.91 0.92

H 0.339 0.377 0.137 4.81 0.91 DIS 0.381 0.4 0.125 4.89 0.95 I/BNP 0.373 0.408 0.134 0.91

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Table 5.13 Average values for hatchling weight and egg shell thickness with regard to the weight of the egg at lay and hatching rates for Farms A and B. Clutch effects were not taken into account.

Number of Crocodiles

Hatched

Egg Weight

(g)

Crocodile Weight (g)

Shell Thickness [Equator (mm)]

Hatched/Infertile

Shell Thickness [End (mm)]

Hatched/Infertile Farm A

11/81 - 13.6% 0/11 - 0% <70 NA NA / 0.41 0 / 0.376 50/81 - 61.7% 21/50 - 42% 70-80 47.6 0.336 / 0.384 0.303 / 0.354 20/81 - 24.7% 5/20 - 25% >80 51.9 0.351 / 0.357 0.327 / 0.331

Farm B 39/136 - 28.7% 12/39 - 30.8% <70 42.5 0.357 / 0.406 0.324 / 0.372 84/136 - 61.8% 38/84 - 45.2% 70-80 46.9 0.357 / 0.378 0.333 / 0.343 13/136 - 9.6% 7/13 - 53.8% >80 51.2 0.349 / 0.457 0.321 / 0.412

The albumen and yolk content plus shell weight for a number of the freshwater eggs on Farm B were measured. Average results are shown in Table 5.14. Previous workers have estimated that the eggshell and membrane for C.johnstoni comprise 11.9 ± 0.3% of the total egg weight. The shell alone was measured and it was found that for infertile eggs; the average shell weight was 9.5% (8.1 to 11.2%). For hatched eggs, it was found the average shell weight was 6.6% (5.4 to 7.4%), again showing the degradation of the shell during the incubation process.

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Table 5.14 Averages for shell thickness, shell weight and egg contents for eggs where data was available for 12 clutches from Farm B. Hatch to infertile (H/I) values are shown where applicable.

Shell thickness (mm) Croc. Clutch Status Equator End Membrane

Albumin (g)

Yolk (+VM)

(g)

Shell weight

(g) Total

(g)

Egg weight at

collection (g)

Shell wt/ egg wt

(%) B1 8/9 H 0.335 0.311 0.137 ND ND 5.34 ND 80 6.7 B2 8/10 I 0.361 0.32 0.154 21.9 30.7 8.1 61.7 72.8 11.1

3/12 H 0.412 0.356 0.143 ND ND 4.2 ND 78.7 5.4 B3 1/12 I 0.43 0.405 0.117 23.7 36.4 7.3 67.4 79.4 9.2

B4 9/11 H 0.328 0.3 0.135 ND ND 4.42 ND 63.3 7 B5 11/12 I 0.345 0.322 0.119 23 25.9 7.7 58.4 68.5 11.2 B6 1/12 H 0.403 0.4 0.16 ND ND 4.58 ND 72.3 6.3 B7 3/10 H 0.439 0.374 0.143 ND ND 4.43 ND 67.9 6.5 B8 2/5 H 0.405 0.36 0.139 ND ND 5.59 ND 78.4 7.1

2/18 H 0.381 0.33 0.156 ND ND 4.75 ND 63.9 7.4 B9 6/18 I 0.449 0.409 0.137 23.5 28.4 5.82 59.9 66.4 9 10/12 H 0.355 0.322 0.126 ND ND 4.6 ND 68.4 6.6

B10

1/12 I 0.423 0.39 ND 23.3 31.2 5.8 60.3 71.9 8.1 2/10 H 0.413 ND 0.137 ND ND 3.7 ND ND ND B11 4/10 I 0.418 0.36 0.13 22.7 25.8 5.2 51.9 60.4 8.3 4/15 H 0.392 0.353 0.129 ND ND 6 ND ND ND B12 2/15 I 0.452 0.419 ND ND ND 7.3 ND ND ND

Calculation of the size and number of pores in the eggshells was not attempted at this time but shells have been cleaned and stored for future reference. Bacterial and fungal contamination of farmed freshwater crocodile eggs The bacterial and fungal contamination of 81 freshwater crocodile eggs from Farm A (7 clutches) and 136 freshwater crocodile eggs from Farm B (12 clutches) was examined with reference to infertility, embryonic death and hatchability. The bacterial and fungal content of 7 freshwater crocodile nests from Farm B was also determined. Eggs were received from the farms within one week of lay and placed into converted chicken egg incubators that allowed for the placement of approximately 30 eggs over three trays. The eggs were not cleaned or washed before placement. An air stone was used in a tray of water at the bottom of each incubator to maintain humidity over 98%. The heating lamp was arranged at the top of the incubator and this led to greater moisture levels in the bottom trays and decreased moisture levels in the top trays. This was particularly noticeable as the eggs were placed on vermiculite during the incubation period. The vermiculite in the lower trays by the end of the trial was soaking wet. The temperature was maintained at 32oC. Egg length, egg width, bandwidth and egg weight were measured on receipt. Bandwidth and egg weight were then measured at approximately two week intervals until hatching date. The eggs from the three infertile clutches A6, B2 and B5 were removed after 4 weeks for examination. All other infertile eggs remained in the incubator, until the fertile eggs

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commenced hatching, so that comparison of shell thickness could be made (see Shell and membrane thickness). Eggs were submitted to the laboratory for processing. Eggs were first washed in a sterile stomacher bag with 10mL of buffered peptone water (BPW). They were then massaged until the shell appeared clean. The liquid was drained off and one drop inoculated onto plates of sheep blood agar (SBA), MacConkey agar (MA), brilliant green agar (BGA), lactose-maltose-glycerol agar (LMG), Sabourad agar (SDA) and mycosel agar (MAT). These agar plates along with the remainder of the BPW were incubated at 37oC for 2 days (SBA, MA, BGA, LMG) while the SDA and MAT plates were incubated at 28oC for 7-10 days. Washed eggs were then soaked in iso-propanol and flamed before being cut open with sterile scissors and forceps and the contents poured into sterile Petri dishes. Cracked eggs or eggs split open by excess water uptake were gently washed in the iso-propanol and allowed to dry before opening. Eggs from which a hatchling emerged were carefully washed on the outside for culture, and swabs were then taken from the inner contents. The swabs were added to 10mL of BPW and then inoculated onto the media listed above. The egg contents were mixed and samples taken onto the same set of agar plates and broth. Incubation was as mentioned above. Observations on the state of the albumen and yolk were noted and comments made on the presence of any embryos visible to the native eye. MED and LED embryos were measured where possible. Bacteria isolated were identified by conventional tests, or by kit determination (API20E, API20NE, Microbact24E, etc). Salmonella species were sent to IMVS in Adelaide for further typing. Fungi were identified by the morphological properties of the culture growth and their microscopic appearance. A summary of the bacteria and fungi isolated from the eggs from Farm A and Farm B are shown in Table 5.15 and Table 5.16 respectively.

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Table 5.15 Distribution of bacteria and fungi on freshwater crocodile eggs from Farm A

Bacterium

Clutch A1 11 eggs

11 eggs tested shell=yolk (%)

10 hatch

Clutch A2 13 eggs

10 eggs tested shell=yolk (%)

2 H; 6 DIS

Clutch A3 11 eggs

10 eggs tested shell=yolk (%)

8 H; 1 DIS

Clutch A4 (a&b)

13 eggs 13 eggs tested shell=yolk (%) 2 hatch; 1 DIS

Clutch A5 18 eggs

17 eggs tested shell=yolk (%)

4 H; 4 DIS

Clutch A6 15 eggs

15 eggs tested shell=yolk (%)

All infertile Achromobacter sp. 36=9 20=0 0=10 15=8 12=0 0 Acinetobacter sp. 9=9 0 0 8=0 0 7=0 Aeromonas caviae 0 0 0 0 0 0 Aeromonas hydrophila 9=0 0 10=10 0=8 0=12 0=13 Aeromonas sobria 0 0 10=0 0=8 0 0 Alcaligenes faecalis 50=0 0 0 6=6 7=0 Alcaligenes sp. 9=0 0 0 0 6=0 0 Bacillus sp. 9=0 30=0 40=0 100=0 59=0 93=0 Citrobacter freundii 18=9 10=20 30=10 15=31 18=0 7=7 Citrobacter sp. 0 0 0 8=0 12=0 0 Edwardsiella tarda 0 0 0 0 0 13=20 Enterobacter aerogenes 0 0 0 0 0 13=0 Enterobacter agglomerans 0 0=20 0 0 0 0 Enterobacter cloacae 27=27 90=70 40=30 69=62 76=65 73=47 Escherichia coli 0 0 40=10 0=8 0 0 Flavobacterium sp. 0 0 0 0 0 13=0 Klebsiella oxytoca 64=27 0 20=10 8=0 6=0 7=0 Klebsiella sp. 0=9 0=10 20=0 0 0 0 Moraxella sp. 18=0 20=0 0 0 0 7=0

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Bacterium

Clutch A1 11 eggs

11 eggs tested shell=yolk (%)

10 hatch

Clutch A2 13 eggs

10 eggs tested shell=yolk (%)

2 H; 6 DIS

Clutch A3 11 eggs

10 eggs tested shell=yolk (%)

8 H; 1 DIS

Clutch A4 (a&b)

13 eggs 13 eggs tested shell=yolk (%) 2 hatch; 1 DIS

Clutch A5 18 eggs

17 eggs tested shell=yolk (%)

4 H; 4 DIS

Clutch A6 15 eggs

15 eggs tested shell=yolk (%)

All infertile Morganella morganii 0 0 10=0 0 0 0 nocardioform 0 0 0 0 0 0 Proteus vulgaris 0 10=10 0 0=8 0 0=7 Providencia rettgeri 0 0 10=0 0=8 0 0 Pseudomonas aeruginosa 100=55 60=20 90=90 46=8 29=0 0 Pseudomonas diminuta 0 0 0 0 0 0 Pseudomonas fluorescens 27=9 30=10 0=20 15=8 35=6 0 Pseudomonas paucimobilis 0 0 0 0 0 0 Pseudomonas putida 9=9 20=10 0 8=0 24=18 7=0 Pseudomonas sp. 0=27 30=40 20=10 0 0=6 7=0 Pseudomonas stutzeri 36=9 20=30 40=20 23=0 41=24 20=13 Salmonella typhimurium 9=0 0 10=0 0 0 0 Salmonella arizonae 0 0 20=10 0 0 0 Salmonella IIIb ser 35:k:z53 0 0 0 0 0 0 Salmonella IIIb ser 50:r:z35 0 0 0 0 0 0 Serratia marcescens 0=9 10=0 10=30 15=8 12=24 33=7 Shewanella putrifaciens 0 0 0 0 0 0 Shewanella sp. 0 0 0 0 0 0 Vibrio cholerae (non01) 0 0 0 0 0 0=7

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Fungi

Clutch 1 11 eggs

11 eggs tested shell=yolk (%)

10 hatch

Clutch 2 13 eggs

10 eggs tested shell=yolk (%)

2 H; 6 DIS

Clutch 3 11 eggs

10 eggs tested shell=yolk (%)

8 H; 1 DIS

Clutch 4 13 eggs

13 eggs tested shell=yolk (%)

2 hatch

Clutch 5 18 eggs

17 eggs tested shell=yolk (%)

4 H; 3 DIS

Clutch 6 15 eggs

15 eggs tested shell=yolk (%)

All infertile Acremonium sp. 0 20=0 10=0 0=8 29=0 7=0 Aspergillus flavus 18=0 70=0 20=0 69=0 47=0 20=0 Aspergillus fumigatus 0 0 0 0 12=0 0 Aspergillus niger 0 0 0 0 29=0 73=0 Aspergillus sp. 0 20=0 20=0 15=0 24=0 47=0 Aspergillus terreus 0 0 0 0 0 0 Chaetomium sp. 0 0 0 8=0 0 0 Cladosporium sp. 0 0 10=0 8=0 6=0 0 Cunninghamella sp. 0 0 0 0 0 7=0 Curvularia sp. 0 0 0 0 0 0 Fusarium solani 18=9 40=80 20=10 85=62 82=71 73=53 Mucor sp. 9=0 0 0 0 0 27=0 Paecilomyces lilacinus 0 0 20=0 23=8 12=6 27=0 Paecilomyces sp. 0 30=0 20=0 15=0 35=0 13=0 Penicillium sp. 36=0 90=10 20=0 46=0 94=0 93=0 Rhizopus sp. 55=0 10=0 10=0 8=0 6=0 7=0 Scedosporium apiospermum 0 0 0 23=0 6=0 7=0 Scopulariopsis sp. 0 10=0 0 8=0 18=0 0 Syncephalastrum sp. 0 0 0 0 0 0 Trichoderma sp. 0 0 0 8=0 0 47=0 yeast 0 0 0 0 0 0 H = HATCH; DIS = Dead in Shell

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Table 5.16 Distribution of bacteria and fungi on freshwater crocodile eggs from Farm B

Bacterium

Clutch 1 9 eggs

9 eggs test shell=yolk

% 8 H; 1 DIS

Clutch 2 10 eggs 10 eggs

test shell=yolk

% All

infertile

Clutch 3 12 eggs

9 eggs test shell=yolk

% 3 H; 7 DIS

Clutch 4 11 eggs 11 eggs

test shell=yolk

% 9 H; 1 DIS

Clutch 5 12 eggs 12 eggs

test shell=yolk

% All

infertile

Clutch 6 12 eggs 10 eggs

test shell=yolk

% 3 H; 4 DIS

Clutch 7 10 eggs

3 eggs test shell=yolk

% 9 hatch

Clutch 8 5 eggs

2 eggs test shell=yolk

% 4 hatch

Clutch 9 18 eggs 18 eggs

test shell=yolk

% 2 hatch

Clutch 10 12 eggs 12 eggs

test shell=yolk

% 11 hatch

Clutch 11 10 eggs 10 eggs

test shell=yolk

% 2 hatch

Clutch12 15 eggs 14 eggs

test shell=yolk

% 6 H; 2 DIS

Acinetobacter sp. 0 70=0 11=0 0 33=0 10=0 0 0 11=0 0 0 14=7 Aeromonas caviae 0 0 0 0 0 0=10 0 0 0 0 0 0 Aeromonas hydrophila 11=0 0=10 0 0 0 0=20 0 0 0 0=8 0=10 7=7 Aeromonas sobria 0 0 11=11 0 0 0 0 0 0 0 10=0 0 Alcaligenes faecalis 44=0 0 22=11 9=18 8=0 30=0 100=0 50=0 22=11 25=0 40=0 50=14 Alcaligenes sp. o 0 0 0 0 0 0 0 11=0 17=0 10=0 21=14 Bacillus sp. 22=22 80=0 44=0 18=0 58=0 70=0 33=0 0 39=0 17=0 80=0 93=7 Citrobacter freundii 11=11 10=10 22=11 9=9 0=17 10=10 0 0 28=0 17=8 0 7=14 Citrobacter sp. 0=11 0 11=11 0 0 0=10 0 0 6=0 0 0 0=7 Edwardsiella tarda 0 0 0 0 0=33 0 0 0 0 0 0 0 Enterobacter aerogenes 0 0 0 0 8=0 0 0 0 0 0 0 0 Enterobacter agglomerans 0 0 22=11 0=9 17=0 0 0 0 17=11 8=25 o 14=0 Enterobacter cloacae 22=0 0 89=44 55=9 0=8 70=40 100=0 100=50 78=28 50=42 100=70 100=86 Escherichia coli 11=0 0 22=0 9=9 0 0=10 0=33 0 6=6 0 0 0=7 Flavobacterium sp. 0 0 0 9=0 0 0 0 0 0 8=0 0 0 Klebsiella sp. 0 0 0 9=0 0 0 0 0 0 0 0 0=7 Morganella morganii 0 0 0 0 0 0=20 0 0 0 0 0 0

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Bacterium

Clutch 1 9 eggs

9 eggs test shell=yolk

% 8 H; 1 DIS

Clutch 2 10 eggs 10 eggs

test shell=yolk

% All

infertile

Clutch 3 12 eggs

9 eggs test shell=yolk

% 3 H; 7 DIS

Clutch 4 11 eggs 11 eggs

test shell=yolk

% 9 H; 1 DIS

Clutch 5 12 eggs 12 eggs

test shell=yolk

% All

infertile

Clutch 6 12 eggs 10 eggs

test shell=yolk

% 3 H; 4 DIS

Clutch 7 10 eggs

3 eggs test shell=yolk

% 9 hatch

Clutch 8 5 eggs

2 eggs test shell=yolk

% 4 hatch

Clutch 9 18 eggs 18 eggs

test shell=yolk

% 2 hatch

Clutch 10 12 eggs 12 eggs

test shell=yolk

% 11 hatch

Clutch 11 10 eggs 10 eggs

test shell=yolk

% 2 hatch

Clutch12 15 eggs 14 eggs

test shell=yolk

% 6 H; 2 DIS

nocardioform 0 0 0 0 0 10=0 0 0 6=0 0 80=0 21=0 Proteus vulgaris 0 0 0 0 0 0=10 0 0 0 0 0 7=7 Providencia rettgeri 0 0 0 0 0 0=10 0 0 0 0 0 0 Pseudomonas aeruginosa 11=0 40=0 0 0 0 20=0 0 0 0 8=0 10=0 21=0 Pseudomonas diminuta 11=0 0 11=0 0 0 0 0=33 0 0 0 0 7=0 Pseudomonas fluorescens 0 0 33=33 46=27 0=8 50=60 33=67 50=50 56=56 25=25 70=40 64=57 Pseudomonas paucimobilis 0 0 11=0 0 0 0 0 0 0 0 0 0 Pseudomonas putida 0 0 11=0 18=9 0 20=0 0 0 0=6 0 10=0 0=7 Pseudomonas sp. 0 50=40 11=0 0 33=0 40=0 0 0 17=0 0=8 0 14=7 Pseudomonas stutzeri 56=44 0 11=0 27=27 0 10=0 33=0 0 11=6 8=0 0 14=7 Salmonella IIIb ser 35:k:z53 78=89 0 0 0 50=58 20=50 0 0=50 0 100=92 0 7=14 Salmonella IIIb ser 50:r:z35 0 0 11=22 82=91 0 0 0 0 83=78 0 20=0 7=7 Serratia marcescens 0 0=40 22=22 9=0 8=0 0 0 0 0 42=33 10=0 0 Shewanella putrifaciens 0 0 11=0 9=0 0 0 0 0 0 0=8 0 0 Shewanella sp. 0 30=10 0 0 42=17 0 0 0 0 0 0 0 Vibrio cholerae 0 0 11=0 0 0=8 0=10 0 0 6=11 0 0=20 0

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Bacterium

Clutch 1 9 eggs

9 eggs test shell=yolk

% 8 H; 1 DIS

Clutch 2 10 eggs 10 eggs

test shell=yolk

% All

infertile

Clutch 3 12 eggs

9 eggs test shell=yolk

% 3 H; 7 DIS

Clutch 4 11 eggs 11 eggs

test shell=yolk

% 9 H; 1 DIS

Clutch 5 12 eggs 12 eggs

test shell=yolk

% All

infertile

Clutch 6 12 eggs 10 eggs

test shell=yolk

% 3 H; 4 DIS

Clutch 7 10 eggs

3 eggs test shell=yolk

% 9 hatch

Clutch 8 5 eggs

2 eggs test shell=yolk

% 4 hatch

Clutch 9 18 eggs 18 eggs

test shell=yolk

% 2 hatch

Clutch 10 12 eggs 12 eggs

test shell=yolk

% 11 hatch

Clutch 11 10 eggs 10 eggs

test shell=yolk

% 2 hatch

Clutch12 15 eggs 14 eggs

test shell=yolk

% 6 H; 2 DIS

(non01)

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Fungus

Clutch 1 9 eggs

9 eggs test shell=yolk %

8 H; 1 DIS

Clutch 2 10 eggs 10 eggs

test shell=yolk

% All

infertile

Clutch 3 12 eggs

9 eggs test shell=yolk

% 3 H; 7 DIS

Clutch 4 11 eggs 11 eggs

test shell=yolk

% 9 H; 1 DIS

Clutch 5 12 eggs 12 eggs

test shell=yolk

% All

infertile

Clutch 6 12 eggs 10 eggs

test shell=yolk

% 3 H; 4 DIS

Clutch 7 10 eggs

3 eggs test shell=yolk

% 9 hatch

Clutch 8 5 eggs

2 eggs test shell=yolk

% 4 hatch

Clutch 9 18 eggs 18 eggs

test shell=yolk

% 2 hatch

Clutch 10 12 eggs 12 eggs

test shell=yolk

% 11 hatch

Clutch 11 10 eggs 10 eggs

test shell=yolk

% 2 hatch

Clutch12 15 eggs 14 eggs

test shell=yolk

% 6 hatch

Acremonium sp. 11=22 0 0 9=0 0 0 0 0 17=11 17=0 10=0 21=0 Aspergillus flavus 0 10=0 22=0 0 17=0 0 0 0 22=0 17=0 70=0 50=0 Aspergillus fumigatus 0 0 0 0 8=0 0 0 0 0 0 0 0 Aspergillus sp. 0 10=0 0 9=0 8=0 0 0 0 6=0 0 0 7=0 Aspergillus terreus 0 0 0 0 17=0 0 0 0 6=0 0 10=0 21=0 Chaetomium sp. 0 0 0 0 0 0 0 0 0 0 0 7=0 Cladosporium sp. 0 0 11=0 0 0 0 0 0 17=0 0 0 0 Cunninghamella sp. 0 10=0 0 0 42=0 10=0 0 0 0 0 30=0 7=0 Curvularia sp. 0 0 0 0 8=0 0 0 0 0 0 0 0 Fusarium solani 22=11 50=70 22=22 9=9 50=17 70=30 67=0 0 61=11 25=8 60=20 71=21 Mucor sp. 0 10=0 0 0 0 0 0 0 0 0 0 0 Paecilomyces lilacinus 44=56 0 0 9=36 67=67 30=30 0=33 0 56=44 25=17 90=40 36=7 Paecilomyces sp. 0 30=0 11=0 18=0 33=0 20=0 33=0 0 17=0 8=0 30=0 0 Penicillium sp. 0 100=0 33=0 64=9 100=0 50=0 33=0 50=0 61=0 58=0 60=0 50=0 Scedosporium apiospermum 0 10=0 0 0 0 0 0 0 0 0 0 7==0 Scopulariopsis sp. 0 0 11=0 0 0 0 0 0 0 8=0 0 7=0 Syncephalastrum sp. 0 0 0 0 0 0 0 0 0 0 30=0 0 Trichoderma sp. 0 0 0 9=0 0 0 0 0 6=0 0 0 7=0 yeast 0 0 11=0 0 0 0 0 0 0 0 0 7=0

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The isolations from the interior of hatched eggs can be misleading as the presence of the organisms isolated could have occurred after the hatchling exited the shell. The more interesting data comes from infertile eggs and eggs where embryonic death occurred. The most frequently isolated bacteria from the external egg shell on both farms were Enterobacter cloacae and Citrobacter freundii (which are of faecal origin and commonly found in the intestinal tract of crocodiles) and Alcaligenes faecalis, Bacillus spp. and members of the Pseudomonas genus, notably Pseudomonas aeruginosa, Pseudomonas fluorescens, Pseudomonas putida and Pseudomonas stutzeri (which are found frequently in the soil and water environment). As can be seen from Tables 5.15 and 5.16, excepting the Alcaligenes faecalis and Bacillus spp., these were also the major organisms found inside the egg. The distribution of the bacteria over the two farms is not unsimilar. Ps. fluorescens, Salmonella IIIb ser 35:k:z53 and Salmonella IIIb ser 50:r:z35 were major isolates from infected and DIS eggs from Farm B but not Farm A. The Salmonella results will be discussed separately in the next section. The type and concentration of the bacteria isolated is generally an indication of presence of these organisms in the nesting material. Nesting material was obtained from 7 freshwater crocodile nests from Farm B. Unfortunately, these could not be correlated to the clutches received from that Farm. The results, as shown in Table 5.19 indicate the presence of Enterobacter cloacae, Citrobacter freundii, Serratia marcescens, Pseudomonas putida and Pseudomonas stutzeri as common contaminants of these nests. Pseudomonas fluorescens was only isolated from one nest. When the status of the egg was taken into consideration (infertile [clean or infected]; eggs where the band did not progress [clean or infected]; and dead-in-shell [clean or infected]), it is interesting to note that the contents from inside the infected eggs from the three categories yielded a higher percentage of Aeromonas sp., Citrobacter freundi, Enterobacter cloacae, Pseudomonas fluorescens and Pseudomonas stutzeri than the clean eggs (Table 5.17). The most frequently isolated fungi from the external egg shell on both farms were Acremonium sp., Aspergillus flavus, Fusarium sp. (notably Fusarium solani) and Paecilomyces lilacinus (Table 5.18). These are not uncommon inhabitants of soil. From the 7 nesting material samples, these fungi were also present but easily outgrown by the zygomycetes Cunninghamella spp. and Rhizopus spp. (Table 5.19). Except for one case where Penicillium sp. was found, the only fungi found in the contents of the freshwater crocodile eggs were Fusarium solani and Paecilomyces lilacinus. As with the bacterial content of the eggs, these two species of fungi were more prevalent in infertile-infected, BNP and DIS eggs than in infertile, clean eggs. The contamination of the eggs by these fungi was dependant on the nest flora. Fusarium sp. were more prevalent on Farm A while Paecilomyces lilacinus were more prevalent on Farm B (Table 5.18)

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Table 5.17 Common bacterial isolates from the inside of eggs that were infertile (clean), infertile (infected), had bands that did not progress (BNP) and dead-in-shell (DIS) from broth Farm A and Farm B

Status Total Eggs Ent.cloacae Cit.freundii Ps.aeruginosa Ps.fluorescens Ps.stutzeri

Serratia spp.

Proteus sp.

Aeromonas sp. Ed.tarda

FARM A

Infertile - clean 13 2 (15.4%) 1 (7.7%) 1 (7.7%) 2

(15.4%) 1 (7.7%) Infertile - infected 20 16 (80.0%) 3 (15.0%) 1 (5.0%) 1 (5.0%)

2 (10.0%) 1 (5.0%) 4 (20%) 2 (10.0%)

BNP - infected 8 7 (87.5%) 4 (50.0%) 2 (25.0%) 2 (25.0%) 1

(12.5%) 2 (25.0%) 2 (25.0%) DIS - infected 10 8 (80%) 1 (10.0%) 1 (10.0%) 4 (40.0%) 1 (10.0%)

Status Total Eggs Ent.cloacae Cit.freundii Ps.aeruginosa Ps.fluorescens Ps.stutzeri

Serratia spp.

Proteus sp.

Aeromonas sp. Ps.putida

FARM B Infertile - clean 21 1 (4.8%) 4 (19%) Infertile - infected 35 15 (42.9%) 2 (5.7%) 16 (28.6%) 1 (2.9%) 3 (8.6%) 1 (2.9%) 6 (17.1%) 3 (8.6%) BNP - infected 6 3 (50%0 2 (33.3%) 2 (33.3%) 2 (33.3%) 2 (33.3%) 1 (16.7%) DIS - clean 5 1 (20%) 2 (40%) 1 (20%) 2 (40%)

DIS - infected 7 2 (28.6%) 1 (14.3%) 5 (71.4%) 1

(14.3%) 2 (28.6%)

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Table 5.18 Common fungal isolates from the inside of eggs that were infertile (clean), infertile (infected), had bands that did not progress (BNP) and dead-in-shell (DIS) from both Farm A and Farm B

Status Total Eggs Fusarium sp. Paecilomyces

lilacinus FARM A Infertile - clean 13 4 (30.8%) 1 (7.7%) Infertile - infected 19 17 (78.9%) BNP 8 6 (75%) 1 (12.5%) DIS 10 10 (100.0%) FARM B Infertile - clean 20 2 (10.0%) 3 (15.0%) Infertile - infected 35 13 (37.1%) 17 (48.6%) BNP - infected 6 3 (50%) 2 (33.3%) DIS - clean 5 1 (20.0%) DIS - infected 7 4 (57.1%) 1 (14.3%)

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Table 5.19 Results of bacterial and fungal isolations from freshwater crocodile nests (Farm B)

Nesting Materials Bacteria Fungi

Nest 1 Sand Ent.agglomerans Aspergillus glaucus Ent.cloacae Paecilomyces sp. Kl.pneumoniae Penicillium sp. Ps.maltophila Rhizopus sp. Ps.stutzeri

Nest 2 Sand Ent.cloacae Cunninghamella sp. Ps.putida Rhizopus sp.

Ps.stutzeri Nest 3 Sand Ach.xylos.denitrificans Cunninghamella sp.

Ent.cloacae Rhizopus sp. Ps.putida Ps.stutzeri

Nest 4 Sand Cit.freundii Acremonium sp. Ent.cloacae Cunninghamella sp. Ps.putida Fusarium sp. Ser.marcescens

Nest 5 Sand Ent.cloacae Apophysomyces elegans Ps.fluorescens Penicillium sp.

Ps.putida Rhizopus sp. Nest 6 Sand Ent.cloacae Aspergillus sp.

Ps.putida Cunninghamella sp. Ps.stutzeri Penicillium sp.

Nest 7 Sand Cit.freundii Cunninghamella sp. Ent.cloacae Fusarium sp. Ps.putida Paecilomyces sp. Ser.marcescens Trichoderma sp.

Salmonella IIIb isolations from the 12 clutches of Farm B During the identification of bacterial isolates from the shell and/or yolk of the twelve clutches of eggs from Farm B, it was noted that a large number of Salmonella IIIb serotypes were present. Table 5.20 shows that there were just two serotypes identified and that these were distributed by clutch. Salmonella IIIb serotype 35:k:z53 was found only in eggs of clutches B1, B5, B6, B8 and B10. Salmonella IIIb serotype 50:r:z35 was found only in eggs of clutches B3, B4, B9 and B11. Both serotypes were found in eggs of clutch B12. No Salmonella IIIb were found in the eggs of clutches B2 and B7. 53.7% of the 136 eggs were positive for Salmonella IIIb. Of those positive eggs, the organisms were isolated from 72.6% of the shells and 83.6% of the yolks. The latter figure may be misleading as a number of isolates were collected from the inside of the egg after the hatchling had left the egg which means that the inside contents may have been contaminated by external factors.

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The presence of these bacteria was not associated with infected eggs or DIS embryos and they were isolated from hatched eggs as well as infertile eggs. Table 5.21 shows the distribution of both serotypes with regard to the status of the egg – hatched, DIS, infertile (clean) or infertile (infected). Salmonella serotypes belonging to the subspecies IIIb are normally isolated from cold-blooded animals and rarely from humans. They are frequently isolated from healthy reptiles (in particular, snakes and crocodiles) as part of the normal flora of the intestine. They are ubiquitous and can persist very well in the environment (dust, faeces, soil) if well protected from direct sunlight. It is unusual, however, to find such a demarcation of serotype with the clutches. The presence of the salmonellae was no doubt due to a heavy load in the nesting material with transfer to the eggs on or after lay. It was unfortunate that nesting material was not available at the time of collection to ascertain that fact. Nor could it be found out from the farm how the clutches and nests correlated in geography to each other, that is, were all the one serotype present in one portion of the breeding area. Nesting materials were obtained from the same Farm at a later date, but no Salmonella species were isolated (Table 5.19) at that time. There was also no indication of whether these nesting materials came from the same vicinity of the original nests. Table 5.22 lists the isolations of a variety of serotype IIIb salmonellae from crocodiles and humans during the period 1996 and 2000 as taken from the Salmonella Reference Centre Annual Reports for that period. The serotypes chosen were those that have been isolated at this laboratory from both freshwater and saltwater crocodiles. Salmonella serotype IIIb 50:r:z35 has been isolated from diseased crocodiles in Qld and from 3 human cases in NSW. Salmonella serotype IIIb 35:k:z53 was a new isolation for crocodiles in this area.

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Table 5.20 Isolation of Salmonella IIIb from 12 clutches of freshwater crocodile eggs from Farm B

Hatching State (Presence of Salmonella) SalmonellaIIIb SalmonellaIIIb Isolation site ser.35:k:z53 ser.50:r:z35 Clutch

Number Number of

eggs Hatched DIS Infertile Infertile

(infected) BNP BNP

(infected) Number positive (%) Shell Yolk B1 9 8 (8) 1 (1) 0 0 0 0 9 (100%) 0 7 8 B2 10 0 0 6 (0) 4 (0) 0 0 0 0 0 0 B3 12 3 (1) 7 (0) 1 (0) 1 (1) 0 0 0 2 (16.7%) 1 2 B4 11 9 (9) 1 (1) 0 1 (1) 0 0 0 11 (100%) 9 10 B5 12 0 0 4 (3) 7 (7) 0 1 (1) 11 (91.7%) 0 6 7 B6 12 3 (0) 4 (2) 0 3 (2) 0 2 (2) 6 (50%) 0 2 5 B7 10 9 (0) 0 0 1 (0) 0 0 0 0 0 0 B8 5 4 (1) 0 1 (0) 0 0 0 1 (20%) 0 0 1 B9 18 2 (2) 0 5 (4) 9 (9) 1(0) 1 (1) 0 16 (88.9%) 12 14

B10 12 11 (11) 0 1 (1) 0 0 0 12 (100%) 0 12 11 B11 10 2 (0) 0 3 (0) 5 (2) 0 0 0 2 (20%) 2 0 B12 15 6 (3) 2 (0) 0 4 (0) 0 3 (0) 2 (13.3%) 1 (6.7%) 2 3

Total 136 eggs 57 (35) 15 (4) 21 (8) 35 (22) 1 (0) 7 (4) 41 32 53 61 73 of 136 eggs (53.7%) were positive for Salmonella 53 of the 73 eggs (72.6%) had Salmonella on the shell 61 of the 73 eggs (83.6%) had Salmonella in the yolk

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Table 5.21 Distribution of Salmonella IIIb in freshwater crocodile eggs from Farm B

Eggs Hatched DIS Infertile (Clean)

Infertile (Infected) Total

Total eggs 57 15 20 43 136 Number with Salmonella 35 (61.4%) 4 (26.7%) 8 (38.1%) 26 (60.5%) 73 (53.7%) Shell 25 (43.8%) 3 (20%) 7 (33.3%) 18 (41.9%) 53 (39.0%) Yolk 33 (57.9%) 3 (20%) 3 (14.3%) 22 (51.2%) 61 (44.9%) Infected infertile eggs had 51.2% of yolks containing Salmonella Clean infertile eggs had 13.6% of yolks containing Salmonella Two clutches did not have Salmonella isolated from them - in one clutch of 10, 9 hatched and one was infertile while in the other, all 10 were infertile.

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Table 5.22 Distribution of Salmonella arizonae species in humans and reptiles (including crocodiles and snakes, etc) in Australia between 1996 and 2000

1996 1997 1998 1999 2000 18:l,v:z - crocodile (Qld) 1 x liver; 1 x lung 18:l,v:z - crocodile (other) 18:l,v:z - human (Qld) 1 18:l,v:z - human (oth) 35:k:z53 – crocodile (Qld) 12 x egg 37 x egg 35:k:z53 – crocodile (other) 35:k:z53 - human (Q) 35:k:z53 - human (oth) 38:l,v:z53(z54)-crocodile (Q) 38:l,v:z53(z54)-crocodile (ot) 38:l,v:z53(z54)-hum(Q) 38:l,v:z53(z54)-hum(ot) 2 x VIC 48:k:1,5,(7)-crocodile (Qld) 1 x crocodile leg 1 x meat 1xcloaca; 2xstom; 1xkid 48:k:1,5,(7)- (oth) 48:k:1,5,(7)-hum (Qld) 1 4 48:k:1,5,(7)-hum (oth) 1 1 x NSW 48:z52:z – crocodile (Qld) 1 x liver 24 48:z52:z – crocodile (oth) 48:z52:z - hum (Qld) 48:z52:z - hum (oth) 50:k:z – crocodile (Qld) 50:k:z – crocodile (oth) 5 x meat (NT) abdomen (NT) 50:k:z - human (Qld) 50:k:z - human (other) 50:r:z35 - crocodile(Qld) 1 x crocodile jaw 3 x egg; 1 x environ 19 24xegg;1xliv;1xlun;1xskin 50:r:z35 - crocodile(other)

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1996 1997 1998 1999 2000 50:r:z35 - human (Qld) 50:r:z35 - human (oth) 1 x NSW 2 x NSW 50:x:z35 - crocodile(Qld) 50:x:z35 - crocodile(other) 50:x:z35 - hum (Qld) 50:x:z35 - hum (other) 61:l,v:1,5,7[z57]-crocQ 2 x liver 61:l,v:1,5,7[z57]-croco spleen (NT) 61:l,v:1,5,7[z57]-humQ 1 61:l,v:1,5,7[z57]-humo 1 1 x VIC 61:l,v:z35 - crocodile(Qld) 1 x liver 2 x liver; 2 x skin 61:l,v:z35 - crocodile(oth) 1 x liver (NT) 61:l,v:z35 - hum (Qld) 19 26 23 19 5 61:l,v:z35 - hum (oth) 3 4 x NSW; 2 X WA 1 x NSW; 1 x WA 1 x NT; 1 x WA 2 x NSW 61:r:z53 - crocodile(Qld) 9 x eggs 1 x egg 61:r:z53 - crocodile(oth) 61:r:z53 - hum (Qld) 2 1 3 1 61:r:z53 - hum (oth) 1 x NT 61:z52:z53 - crocodile(Qld) 1 x egg 5 x liver; 1 x skin 61:z52:z53 - crocodile(oth) 61:z52:z53 - hum (Qld) 61:z52:z53 - hum (oth) 1 1 x SA 2 x SA 1 x SA 1 x WA

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Summary The observations made during the incubation of 217 freshwater crocodile eggs collected from two farms in northern Queensland indicated that there was a similar pattern to measurements taken in the Northern Territory on eggs collected from freshwater crocodiles in the wild. Wild-collected eggs from Queensland were not available for comparison. Although clutch effects can make the comparison of data difficult, it is still worthwhile to look at the average data for both farms combined for the comparison. Table 5.23 summarizes a portion of the data. The wild NT freshwater crocodiles had larger clutches (13.2 eggs per clutch compared to 11.4 eggs per clutch for captive breeders) and had a fertility rate of 96%. Hatchability of the eggs in the wild was very low, 31.3%, but this was mainly due to loss of eggs through flooding of nests or robbing of nests by wildlife in the area. The farm eggs had a fertility rate of only 57.1% and the hatchability of those eggs was 66.9%. The low fertility rate of the captive breeder eggs could be due to a combination of younger or first-time female breeders, incorrect diet or non-competent male donors. The low hatchability rate was possibly due in part to transport of the eggs during the vulnerable stage of embryo attachment (which may account for the number of eggs where the band did not progress (BNP)); in part due to the large number of dead-in-shell (DIS) eggs that were believed to have occurred because of the inability of a few of the converted chicken incubators to maintain a constant humid environment (a gradient occurred due to the heating lamp positioned at the top of the incubator leading to increased water loss at the top of the incubator and a gain in water levels at the bottom of the incubator); in part due to infection in some of the eggs due to bacterial and fungal contamination; and in part due to the possibility of the oxygen exchange to the eggs being reduced even though the doors were opened daily to enhance such an exchange. It has been reported that C.johnstoni eggs at 30oC consume 2.8 mL O2 per gram per day that would equate to 90 litres of O2 per day. This would mean the entire contents of a large 300 litre incubator being consumed within 16 hours. Although clutch sizes were smaller in the captive situation, the eggs tended to be longer, wider and heavier than their wild counterparts (Table 5.23). This translated into longer and heavier crocodiles at hatch. However, this is no doubt related to the size of the breeder as the width of the egg is an indication of oviduct size that is in turn an indication of the size of the animal. As reported by previous workers, there were positive correlations between egg weight and egg volume and crocodile weight and egg volume. Water loss was multi-factorial. It was the result of clutch factors such as shell thickness, membrane thickness, size and number of pores in the shell and membrane and environmental factors such as the humidity and gaseous availability within the incubator and in contact with the eggs. Irrespective of the variance of humidity and its gradients in the incubators, the clutches varied in their rate of water loss during the incubation period. Clutch A2, for example, had an average water loss of 20.5% (6.1 to 27.2%) with the 2 hatched eggs losing 16.1 and 17%. However, the rate of loss for each clutch was consistent throughout the incubation period. Overall, the water loss rate for the two farms was 5.7%, with hatched eggs averaging 5.3% (with a wide range of +9.3% to 17%). Eggshell thickness varied between the farms and between clutches and is due to a function of the diet, age, and genetics of the breeders. The shells are thicker at the equatorial region and the pore numbers are also greater in this region. Overall for both farms the average thickness at the equator was 0.382 mm and the average thickness at the poles was 0.347 mm (Table 5.24). The ratio of the thicknesses of the pole to the equator regions was consistent throughout the incubation period at 0.88-0.92, which meant a consistent intrinsic degradation of the whole shell during that time. This degradation was also noticed when the shells were weighed at the end of the incubation period.

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Infertile eggs had shells of weight 8-11% of the total weight of the egg while fertile eggs had shell weights of 5-7%. When the data is broken down with regard to hatched, DIS and infertile eggs, the expected decrease in thickness from the infertile eggs (average 0.401 mm at the equator) through to the DIS eggs (average 0.383 mm at the equator) to the hatched eggs (0.365 mm at the equator) is seen due to intrinsic degradation over the incubation period. With the DIS eggs, one would expect a gradual decrease in thickness from the EED through the MED to the LED eggs. However, although there was not a large number of eggs to measure, the results confirm some of the previously published reports in that EED eggs, death is often due to the fact that shells of EED eggs have a thicker outer and more densely calcified layer than either LED or hatched eggs. Table 5.24 shows that the freshwater EED eggs had an average shell thickness both at the poles and the equator that was greater than that of the infertile eggs. The MED eggs had an average shell thickness of equator and poles that was smaller than the LED and hatched eggs indicating that these eggs with such a narrow width would be more susceptible to water loss and infection. The LED eggs had thicknesses similar to hatched eggs. Deaths in these eggs may be due to infection, dehydration, water gain, etc. The shell membrane stayed consistent in thickness throughout and averaged 0.131 mm (0.133 mm for hatched eggs, 0.132 mm for DIS eggs and 0.128 mm for infertile eggs). A variety of bacteria and fungi can be isolated from the nesting materials of the freshwater crocodile (Table 5.19) and therefore available to attach to the exterior of the laid eggs. Some of these organisms are faecal in origin while others are common contaminants of soil and water. It is also possible for some eggs to be contaminated in the oviduct before lay. For the organisms to penetrate into the yolk and/or growing embryo, they first must pass through the shell calcite layers and then through the shell membrane. In crocodile eggs, both of these are quite thick (Table 5.11 and 5.12) but they do contain numerous pores of various sizes so that there can be passage of water and gaseous exchange between the growing embryo and the environment. It is the size and number of the pores and also their length and tortuous route through the shell that makes them a good but not impenetrable barrier to microorganisms. This is noticeable in the infertile eggs that were left in the incubators during the incubation phase. On farms, these eggs would have been discarded as soon as it was determined that no banding was going to occur. Although there were a few infertile eggs that stayed uninfected until they were sampled at the end of the incubation period, most of the infertile eggs were contaminated. A range of bacteria was isolated from the inside of these eggs (Table 5.25). Enterobacter cloacae, Citrobacter freundii and Pseudomonas fluorescens were the most prominent. On Farm B, a mass contamination by two species Salmonella group IIIb was noticed. However, these salmonellae are normally commensals in reptiles and the study showed that the microbes were isolated from infertile and hatched eggs alike. The interesting thing was that the two species were clutch related and therefore probably nest related. The fungal contamination was restricted to two fungi that are well known for their ability to produce infections in reptile eggs, specifically crocodile, snake and turtle eggs. Fusarium solani and Paecilomyces lilacinus have been incriminated in deaths of saltwater crocodiles within 1 to 9 months of hatching. These fungi infect the liver of the hatchling after crossing the shell and membrane from the nesting materials where they are commonly present. They have also been incriminated in the production of skin diseases of juveniles where they produce a plaque-like lesion on the skin with eventual death of the crocodile. The presence of only these two fungi within the egg (Table 5.25) when compared to the variety of fungi isolated on the outside of the egg (Table 5.25) indicates some particular mechanism to penetrate via the pores in the shell and membrane. There are three levels of pore sizes. These are classed as small, medium and large, with more of the smaller sized pores present. The pore sizes have been measured for a number of crocodile species and vary from 0.45 µm up to 3.63 µm. Only 6% of the pores are open at lay. As incubation progresses, more pores open up to allow for the water and gaseous exchanges required and some 22-24% are open

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at hatch. The remainder tend to be clogged with water and solutes. Also, as incubation proceeds, the width of the pores tends to increase. How does this relate to bacterial and fungal penetration of the egg? The general size of the bacteria isolated during this study was 2-3 µm long by 1 µm long. Therefore, the bacteria have no trouble in moving along the pore length. They were motile bacteria and that may or may not be an advantage depending on whether entrance was active (movement through the water in the pores) or passive (taken in with the water in those eggs that gained weight on the bottom trays). The general sizes of the fungi do differ in their hyphal width. The zygomycetes, to which Cunninghamella, Mucor and Rhizopus species belong, have hyphal diameters greater than 4 µm so would have difficulty in traversing the pores. However, the remainder of the fungi isolated have a hyphal width of less than 4 µm and are therefore possible invaders. Instead of the hyphae penetrating the egg, it is possible that conidia (asexual spores) of these fungi could travel the route of the pores. This would be passive transmission as these conidia are not motile. The conidial sizes for the four main fungi are: - Fusarium solani 8-16 x 2-4 µm Paecilomyces lilacinus 2.5-3.0 x 2-2.2 µm Aspergillus flavus 3.5-4.5 µm Penicillium species vary from 2-2.5; 2.5-3; 3.5-4.5 µm It can be seen that while Asp.flavus conidia may have difficulty passaging through the pore channel, the other genera could do so quite easily. However, recent work on the passage of fungi through eggshells has indicated hyphal growth as the main route of entry. Hyphal width is generally narrower than the diameter of its respective conidia. Why only Fusarium solani and Paecilomyces lilacinus have been found to traverse the pores and not the other fungi is interesting. Both Fusarium solani and Paecilomyces lilacinus are strongly proteolytic and are known for their ability to attack keratin. There is the possibility of the production of other exo-enzymes that allow for penetration or easy access for these two fungi that Penicillium and Aspergillus species cannot produce. They all have similar optimum temperature ranges (26oC to 31oC) and the optimum pH ranges are 6.5 to 7.5 (with a wide variance acceptable). It may also be due to some gaseous effect. Aspergillus flavus growth is retarded in gaseous mixtures with 70 to 87% CO2, while Paecilomyces lilacinus grows best under partial O2 pressure. Conclusion Increase of fertility and hatchability of freshwater crocodile eggs can be achieved by genetic selection of the breeders, a balanced diet, collection and transport of the eggs at the appropriate time and washing of the eggs in a suitable disinfectant before incubation. The incubators need to be cleaned thoroughly before and after use and must allow for a reasonable gaseous exchange and maintain a high humidity throughout the incubation period.

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Table 5.23 Comparison of data collected on eggs from captive freshwater crocodiles from northern Queensland compared to data collected on eggs from wild freshwater crocodiles from the Northern Territory

Status Clutch

Size Fertility

(%)

Hatchability of fertile

eggs

Egg Length

(cm)

Egg width (cm)

Egg Weight

(g) Croc

weight (g)

Croc length (mm)

Qld captive

11.4 (5-18) 57.1 66.9

6.99 (6.21-7.76)

4.23 (3.82-4.57)

73.9 (59.1-88.8)

47.4 (37.1-57.5)

246.8 (220-267)

NT wild

13.2 (4-21) 96 31.3 6.64 4.19

68.2 (50-86)

42 (33-56) 244

Table 5.24 Average shell thickness readings for the freshwater crocodile eggs from both farms

Category

Thickness at pole (mm)

Thickness at equator (mm)

Ratio (Pole/Equator)

Overall 0.347 0.382 0.91 Hatch 0.329 0.365 0.9 (0.25-0.36) (0.267-0.393) DIS 0.353 0.383 0.92

(0.267-0.44) (0.31-0.447) LED 0.333 0.363

MED 0.296 0.354 EED 0.384 0.413

Infertile 0.364 0.401 0.91 (0.286-0.438) (0.328-0.457)

RATIO (HATCH/INFERTILE) 0.9 0.91 Ratio (DIS/Infertile) 0.97 0.96

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Table 5.25 The more prevalent bacteria and fungi isolated from infertile and DIS eggs from freshwater crocodiles Egg Status Yolk (bact) Shell (fungi) Yolk (fungi) A1/4 I Ent.cl. Fus/Pen/Rhiz 0

A2/2 MED C.fr./Ent.cl./Ps.st Pen Fus A2/3 EED Ent.cl./Ps.pu Asp.fl/Fus/Pen Fus A2/4 BNP/I C.fr./Ent.cl./Prot. Acr/Asp.fl/Pae/Pen 0 A2/5 MED Ent.cl/Ps.st Asp.fl/Fus/Pen Fus A2/6 LED Ent.cl/Ps.st As p.fl./Pen Fus

A2/10 I/I Ent.cl. Asp.fl/Fus/Pen Fus A2/11 MED Ent.aggl. As p.fl./Pen Fus A2/12 I/I Ps.aer. mix/Asp.fl./Fus Fus/Pen A2/13 EED Ent.aggl./Kl.oxy. Asp/Pae/Pen Fus A3/3 EED Ent.cl/Ps.aer Asp.fl. Fus A3/7 I/I Ent.cl. 0 ND

A3/10 BNP/I Aer.hyd./C.fr./Ps.aer. Fus/Rhiz 0 A4/1 I/I Ent.cl. Asp.fl/Fus/Rhiz Fus A4/2 I/I Ent.cl. Asp.fl./Fus/P.lil/Pen Fus A4/5 I/I C.fr. Asp.fl./Fus Fus A4/6 BNP/I C.fr./E.c./Ent.cl. Asp.fl./Fus/P.lil Fus A4/7 I/I C.fr./Ent.cl. Asp.fl./Fus/Pen/Pae 0 A4/8 I 0 Asp.fl./Fus/Pen/Pae 0 A4/9 I/I Ent.cl. Fus/Pen Acr/Fus

A4/10 EED Aer.sob./Ent.cl. Asp.fl/Fus/P.lil 0 A4/11 BNP/I Ent.cl./Prot. Asp.fl/Fus/Pen Fus A4/12 I/I Aer.hyd. Asp.fl/Fus/Pen Fus A4/13 BNP/I C.fr./Ent.cl./Ps.aer. Fus Fus/P.lil A5/2 BNP/I Aer.hyd.//Ent.cl. Acr/Asp.fl./Fus/Pa/Pen Fus A5/3 I/crushed Ent.cl./Ser.mar. Asp.fu./Fus/Pen Fus A5/5 I/I Ent.cl./Ps.st. Fus/Pen Fus A5/8 EED Ent.cl/Ps.st Asp/Fus/Pen/Pae Fus A5/9 I/I Ent.cl./Ps.pu. Asp.nigr/Fus/Pen Fus

A5/10 EED Ent.cl./Ser.mar. Asp.fl/Fus/P.lil Fus/P.lil A5/11 I 0 mix/Fus 0 A5/12 I/I Aer.hyd./Ent.cl. Asp.fl./Fus/Pen Fus A5/13 LED Ent.cl. Asp.fl/Fus/Pen Fus A5/14 I/I Ps.pu. mix/Fus/P.lil Fus A5/15 I/I Ent.cl. mix/Fus Fus A5/16 MED Ent.cl. Asp/Fus/Pen/Rhi Fus A5/17 I Ps.st./Ser.mar. mix/Fus Fus A5/18 I Ps.fl./Ps.pu. Fus/Pae/Pen 0 A6/1 BNP/I Ent.cl./Ps.st./Ser.liq. Fus Fus A6/2 BNP/I Ent.cl/Ps.st. mix/Fus Fus A6/3 I 0 mix/Fus 0 A6/4 I Ed.tarda mix Fus A6/5 I/I C.fr./Ent.cl./Prot. Asp.nigr/P.lil/Pen 0 A6/6 I/I Ent.cl mix/Fus 0 A6/7 I 0 mix/Fus 0 A6/8 I 0 mix 0 A6/9 I/I Aer.hyd./Ed.tarda/Ent.cl. mix/Fus/P.lil Fus

A6/10 I/I Aer.hyd./Ent.cl./V.ch. Asp.nigr/Fus/Muc/Pen Fus A6/11 I 0 mix/P.lil 0

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Egg Status Yolk (bact) Shell (fungi) Yolk (fungi) A6/12 I 0 mix/Fus/P.lil Fus A6/13 I 0 Asp/Fus/Pen 0 A6/14 I/I Haf.alv./Ser.mar. Asp/Fus/Pen Fus A6/15 I/I Ed.tarda/Ent.cl. Asp/Fus/Pen 0 B1/2 LED 0 Fus/P.lil P.lil B2/1 I Ser.mar mix 0 B2/2 I/I Ser.mar mix Fus B2/3 I Pseudomonas Fus/mix 0 B2/4 I/I Aer.hyd Fus/mix Fus B2/5 I 0 Asp/Pen Fus B2/6 I 0 Mucor/Pen 0 B2/7 I/I Ser.mar Fus/Pen Fus B2/8 I 0 Fus/Pen Fus B2/9 I Ps./Ser. Paec/Pen 0

B2/10 I/I Pseudomonas Pen Fus B3/1 LED 0 Asp.fl. 0 B3/2 MED Ent.cl./Ser.mar. Fus/Scop Fus B3/3 LED C.fr./Ps/fl. 0 0 B3/6 LED ND ND ND B3/8 MED Ent.cl./e.c./Ps.aer./Ps.fl. Fus Fus B3/9 MED Ent.cl/Ps.fl Paec/Pen 0

B3/10 MED C.fr./Ps.aer./Salm.ar. Fus 0 B3/11 I/I (hole) Aer.sob/Ent.cl/Sal.ar.50 Pen 0 B4/6 LED Ps.fl/Sal.ar.50 Fus/Trichoderma P.lil

B4/10 I/I Ps.fl/Ent.cl/Sal.ar.50 Pen Fus B5/1 I 0 mix 0 B5/2 I/long crack Ent.cl/Sal.ar.35 Curv/Pen P.lil B5/3 I/I C.fr/Ed.tarda mix/Fus/P.lil P.lil B5/4 I/I C.fr/Ed.tarda Fus/P.lil P.lil B5/5 I/I Shew/Vib.ch/Sal.ar.35 Fus/P.lil Fus/P.lil B5/6 I/I Sal.ar.35 mix/P.lil P.lil B5/7 I 0 Fus/P.lil 0 B5/8 I/I Ps.fl/Sal.ar.35 mix/Fus/P.lil Fus/P.lil B5/9 I Sal.ar.35 mix/P.lil 0

B5/10 BNP/long

crack Ed.tarda/Sal.ar.35 Cunn/Pen Fus/P.lil B5/11 I/I Ed.tarda/Sal.ar.35 Cunn/Pen/P.lil P.lil B5/12 I 0 mix 0 B6/1 BNP/I Aer.cav.Sal.ar.35 Fus/P.lil P.lil B6/2 EED E.c/M.m/Ps.fl/Sal.ar.35 Pen 0 B6/3 I/I Aer.hyd/Ent.cl/M.m/Sal.ar.35 Fus/P.lil P.lil B6/4 MED Ps.fl Fus Fus B6/5 I/I Aer.hyd/Ent.cl/Sal.ar.35 Fus 0 B6/7 I/crack Ps.fl Fus P.lil B6/8 BNP/I Ent.cl/Ps.fl/Sal.ar.35 Fus/P.lil Fus

B6/10 EED C.fr/Prot. Cunn/Fus Fus B6/11 EED Ps.fl/Vib.ch. Paec/Pen 0 B7/10 I/I E.c/Ps.fl mix 0 B9/2 I/I Ent.cl/Ps.fl/Sal.ar.50 mix/Fus/P.lil P.lil B9/3 I/I Ent.cl/Ps.fl/Sal.ar.50 Fus/P.lil P.lil B9/5 BNP Al.fae/Ent.cl Fus 0

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Egg Status Yolk (bact) Shell (fungi) Yolk (fungi) B9/6 I 0 Fus/P.lil/Pen P.lil B9/7 I Ps.fl./Sal.ar.50 Fus/P.lil/Pen P.lil B9/8 BNP/I Ent.cl/Ps.fl/Ps.st/Sal.ar.50 mix/Fus/P.lil 0 B9/9 I Ps.fl. 0 0

B9/10 I/hole Vib.ch./Sal.ar.50 mix P.lil B9/11 I 0 Pen P.lil B9/12 I/I Ps.fl/Vib.ch./Sal.ar.50 mix 0 B9/13 I/I E.c/Ent.aggl/Sal.ar.50 mix Fus B9/14 I/I Ps.fl./Sal.ar.50 mix P.lil B9/15 I/I Ps.fl./Sal.ar.50 Pen 0 B9/16 I/I Ps.fl./Sal.ar.50 ND Fus/P.lil B9/17 I/I Sal.ar.50 Fus/P.lil P.lil B9/18 I Ps.fl./Sal.ar.50 Clad/p.lil 0 B10/3 I Ent.cl/Ps.fl. Pen 0 B11/1 I 0 Asp/Fus/P.lil 0 B11/2 I/I Aer.hyd/Ent.cl/Vib mix/Fus/P.lil Fus B11/3 I 0 Asp/Fus/P.lil 0 B11/4 I/I Ent.cl./Ps.fl Asp/Pen/P.lil 0 B11/5 I/crack Ent.cl./Ps.fl mix/P.lil P.lil B11/6 I/smashed Ent.cl./Ps.fl/Vib.ch mix/Fus/P.lil Fus B11/7 I 0 mix/Fus/P.lil P.lil B11/8 I/I Ent.cl. Asp/P.lil P.lil B12/1 BNP/crack C.div./C.fr./E.c./Ent.cl. mix 0 B12/2 I/I Aer.hyd./Ent.cl./Ps.fl. Fus Fus B12/3 I/I Ent.cl./Ps.fl Fus/Pen 0 B12/4 I/I Ent.cl./Ps.fl./Ps.pu Cunn 0 B12/5 BNP/I C.fr./Ent.cl./Kl./Prot. mix/Fus/P.lil 0 B12/8 EED Ent.cl. mix/Fus/P.lil 0

B12/11 I/crack Ps.fl./Ps.st. Rhizopus Fus B12/13 BNP/I Aer.hyd/Prot./Ps.fl. Acr/Fus/P.lil Fus B12/14 Dead at hatch Ac./Ach./Ent.cl. Fus/Pen 0

Ac. - Acinetobacter spp.; Ach. - Achromobacterium spp. Aer.hyd. - Aeromonas hydrophila; Aer.sob. - Aeromonas sobria Al.fae - Alcaligenes faecalis C.div. - Citrobacter diversus; C.fr. - Citrobacter freundii Ed.tarda - Edwardsiella tarda; E.c - Escherichia coli Ent.aggl.-Enterobacter agglomerans; Ent.cl.-Enterobacter cloacae Kl. - Klebsiella spp.; Kl.oxy. - Klebsiella oxytoca Prot. - Proteus spp. Ps.aer.-Pseudomonas aeruginosa;Ps.fl -Pseudomonas fluorescens Ps.pu. - Pseudomonas putida; Ps.st. - Pseudomonas stutzeri Sal.ar.35 - Salmonella IIIb 35; Sal.ar.50 - Salmonella IIIb50 Ser.mar. - Serratia marcescens; Ser.liq. - Serratia liquifaciens

Acr - Acremonium sp Asp - Aspergillus spp. Asp.fl - Aspergillus flavus Asp.nigr - Aspergillus niger Cunn - Cunninghamella sp Curv - Curvularia spp. Fus - Fusarium solani mix - mix of other fungi Paec - Paecilomyces spp. P.lil - Paecilomyces lilacinus Pen - Penicillium spp. Rhi - Rhizopus spp.

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References Berrang ME, Frank JF, Buhr, RJ, Bailey, JS & Cox NA 1999, ‘Eggshell membrane structure and

penetration by Salmonella Typhimurium’, Journal of Food Protection 62(1): 73-76. Buenviaje GN, Ladds, PW and Martin, Y 1994, ‘Disease-husbandry associations in farmed

crocodiles in Queensland and the Northern Territory’, Australian Veterinary Journal 71: 165-173.

Deeming DC & Ferguson MWJ 1990, ‘Methods for the determination of the physical characteristics

of eggs of Alligator mississippiensis: a comparison with other crocodilian and avian eggs’, Herpetological Journal 1: 458-462.

Ferguson MWJ 1982, ‘The structure and composition of the eggshell and embryonic membranes of

Alligator mississippiensis’, Trans. Zool. Soc. Lond. 36: 99-152. Gonzalez A, Satterlee, DG, Moharer, F & Cadd, GG 1999, ‘Factors affecting ostrich egg

hatchability’, Poultry Science 78: 1257-1262. Grigg G & Beard L 1985, ‘Water loss and gain by eggs of Crocodylus porosus, related to incubation

age and fertility’, in “biology of Australasian Frogs and Reptiles”, ed. Grigg et al., Royal Zoological Society of NSW, pages 353-359.

Heard DJ, Jacobson, ER, Clemmons, RE and Campbell GP 1988, ‘Bacteremia and septic arthritis in a

West African dwarf crocodile’, JAVMA 192(10): 1453-1454. Hibberd E 1991, ‘Mycoses in crocodiles’, In “Proc.Intensive Trop.Anim.Prod.Seminar (ITAPS),

Townsville, pp 216-223. Huchzermeyer FW, Henton, MM, Riley, J & Agnagna, M 2000, ‘Aerobic intestinal flora of wild-

caught African dwarf crocodiles Osteolaemus tetraspis’, Onderstepoort Journal of Veterinary Research 67: 201-204.

Joanen T & McNease L 1981, ‘Incubation of alligator eggs’, First Ann. Alligator Production

Conference, Gainesville, Florida Joanen T & McNease L 1991, ‘Managing the alligator egg and hatchling’, Procs. Intensive Tropical

Animal Production Seminar, Townsville, Australia, pp 193-205. Kern MD & Ferguson MWJ 1997, ‘Gas permeability of American alligator eggs and its anatomical

basis’, Physiological Zoology 70(5): 530-546. Ladds PW 1989, ‘Management and diseases of farmed crocodiles’, in Proc.Intensive

Trop.Anim.Prod.Seminar (ITAPS), Townsville. Madsen M, Hangartner, P, West, K & Kelly P 1998, ‘Recovery rates, serotypes, and antimicrobial

susceptibility patterns of salmonellae isolated from cloacal swabs of wild Nile crocodiles (Crocodylus niloticus) in Zimbabwe’, J. Zoo Wildlife Med. 29(1): 31-34.

Manolis SC, Webb, GJW and Dempsey KE 1987, ‘Crocodile egg chemistry’, in “Wildlife

Management: Crocodiles and Alligators”, ed. Webb et al., Surry Beatty and Sons Pty Ltd, Chapter 46, pages 445-472.

Manolis SC, Webb, GJW, Pinch, D, Melville, L & Hollis, G 1991, ‘Salmonella in captive crocodiles

(Crocodylus johnstoni and C. porosus)’, Australian Veterinary Journal 68 (3): 102-105.

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Misra PR, Kumar, D, Patnaik, GM, Ramon, RP and Sinha, A 1993, ‘Bacterial isolates from apparently healthy and diseased crocodiles (Gavialis gangeticus)’, Indian Veterinary Journal 70: 375-376.

Rickard MW, Thomas AD, Bradley S, Forbes-Faulkner J & Mayer RJ 1995, ‘Microbiological

evaluation of dressing procedures for crocodile carcases in Queensland’, Aust. Vet. J. 72: 172-176.

Schumacher J & Cardeilhac PT 1990, ‘Mycotic infections of egg membranes in the American

alligator (Alligator mississippiensis)’, in “IAAAM Proc.”, ed. Francis-Floyd R, vol.21, pages 138-140.

Sinha RP, Roy, BK & Chaudary, SP 1987, ‘Gastroenteritis in a crocodile (Crocodylus palustris)’,

Indian Veterinary Journal 64:69-70. Thomas AD, Forbes-Faulkner, JC, Speare, R & Murray C 2001, ‘Salmonelliasis in wildlife from

Queensland’, Journal of Wildlife Diseases 37(2): 229-238. Thomas AD, Sigler L, Peucker S, Norton JH & Nielan A 2001, ‘Chrysosporium anamorph of

Nanizziopsis vriesii associated with fatal cutaneous mycoses in the salt-water crocodile (Crocodylus porosus)’, Medical Mycology 40:143-151.

Thorbjarnarson JB & Hernandez G 1993, ‘Reproductive ecology of the Orinoco crocodile

(Crocodylus intermedius) in Venezuela I. Nesting ecology and egg and clutch relationship’, J. Herpetology 27(4): 363-370.

Webb GJW, Buckworth, R & Manolis, SC 1983, ‘Crocodylus johnstoni in the McKinley River, N.T.

VI. Nesting biology’, Australian Wildlife Research 10: 607-637. Webb GJW, Manolis, SC, Dempsey, KE & Whitehead PJ 1987, ‘Crocodilian eggs: A functional

overview’, in “Wildlife Management: Crocodiles and Alligators”, ed. Webb et al., Surry Beatty and Sons Pty Ltd, Chapter 43, pages 417-422.

Whitehead PJ 1987, ‘Respiration of Crocodylus johnstoni embryos’, in “Wildlife Management:

Crocodiles and Alligators”, ed. Webb et al., Surry Beatty and Sons Pty Ltd, Chapter 47, pages 473-497.

Wink CS & Elsey RM 1994, ‘Morphology of shells from viable and nonviable eggs of the Chinese

alligator (Alligator sinensis)’, Journal of Morphology 222: 103-110. Wink CS, Elsey RM & Bouvier M 1990, ‘Porosity of eggs shells from wild and captive, pen-reared

alligators (Alligator mississippiensis)’, Journal of Morphology 203: 35-39.

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6. Capture The development of electrical stunning equipment has progressed significantly since it was first reported (RIRDC Pub. No.00/105). Several stunning wands have been developed in recent years with improvements with each new model and the original mains power and belt pack units have been replaced by a backpack unit (see picture below). The unit is enclosed in a clear plastic Perspex box and housed in a canvass backpack. The unit is waterproof and will float if dropped into the water. The equipment is now considered an essential piece of farm equipment to ensure the safe and efficient capture of crocodiles (Jason Lever, pers. comm.) The equipment has a high adoption rate amongst farmers in Australia with most farms now using the stunner to capture animals. In recent years the technology has been exported to several countries around the world including South Africa, Zimbabwe and Spain. Producers in the USA have also shown keen interest after an equipment demonstration at Darwin in 2004 at CSG who hosted an international conference.

This project was developed in two phases. The first phase was to develop the equipment, and to trial and test it’s usefulness under commercial conditions. This phase has been completed and detailed in RIRDC Pub No 01/123. The second phase relates to the issue of animal welfare and the effects stunning may have on the animal. A trial was conducted at Koorana Crocodile Farm, Rockhampton Queensland. The aim was to compare and evaluate the stress responses and welfare of crocodiles to electrical stunning as opposed to the traditional capture and restraint method of noosing and roping crocodiles. The trial involved 99 estuarine crocodiles, C. porosus. Animals had a mean total length of 1.96 with a range of 1.56 to 2.24metres. Animals were housed individually in fibre-glass pens. For animals captured using the stunning equipment a 110 V charge was delivered for 3-11 seconds (average 6.1seconds) via a set of metal forks applied to the back of the animal's neck. Crocodiles were rapidly immobilized during stunning, which was followed by 5-10s of rigor and tail twitching after which the animals were completely relaxed with legs splayed backwards, parallel to the body. Crocodiles remained incapacitated for 5-10 minutes, allowing animals to be handled, examined and measured. Another group of crocodiles that were noosed and restrained using standard handling methods was used for comparison. This method of capture involved noosing the animal’s top jaw and allowing the animal to roll and thereby tangling both jaws around the rope. A second rope was used to secure the jaws closed. The time taken for this method ranged from 1.6-15.5 minutes from capture to sampling and returning animals to pens (average 8.3minutes). When capturing using this method, animals would struggle, thrash and roll about and generally exert short sharp bursts of intense activity. Blood samples were taken via the cervical sinus located at the back of the head from stunned and noosed animals immediately upon immobilization or capture and after 30 min, 1, 4, 12, 24 and 48 hours of recovery. Blood samples were analysed for the stress hormone corticosterone, as well as

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glucose and lactate levels. Following noosing and restraint, there were large increases in blood glucose, lactate and corticosterone levels and recovery back to resting levels occurred between 8-24 hours after capture. With the stunned animals, there were no increases in blood glucose or corticosterone levels from resting levels, however lactate levels did increase but the increase was much lower than for noosed animals. Recovery to resting levels of lactate was within 4 hours for the stunned crocs. The results conclude that the stress response of stunned animals was significantly reduced compared to manually captured crocodiles and that recovery was more rapid for the stunned animals. The results imply that immobilization by electro-stunning is much less stressful for the crocodiles. These results support the observations made by researchers and industry that stunned animal’s return to feed and water sooner than animals caught using traditional methods. A full report on this study “Comparison of Stress Induced by Manual Restraint and Immobilisation in the Estuarine Crocodile, Crocodylus porosus” can be found in the Journal of Experimental Zoology 298A:86-92 (2003). Clearly this equipment would not have been developed without the collaborative working relationship between DPI&F, RIRDC and industry. Several significant benefits flow from this work. Some of those benefits are of a welfare nature and some of an economic nature. Benefits include:

• animals are less stressed who stunning is applied as opposed to traditional rope capture techniques

• reduced stress means animals resume normal activities more rapidly; they commence eating sooner which mean less interruption to growth rates

• there is less staff down time when stunners are used because injuries are significantly reduced

• prior to the development of stunners crocodiles would be shot at harvest time without any knowledge of skin quality prior to capture

• the action of discharging firearms in confined spaces is in itself risky; stunning eliminates this risk

• stunning allows selective harvesting because animals with damaged skin can be returned to enclosures to recover; the price difference between first and second quality skins is significant.

• several major Australian producers have installed individual growout pens at considerable cost, some have invested millions of dollars. Without stunners the task of removing crocodiles from these pens without damaging skins would be almost impossible

• as a result of changes to handling practices more animals can now be safely handled in one day’s work.

In essence stunning equipment has reduced animal stress, increased human safety, conferred economic benefits and encouraged investment with confidence.

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7. Economics CrocProfit CrocProfit is a software package developed by Bill Johnston, DPI&F (Queensland) in consultation with Steve Peucker, Rob Jack and Bernie Davis. Experienced commercial crocodile producers were also consulted regarding content and operations which are relevant only to the crocodile industry. CrocProfit is a complete information package for crocodile farmers and potential investors which includes a CD which contains reference material and a comprehensive list of contacts for related State Governments and industry associations for any questions or assistance that may be required. In conjunction with the reference material, the farm model provided allows potential investors to evaluate the economics of crocodile farming, using their own input parameters, before any investment or construction occurs. The model covers all aspects of farming in a comprehensive fashion. The model is based upon the cost-benefit analysis technique. Cost-benefit analysis is a conceptual framework for the economic evaluation of projects, in this case, crocodile farming projects. This approach differs from financial appraisal in that it considers all gains and losses. The basic premise of cost-benefit analysis is to assist you to make a decision in regard to the allocation of resources. In particular, CrocProfit helps investors to make decisions about whether or not to invest in crocodile farming. Existing farmers can also use CrocProfit. Once the data are entered into the model a farmer can use the computer version of his farm to determine the impact of different management decisions. For example, the farmer may wish to know how introducing new diets will affect annual production and profitability. The model is easy to operate. It is simply a matter of entering data into the input cells of the model you have selected. It is better to be as accurate as possible with data entered in order to get the best possible results. Risk analysis is also included to ensure that all possible economic outcomes are considered. The model provides a static figure on the summary page (annual return). When probabilities (uncertainty) are added to production and price the range of economic outcomes are delivered (cumulative probability distribution). Investors or potential farmers must accept that they can receive a wide range of returns (including negative ones). Once all the data are entered into the model you can view the summary statistics for the farm. All the statistics used are explained in the model itself. The program runs the farm over a 20-year period. The output includes the expected annual returns, when the farm is paid off and the interest rate at which you can borrow funds to invest in the project. The summary statistics will also provide a break down of costs on a per animal basis. The models are set up in Microsoft Excel as a spreadsheet. All the sheets contained in the model are labelled for easy reference and there are buttons in the menu for easy movement between sections. The basic premise for the operation of the model is that there are two types of cells; the "data entry cell" is coloured yellow and the "data secured cell" in coloured red. The yellow cells allow you to enter data and the red cells are locked because they contain calculated answers. CrocProfit can be purchased from DPI&F (Queensland) by contacting the DPI Call Centre on 132523 (local call within Queensland) or 1800 816 541 (within Australia) or +61 7 3239 3163 (overseas), email [email protected] or by going to the DPI website www.dpi.qld.gov.au.

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8. Extension 17th Working Meeting of the IUCN-SSC Crocodile Specialist Group The 17th Working Meeting of the Crocodile Specialist Group (CSG) was held in Darwin from the 27th to 29th May 2004. CSG is an international crocodile specialist body with approximately 350 members in eight regions throughout the world. The organization’s primary role has been crocodile conservation. However, this year more emphasis was given to commercial production because of financial support from RIRDC. The notion of sustainable production is by no means new to the international crocodile community and the links between conservation and commercial farming are strengthening. Steve Peucker, Rob Jack, Bernie Davis and Robert van Barneveld represented DPI&F/RIRDC. DPI&F also had Bill Johnston and David Lohan representing them. Robert van Barneveld and Steve Peucker presented a paper on the development of manufactured feed for commercial crocodiles. This paper outlined past, present and future research directions in encouraging these fussy feeders to accept pellets as their primary source of food. If this objective can be achieved it will reduce production costs and improve feed efficiency. Much interest was shown in this paper not only by Australian producers but also by producers from South Africa, Zimbabwe and Papua New Guinea. In addition to this paper, DPI&F staff presented posters on the following topics: 1. CrocProfit, a software package developed by Bill Johnston of DPI&F in conjunction with

crocodile R&D staff and commercial crocodile producers 2. use of BIA to determine body condition of farmed crocodiles 3. development of various types of crocodile stunning equipment covering a range of models from

early prototypes to current commercial models. In addition to the poster on stunning equipment Rob Jack and Steve Peucker demonstrated the equipment for CSG delegates on animals at Crocodylus Park, a Darwin crocodile farm. Delegates to CSG include biologists, wildlife managers, government officials, commercial crocodile producers, conservationists, skin traders, tanner and leather goods fashion experts. Any opportunity to address and discuss the commercial aspects of crocodile production with such people is seen as most important. Crocodile research seminar The conference was held on the 13th November 2003 in Cairns. The primary purpose of the conference was to concentrate leading industry researchers and industry stakeholders together. This was the first time that a ‘combined’ industry conference has been held; involving major RIRDC funded research for the crocodile farming industry. The conference featured eight RIRDC funded projects plus an additional three speakers, including producer Mrs Lillian Lever from Koorana Crocodile Farm who spoke on single pen accommodation. Researchers presented results and information from all RIRDC funded (see Table 8.1) crocodile research projects that are current or have been recently completed. Topics included nutrition, diagnosis and management of fungal diseases, skins processing, marketing, incubation, genetics and husbandry. The conference presented an opportunity for vigorous discussions of results that have been achieved to date and also allowed producers to meet researchers on a face-to-face basis.

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The following RIRDC funded crocodile industry researchers were invited to speak at this conference along with the three additional speakers. Table 8.1 Speaker agenda for Cairns Seminar – November 2003 Researcher Organisation Project Mr Charlie Manolis Wildlife Management

International, Darwin Improving the quality of Australian crocodile (C. porosus) skins Production implications of trace element concentrations in crocodile eggs and tissues

Mr Brendan Goulding Department of Primary Industries and Fisheries, Brisbane

Desktop analysis of the export and domestic market for the skins and leather products of newly emerging animal industries

Ms Sally Isberg University of Sydney, Sydney

A genetic improvement program for farmed saltwater crocodiles

Dr Stephen Hawkins CSIRO, Melbourne Improved preservation and early stage processing of Australian crocodile skins

Dr Robert van Barneveld Barneveld Nutrition P/L, Brisbane

On-farm research of pelleted feed for crocodiles

*Mrs Lillian Lever Koorana Crocodile Farm. Rockhampton

Single pen accommodation for skin quality improvement

*Mr Tom Dacey Wet Tropics Authority, Cairns

CSG 2004 Meeting

*Dr Mark Read Environmental Protection Agency, Cairns

Satellite tracking of wild crocodiles

Dr Annette Thomas Department of Primary Industries and Fisheries, Townsville

Fungal disease of crocodiles and their control

Mr Steve Peucker Ms Beth Symonds

Department of Primary Industries and Fisheries, Townsville University of Queensland, Brisbane

Using a Bioimpedance Analyser to measure the body composition of farmed estuarine crocodiles

*non RIRDC project speakers Speakers travelled from Darwin, Sydney, Melbourne, Rockhampton, Cairns and Brisbane to attend the conference. Producers travelled from Darwin and regional Queensland to attend. Conference organizers fostered a climate of open discussion on research issues, which involved both researchers and industry to their mutual benefit. Some outcomes from this seminar were: • appreciation by industry of RIRDC research involvement in the crocodile industry • positive feedback on all research projects from clients and especially established producers • illustration of the wide variety of current research • presentation of benefits to industry that have already been achieved recently and anticipated future

benefits. Crocodile producer, Mrs Lillian Lever, from Koorana Crocodile Farm, Rockhampton displayed a single pen accommodation that her company has developed. These pens are manufactured from ploy

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propylene plastic. There is a trend in the industry to use single animal pens to finish animals prior to harvesting to maintain their skin quality. Mr Charlie Manolis from Wildlife Management International, Darwin discussed the influence of incubation temperature and male genetics in determining scale row patterns. Both incubation temperature and male genetics are thought to influence scale patterns, which in turn influence the value producers receive for their skins. Ms Sally Isberg, University of Sydney, presented a paper on a genetic improvement program for farmed crocodiles. The program identifies a number of breeding objectives thus creating a crocodile selection index (crocPLAN). Relative economic values are provided thus aiding in determining the amount of emphasis placed on the selection objective (CrocIndex). Once fully understood by industry this research offers the industry a blue print for selecting future breeders and superior progeny. This may eventually lead to less reliance on wild stocks. Reports of the completed projects were on display along with an RIRDC order forms. Consideration should be given to holding this event on a bi-annual or annual basis. The timing of the conference could also coincide with the milestones/results reporting of the different projects. The conference could be held on an alternating basis in Cairns or Darwin because these are the two primary locations for commercial crocodile production in Australia. Introduction to crocodile farming in Queensland seminars A number of seminars were given by DPI&F staff as an Introduction to crocodile farming in Queensland. This project’s target audience was potential crocodile farming investors in far north Queensland including some indigenous communities within the region. Interested parties who attended this seminar were members of the Hopevale and Yarrabah Aboriginal Councils and other individuals from Dimbulah and Townsville. The main objective of this work is to establish a sustainable crocodile farming industry in north Queensland. A half-day seminar for potential stakeholders with presentations from government and industry representatives was held on the 30th October with a CrocProfit workshop on 31st October (see attached agenda). The half-day seminar offered information on technical requirements for farming crocodiles, the marketing of crocodile skin and meat products and a description of the requirements (environmental laws and licences) needed to establish a crocodile farm. Current industry situation and possible future directions were also discussed so that potential investors could make an informed decision on entering the industry. A tour of Peter Fisher’s Melaleuca Crocodile Farm near Mareeba was also undertaken on the afternoon of the 30th October. The CrocProfit workshop on day two offered participants an insight into the computer program Crocprofit and how it could assist potential producers and investors. The expected outcome of this workshop was that interested parties would gain an insight into the requirements for farming crocodiles in Queensland. To this end, positive feedback was received from all participants on content and usefulness of presentations. It was clear that this workshop left interested parties in a better position to determine the feasibility of establishing a crocodile farming operations. There is strong interest from all those that attended and Steve Peucker will follow up to see how things are progressing and if any further assistance is required.

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The agenda for these seminars can be seen below.

An introduction to crocodile farming in Queensland

Brother’s Leagues Club, 99-105 Anderson Street, Manunda, Cairns

Thursday 30th October 2003

9.15 am Welcome and introduction Steve Peucker, DPI&F, Townsville

9.30 am DPI research facilities Steve Peucker, DPI&F, Townsville

9.45 am Environmental issues and crocodile farming Mark Cavicchiolo, EPA, Cairns

10.15 am Licensing requirements for farming crocodiles in Queensland Crocodile Management Plan

Russell Best, EPA, Cairns

10.45 am Morning Tea 11.15 am Tourism and crocodile farming John Lever, Koorana Crocodile Farm

11.45 am Establishing a farm – what to look for and things to consider

Peter Fisher, Melaleuca Crocodile Farm

12.15 pm General husbandry of farmed crocodiles Steve Peucker, DPI&F, Townsville

12.30 pm Future of the industry

Bernie Davis, DPI&F, Townsville

Thursday 30th October – Afternoon

For interested participants - Visit to Peter Fisher’s crocodile farm, Mareeba

Friday 31st October 2003 CrocProfit

9.00 am Introduction – Bill Johnston 9.15 am Morning Tea 9.30 am Learning CrocProfit 12.00 pm Lunch 12.45 pm Using CrocProfit to predict outcomes from management changes. 2.00 pm Close What is CrocProfit? CrocProfit is an Excel spreadsheet program designed to provide valuable management assistance. It is a decision tool for investors wishing to start a crocodile farm, or for existing producers to monitor progress and assess changes in their business. It covers all aspects of the farming operation and if utilised effectively will be invaluable in the day-to-day management of the farm.

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What will be involved? If possible please bring your own laptop computer. This will be a hands-on exercise that would work best if you bought along your own figures.

If you are not able to supply your own computer please let us know in advance and we will endeavour to bring one for you to use on the day. Also if you do not have your own figures we can suggest possible scenarios for the exercise. On-farm visits Crocodile farming as an emerging industry requires and involves on-farm demonstrations. This is true in particular of the areas involving stunning equipment, pellet manufacture and feed trials to gauge pellet acceptability by commercially farmed animals. Two other areas of note have emerged in 2004 during the course of farm visits to the Northern Territory. These are the development of individual pens for animals towards the final stages of their grow-out life and the request for assistance to develop solutions/ treatments to speed up recovery from bites and scratches which could result in the downgrading of skins. In summary farm visits have been used to: 1. build rapport and trust between researchers and producer clients 2. show producers how use improved stunning equipment to capture crocodiles 3. gauge if stunning is more welfare friendly to crocodiles than the traditional pole/rope method of

capture 4. allow crocodile team members to observe what producers are doing by way of individual pen

accommodation for crocodiles which is an industry initiative and is being researched by producers rather than research organizations

5. place R&D members in a better position to discuss developments with other producers 6. allow for the inspection and discussion about animals under going on-farm pelleted feed trials. 7. demonstrate that pelleted feed can deliver benefits but the practice is somewhat complex and

needs further adjustment with their assistance.

Crocodile Research and Development Bulletin – Volume 3 This document represents the third Crocodile Research/Development Bulletin released by DPI&F. The format is different from previous Bulletins in that it will allow readers to add further information to the various topics as it becomes available. Other features of Bulletin No. 3 are the inclusion of CD's containing: • CrocProfit and CROCTEL. CrocProfit (prototype) is an Excel spreadsheet program designed to

provide valuable management assistance. It is a decision tool for investors and established farmers alike. CROCTEL is a program that compiles statistics on production parameters and in particular as it relates to breeder animals.

• CrocInfo which contains the Crocodile Research Bulletin No.2 and "Crocodile Capers"

Newsletter (Issues 1-7).

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A range of topics were addressed including housing, effluent management and genetics. Some of the information might be of a preliminary nature however the Bulletin represents an attempt to consolidate a cross section of information in one document making it readily available to clients. Crocodile Capers newsletter Crocodile Capers has proved popular with both Australian and international crocodile industry stakeholders and has been recognised as a “very useful technical source”. Articles on crocodile research outcomes have been presented from various research organisations including DPI&F, University of Queensland and CSIRO. The newsletter is now emailed to most readers which is a faster and more efficient distribution method. Past and current issues of Crocodile Capers can now also be found on the DPI&F website.

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9. Discussion of results Nutrition Pelleted feeding, as the highest industry priority, has been pursued in the research period 2003/2004. Originally the objective was to feed crocodiles manufactured feed from the day they are hatched until the day they are harvested. This has proven to be an elusive goal in part. Results have varied from clutch to clutch with an occasional group taking manufactured feed. Conversely other clutches have chosen to withhold from feeding to the point of starving, while others have picked over pellets, consumed some but never consumed sufficient to promote the animals’ growth and maintain their welfare. Newly hatched crocodiles have been generally reluctant to accept pellets. The non-acceptance of pellets for newly hatched crocodiles should not be seen as an issue. Animals in their early life consume so little feed that any savings by way of reducing feed intake by using a more concentrated diet is of little consequence. Further, the margin of error at this early stage of the animal’s life is so small that low(er) intake can have serious adverse results. It is concluded that producers should start newly hatched crocodiles on meat diets to ensure a robust start for animals. Grower animals are another issue when it comes to manufactured pellet diets. They accept pellets and grow well on them. The link between pelleted feed and skin quality is not well understood at this juncture but is the subject of ongoing investigation. Success has been achieved in having grower crocodiles take manufactured pellets as their sole source of feed. Further, animals have grown well on this feed and achieved high welfare standards. Animals have proven to be leaner than those animals fed meat offal diets. This achievement is most desirable in the eyes of producers and tanners as lean animals tend to produce long narrow(er) skins which are best suited to the manufacturing trade. Grower animals consume larger quantities of food and this is where feed cost economies are important. Manufactured feed has feed efficient advantages over offal diets leading to transport and storage savings. It seems size rather than age is the trigger for crocodiles to take pellets as their sole source of food. This area is under further investigation so refinements can be built into advice to producers. Further work needs to be done on identifying the best least cost diets which will promote animal growth and this work is underway. In summary it seems that starting hatchling animals on manufactured pelleted feed is not universally achievable. However, grower animals will consume manufactured feed and it is now a matter of refining practices. A comprehensive paper on nutrition is attached as Appendix A with the title Crocodile Nutrition Research – Strategic Directions 2002-2005. Bioelectrical Impedance Analysis (BIA) BIA techniques have been used in human populations but the technology is very new to the crocodile industry. The purpose of this work in crocodile R&D is to measure body fat content. Comparisons are being made between diets researched at the Townsville crocodile complex. Work is in its infancy but early results look promising.

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Skin Quality The link between manufactured diet and skin quality is being studied. Some skins have been placed with CSIRO’s leather research unit based in Melbourne. No results are available to date. Bite treatment Clearly, the treatment of and healing of bites and scratches are linked to skin quality. This work is in the proposed category at this stage and can be conducted at the Oonoonba Veterinary Laboratory in Townsville. This location has the advantage of easy dialogue between laboratory and crocodile R&D staff, as they are all located at the same centre. Single pens Single pens are an industry initiative. No claim is made that development in this area is an outcome of the R&D team. However, it is important to recognise the importance of these pens in contributing to the production of first quality skins. Stunning equipment has been linked to the development of these pens because stunning equipment facilitates the easy(ier) handling of animals housed under these conditions. Stunning equipment Stunning equipment is highly developed and its use has been linked successfully to the development of individual pens. Of significance in the further development of stunning equipment has been the recognition that stunning equipment operated on the same electrical impulses as the human heart (50 mHz). To avoid any problems if an electrical shock occurred to the operator the electrical impulse of stunners has been altered to 400 mHz. Microorganism study of eggs from freshwater crocodiles (C. johnstoni) Prior to the completion of this work little had been done to gain an insight into the impact of microorganisms both on hatchability and hatchling viability. This research has advanced industry knowledge in this area, however the reported research relates to freshwater crocodiles which are of little commercial importance. Research completed on saltwater crocodiles which are of commercial significance needs to be reported so that producers can take full advantage of these important results.

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10. Implications The objective of the crocodile R&D program is to contribute to the sustainability and development of the Australian commercial crocodile industry. Without R&D support the industry will make some technical advancement but it will be slow and remain the subject of closely guarded intellectual property. In other words some producers may advance their cause but the industry generally will not benefit. A major R&D contribution has been the development of electrical stunning equipment to capture crocodiles. This equipment allows: 1. producers to capture more animals in a day’s work. 2. safer handling of these animals resulting in less down time due to injury. 3. animals to be captured, inspected and returned to pens in cases where skins had been damaged.

Previously, all animals would be shot at harvest time. If an animal had a damaged skin this lead to down grading and lower returns.

4. for the expansion of single pen accommodation because animals can now be easily caught

without damaging skins. In the absence of stunners, investment in single pen accommodation would have been much lower.

One industry member tried to build his own stunning equipment. This farm-built equipment was seen to be both very dangerous and ineffective. In the United States researchers had tried to build stunning equipment, but these early US models proved ineffective. In the event that nutritional research ceased, the industry would most likely not have the skills or the facilities to bring pelleted feed research to a successful conclusion. Neither would industry have the facilities to test a range of diets to best identify the crocodile’s nutritional requirements and present these diets in a least cost program. The crocodile R&D program has played a vital and important role in industry development. The R&D program should continue into the future because it will continue to deliver outcomes that will help meet the goal of the sustainable and profitable use of resources.

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11. Recommendations The R&D packages used to deliver outcomes for the Australian commercial crocodile industry and government agencies have been several and varied. The combination has been shown to be effective and will continue to be used. As recently as December 2004, DPI&F conducted a review of the R&D program to gauge its effectiveness. They showed the program to be sound after extensive consultation with clients and researchers outside the immediate R&D crocodile team. On the basis that the program has shown to be sound and meets the needs of clients including government agencies who fund R&D the following successful R&D methods will continue to be used. 1. Research on crocodile nutrition will continue to be conducted at DPI & F’s purpose-built

crocodile research centre in Townsville. 2. Some areas of nutritional research will require out-sourcing and a collaborative consultancy

approach with scientists outside DPIF who specialize in aquatic nutritional programs will continue.

3. The consultancy will further research the question of pelleted feed manufacture for crocodiles. 4. The consultancy will also examine and determine which diet formulations best meet the animals’

needs and can be met on a cost/benefit basis. Amino acid profiles for superior diets will then be written into least cost diet programs for distribution to clients.

5. Other nutritional work may involve negotiations with commercial feed millers to produce

concentrates based on these research findings. Producers can readily mix concentrates with ingredients held on farms. Concentrates reduce the risk of making errors in compiling the important micro ingredients of diets.

6. Some work does not lend itself to the Townsville crocodile R&D facility. A case in point is work

covering bites and scratches, which will require the assistance and direction of veterinary staff from Townsville’s Oonoonba Veterinary Laboratory to produce outcomes.

7. Other work will require the support of staff from organizations other than DPI&F. For example

collaborative work between the crocodile R&D team and CSIRO’s animal skin research team will take place. The CSIRO team will examine skins from animals fed pellets as their sole source of food and compare the quality of skins from these animals with animals fed traditional offal meat diets. This work will play an important role in discussions with tanners and manufacturers.

8. On-farm experiments need to be continued as they play a key role in the dissemination of

research results. Results can be refined with feedback from client producers to better suit commercial conditions. Pelleted feeding is an example.

9. Workshops, seminars, technical bulletins and Crocodile Capers need to continue to play a key

role in the dissemination of R&D results. 10. Farm visits will continue to help the program with the dissemination and collection of on-farm

data and experiences. 11. Work covering the micro contamination of eggs from C. johnstoni is important. However, the

work covering research on estuarine crocodiles (C. porosus) eggs needs to be written up and made available to producer client because of the commercial significance of these animals.

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12. DPI&F should invite international skin buyers and garment manufacturers to its Townsville research facility to make them aware that a R&D facility is in place to service the Australian crocodile industry and some of this work is linked to skin quality.

13. Consideration should be given to writing a skin quality standard. The standard should depict

quality colored photographs of different skin types and a short, crisp description of what is represented in each photograph.

The crocodile R&D program is well organized and will continue to use tried and tested methods. At the same time researchers are always on the looking for further opportunities to improve their research and development skills.

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Appendix A. Crocodile Nutrition Research - Strategic Directions 2002-2005

Developed in conjunction with the Queensland Department of Primary Industries and Fisheries,

PO Box 1085 Townsville, QLD, 4810.

January, 2003 (Last modified: November, 2003)

Prepared by Dr Robert van Barneveld

Barneveld Nutrition Pty Ltd and the BECAN Consulting Group,

19-27 Coonan Rd, South Maclean, Qld, 4280. Ph: 07 5547 8611. Fax: 07 5547 8624.

E-mail: [email protected]

Visions • To contribute to the sustainability of the farmed crocodile sector by addressing the needs

identified by commercial producers. • To reduce the nutrition proportion of farmed crocodile (all life stages) production costs by

improving the supply of nutrients and the efficiency of use of nutrients and reducing the cost of diets. This vision is also reflected in the RIRDC R&D plan for new animal products 2002-2005.

Introduction Commercial crocodile production currently relies on combinations of fresh meat for the supply of nutrients to all production phases. Not only is fresh meat unlikely to represent an optimal supply of nutrients for efficient crocodile growth, but supply, storage and handling can be difficult and costly. To date, crocodile nutrition research undertaken at the Queensland Department of Primary Industries and Fisheries crocodile research facility in Townsville has addressed a number of aspects of crocodile nutrition with a view to reducing the production costs attributable to nutrition. These include: • Growth and preference experiments with diets consisting of single types or blends or raw meat.

Unfortunately, all this research served to demonstrate is that different forms of meat have different nutrient contents and hence will induce varying growth responses.

• Development of manufacturing capacity for the production of semi-moist feeds for crocodiles at

the Queensland Department of Primary Industries and Fisheries facilities at the Oonoonba Veterinary Laboratories. The team at DPI&F now possess unique skills for the production of semi-moist feeds.

• A range of growth experiments comparing the performance of crocodiles fed semi-moist

manufactured feeds varying in nutrient and ingredient composition. • Preference experiments with individually housed crocodiles. Outcomes from this research may

have been influenced by the pre-trial feeding regime of the crocodiles.

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• Provision of semi-moist manufactured diets for hatchling and grower crocodiles for assessment on commercial enterprises.

• Development of breeder diets for commercial assessment. It is interesting to note that in 1998, a crocodile nutrition workshop convened in Townsville defined a starting composition for a manufactured pellet based on previous experience with the following features deemed desirable:

Dry matter content 60% Crude protein content (as fed) 35-40% Crude fat content (as fed) 16% Fresh product: 20-50% Binders: Nil Cost: $1.00-$1.50/kg.

Research conducted to date has revealed that crude fat content should be less than 5% and that fresh product is not essential for grower crocodile consumption, hence the initial features of manufactured feeds have changed through the conduct of research. In summary, crocodile nutrition research conducted to date has resulted in the development of manufactured diets with no fresh product needed that will support growth of crocodiles at an acceptable rate. A research program is now needed to capitalize on these initial investigations with a view to reducing the nutritional costs of producing crocodiles. Mission To optimize the nutrition of farmed crocodiles through research and development aimed at reducing diet costs, improving the efficiency of nutrient supply and the efficiency of nutrient utilization. Nutritional drivers of profitability The following outlines the primary nutritional drivers to profitability in any intensive animal production enterprise and should form the basis for the development of a nutrition research program: Profit = (Revenue – Costs of production) x volume Feed costs Non-feed costs Feed cost ($/kg) Feed conversion efficiency Ingredient supply Feed intake Diet specifications Variation in feed intake and FCE Manufacturing costs Feed utilization Transport costs Storage costs

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Feed costs Ingredient supply The range of ingredients available, their maximum inclusion level and their capacity to supply protein and energy for use in metabolism will have the greatest influence on the cost of manufactured diets for crocodiles. Strategies 1. Characterise the digestible amino acid and energy content of a range of feed ingredients for

crocodiles in different production phases. The initial research focus will be on the grower phase where the greatest potential exists to improve production efficiency through reduced improved nutrition.

2. Review existing literature or conduct independent research to define the enzymic and

microbiological profiles of the crocodile gut to determine the most suitable ingredients for use in manufactured crocodile diets.

3. Determine the proportion of dietary protein utilized as energy in farmed crocodiles and alternative

energy sources. Key performance indicators • A comprehensive database on the nutritional quality of a range of feed ingredients suitable for use

in manufactured crocodile diets. • Definition of the digestive capacity of farmed crocodiles in various production phases. • An understanding of the fate of dietary protein in crocodile nutrition. Diet specifications Definition of the nutrient requirements of farmed crocodiles in various production phases will assist with reducing the cost of manufactured diets, will improve the efficiency of use of nutrients in manufactured diets and will reduce the proportion of nutrients excreted in waste. While protein and energy supply will be a focus, other diet specifications such as calcium:phosphorus ratio and the strategic use of feed additives requires examination. Strategies 1. Conduct growth experiments to determine the response of crocodiles to changes in dietary

protein, fat and energy supply. This will form the basis of subsequent experiments by defining the degree of accuracy required when formulating diets.

2. Assess the performance of individually housed and group housed crocodiles when they are fed

manufactured diets to quantify the influence of social interactions on growth performance. 3. Assess the degree of variation in growth performance and feed intake of individually-housed

crocodiles to determine the influence of other factors, such as genetic potential, of farmed crocodile production.

4. Conduct experiments to define the protein and energy requirements of farmed crocodiles in

various production phases.

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5. Define the role of calcium and phosphorus in crocodile diets in relation to a) skeletal and scutal development, and b) influence on acid-binding and expression of endogenous enzymes and osmotic potentials in the digestive tract.

Key performance indicators • Knowledge of the influence of social interactions vs nutritional inputs on the growth

performance of farmed crocodiles. For example, the influence of genetics on different growth responses of animals from the same clutch (parents) on the same diet.

• Definition of the protein and energy requirements of farmed crocodiles in various production

phases. Manufacturing costs and capacity At present, manufactured diets for crocodiles are in the form of semi-moist pellets produced using cold extrusion. While this form of production may have application on-farm, it is a comparatively inefficient means of production from a distance unless the pellets can be made shelf-stable and durable. This is because semi-moist pellets produced using cold extrusion often require freezing until use, have a high water activity, have diminished quality following bagging (fines, distortion in pellet shape), and at least 25% of the transportable weight is water. Strategies 1. Examine the potential for alternative forms of manufactured feed production including the

production of a shelf-stable, semi-moist steam extruded pellet. 2. Develop concentrates that can be used for the convenient on-farm production of semi-moist cold-

extruded pellets as required. 3. Enhance manufacturing capacity to facilitate the production of a wider range of pellet sizes and

pellet forms (eg. floating). Key performance indicators • Commercial availability of shelf-stable, semi-moist steam extruded manufactured pellets for

crocodiles. • Premixes, definition and provision of production equipment and documented procedures for the

on-farm production of manufactured pellets for all production phases of farmed crocodiles. Storage costs Provision of semi-moist pellets and the inclusion of fresh product in these diets necessitates cold storage of the diets. This can add a significant cost to the provision of feed to crocodiles, and represents a risk to the continuity of nutrient supply in the event the refrigeration fails. Eliminating the need for fresh product in manufactured diets and the development of procedures for the utilization of low moisture diets is a potential solution to this problem. Strategies 1. Assess the potential for the use of steam extruded pellets with low moisture content (<10%) in

farmed crocodile production.

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Key performance indicators • Reduction in the transport, storage and handling of manufactured diets used in farmed crocodile

production. • Limited reliance on fresh product for the growth of farmed crocodiles fed manufactured diets. • Minimal moisture content diets for farmed crocodile production. Feed conversion efficiency Feed intake Feed intake is fundamental to all growth and production. Many diet related, environmental and animal factors can influence feed intake and need to be addressed if the nutrition of farmed crocodiles is to be optimized. Strategies 1. Conduct experiments to define those factors influencing the rate of intake and preference of

manufactured diets including the role of diet form and attractants. Attractants worthy of consideration may include chicken tallow, ensiled fish products and those that contain a high proportion of keratin. Some of these are proving to be useful in other forms of aquaculture production.

2. Conduct experiments to develop weaning procedures for hatchling crocodiles and to increase

the rate of acceptance of manufactured diets. 3. Develop feeding strategies to promote the initiation of feeding and the intensity of feeding by

farmed crocodiles at all growth stages when offered manufactured diets. Key performance indicators • Definition of those diet factors that influence the intake of manufactured diets by farmed

crocodiles. • Capacity for rapid weaning of farmed crocodiles onto manufactured diets. • Optimal feeding strategies for manufactured diets. Feed utilisation The efficiency of use of nutrients by farmed crocodiles will depend on the production phase, the feeding strategy, the raw materials used in the diet, feed wastage, and environmental factors. Improvements in the capacity of crocodiles to utilize nutrients from manufactured diets is the single most effective way of improving feed conversion efficiency. Strategies 1. Conduct growth experiments to establish the optimum feeding regime or strategies for crocodiles

offered manufactured diets. Growth experiments should examine differences in performance between feeding every day versus every two and three days.

2. Examine the potential for use of exogenous enzymes and other feed additives to improve feed

utilization and energy yield from non-protein sources.

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3. Develop feed delivery systems that will reduce feed wastage and improve feed utilization. 4. Conduct experiments to define the transit time of diets in crocodiles fed manufactured feeds vs

fresh meat as a means of defining feeding strategies and improving the performance of crocodiles fed manufactured feeds.

Key performance indicators • Improved nutrient utilization from manufactured diets fed to crocodiles so that performance and

product quality exceeds that currently achieved with fresh meat. • Feed delivery systems that minimize feed waste and reduce labour costs. • Identification of feed additives that enhance nutrient utilization by farmed crocodiles. Other considerations Benchmarking and summary data While a research program can be defined based on the primary drivers of profitability, it is important to ensure that the outcomes of the research are realized in a commercial sense. To monitor this, a benchmarking program documenting on-farm performance of crocodiles fed manufactured feeds is recommended. Strategies 1. Compile all information generated to date on the nutrition of farmed crocodiles and the production

of manufactured feeds in an easy to update and reference format. 2. Develop a benchmarking program that monitors the commercial performance of crocodiles fed

manufactured feeds. Key performance indicators • A crocodile nutrition manual that provides an up to date summary of research and outcomes from

crocodile nutrition research and provides a mechanism for primary gaps in the knowledge base. • Published benchmarking data derived from commercial farms that have assessed the performance

of crocodiles fed manufactured feeds. Priorities Based on an industry workshop held in Townsville on August 11, 2003 focussing on the nutrition of farmed crocodiles, the above strategic directions were ratified and the following priorities for research became evident: Priority 1: Feed Intake: Develop feeding strategies to promote the initiation of feeding and the intensity of feeding by farmed crocodiles at all growth stages when offered manufactured diets. Experiment 1: Use of feed attractants to stimulate feeding responses in farmed crocodiles fed manufactured diets. Experiment 2: Transit time of manufactured and fresh meat diets fed to farmed crocodiles.

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Experiment 3: Influence of feeding frequency on the feeding response and feed utilisation of farmed crocodiles fed manufactured feeds. Priority 2: Diet specifications: Define the role of calcium and phosphorus in crocodile diets in relation to a) skeletal and scutal development, and b) influence on acid-binding and expression of endogenous enzymes and osmotic potentials in the digestive tract. Experiment 1: Response of growing crocodiles to dietary calcium and phosphorus content. Experiment 2: Use of organic acidifiers to improve the performance of crocodiles fed manufactured diets. Priority 3: Manufacturing costs and capacity: Examine the potential for alternative forms of manufactured feed production including the production of a shelf-stable, semi-moist steam extruded pellet. Experiment 1: Use of mycotoxin binders in manufactured feeds to control fusaritoxins of both feed and environmental origin. Experiment 2: Influence of feed preservatives (eg. potassium sorbate, propylene glycol, phosphoric acid) on the digestive efficiency of farmed crocodiles fed manufactured feeds. Experiment 3: Potential for semi-moist steam extruded pellets as the primary source of nutrients for farmed crocodiles. Priority 4: Manufacturing costs and capacity: Enhance manufacturing capacity to facilitate the production of a wider range of pellet sizes and pellet forms (eg. floating). Experiment 1: Cold-extrusion of sinking and floating manufactured feeds of various sizes for farmed crocodiles. Priority 5: Ingredient supply: Characterise the digestible amino acid and energy content of a range of feed ingredients for crocodiles in different production phases. Experiment 1: Digestibility of nutrients in feed ingredients with potential for use in manufactured feeds for farmed crocodiles. Priority 6: Benchmarking and summary data: Compile all information generated to date on the nutrition of farmed crocodiles and the production of manufactured feeds in an easy to update and reference format. Experiment 1: Compilation and publication of a crocodile nutrition manual. Priority 7: Benchmarking and summary data: Develop a benchmarking program that monitors the commercial performance of crocodiles fed manufactured feeds. Experiment 1: Initiate a benchmarking program for the use of manufactured feeds in commercial farming operations.

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Appendix B. Development of manufactured feeds for Crocodylus porosus

Robert J. van Barneveld1, Steven Peucker2, Bernie Davis2 and Robert Mayer2

1Barneveld Nutrition Pty Ltd, 19-27 Coonan Road, South Maclean, QLD 4280, Australia

([email protected]); 2 Department of Primary Industries and Fisheries, PO Box 1085, Townsville, QLD 4810,

Australia ([email protected]) ABSTRACT: Potential exists to meet the nutrient requirements for the growth and production of Crocodylus porosus using manufactured semi-moist feeds, thus presenting an opportunity to reduce overall production costs and improve the efficiency of feeding. Research completed to date has demonstrated that juvenile crocodiles can achieve growth rates approaching 16 g/d with feed conversion ratios of 3:1 when fed semi-moist pellets (420 g/kg crude protein; 50 g/kg crude fat; 630 g/kg dry matter) at a rate of 3% of liveweight per day. Manufactured diets do not require the inclusion of fresh meat co-products (kangaroo mince, beef mince, chicken heads etc) to achieve adequate intakes, and attractants appear to have little influence on feeding intensity or total feed intake. A staged introduction of manufactured feeds is required to ensure newly hatched crocodiles are successfully weaned onto manufactured feeds as their primary source of nutrients, but once weaned, no difference in growth or feeding efficiency is observed between feeding every 3, 4 or 5 days. Further research will investigate improved weaning procedures, ingredient characterisation, feeding delivery strategies, feed production methods, and nutrient requirements with a view to reducing the proportion of production costs attributable to feed. INTRODUCTION: Commercial crocodile production in Australia relies primarily on combinations of fresh meat for the supply of nutrients to all production phases. Not only is fresh meat unlikely to represent an optimal supply of nutrients for efficient crocodile growth, but supply, storage and handling can be difficult and costly. In contrast, the composition of manufactured feeds can be manipulated to match diet specifications to the nutrient requirements of the crocodile for a particular production phase, manufactured feeds can be formulated to reduce the content of fresh meat thus improving shelf life and reducing the need for refrigerated storage, and they are generally easy to handle. In addition, the nutrient density of manufactured feeds is often greater than fresh meat and hence the cost per nutrient supplied in every kilogram of manufactured feed is usually less than via fresh meat. When defining the most appropriate approach for the development of manufactured feeds for crocodiles, it is important to maintain a focus on the primary nutritional drivers of profitability in any intensive animal production system (Figure B.1). By maintaining this focus, it becomes clear that the primary nutritional challenges for Crocodylus porosus, where development of manufactured feeds is still in its infancy, include: • Identification of the most suitable diet form taking in to account acceptance by crocodiles in

various production phases, feeding habits, digestive anatomy and physiology, and the shelf-life and handling characteristics of the feed and the most efficient means of manufacturing the feed;

• Selection of the most appropriate ingredients for inclusion in manufactured feeds; • Matching diet specifications to the nutrient requirements of crocodiles in various phases of

production; • Initiation of feeding and the weaning of juvenile crocodiles onto manufactured diets as their

primary food source; • Identification that influence feed intake of manufactured feeds by crocodiles and the most

appropriate feeding strategies.

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Profit = (Revenue – Costs of production) x volume Feed costs Non-feed costs Feed cost ($/kg) Feed conversion efficiency Ingredient supply Feed intake Diet specifications Variation in feed intake and FCE Manufacturing costs Feed utilization Transport costs Storage costs Figure B.1 Primary nutritional drivers of profitability in intensive animal production systems. The aim of this paper is to describe the attributes of manufactured feeds and feeding strategies developed for Crocodylus porosus and results of an extensive research program that underpins this development. Overall, we are seeking to develop manufactured feeds that are: • Acceptable to crocodiles; • Cost-effective; • Capable of promoting efficient production; • Optimal in terms of final product quality; • Easy to manufacture, store and handle. DIET FORM: Based on research undertaken previously in Zimbabwe and local experience, a semi-moist pellet (ie 25% moisture) was deemed to be the most appropriate initial diet form for manufactured crocodile feeds in Australia. Jansen-Van Vuuren (1993) reported that dry diets were poorly accepted by crocodiles, but acceptance was improved when moist pasta-like products were offered. As more experience is gained with feeding manufactured feeds to crocodiles, it is possible that the need for a semi-moist pellet will be reduced and that dry pellets could have application with specific production phases. This was certainly the experience of the salmon industry worldwide that now relies totally on low moisture, high protein, high fat extruded feeds, but started as an industry feeding fresh bait fish and semi-moist feeds (Figure B.2). As well as acceptance, diet form must be matched to the digestive anatomy and physiology and the feeding habits of the target animal. Crocodiles are generally opportunistic feeders and can go for long periods without food with frequency of feeding influenced by body size and environmental temperatures. In addition, crocodiles do not extensively chew their food, have a highly acidic and mechanically active stomach environment and comparatively short intestines and colon. As a consequence, the diet form needs to be such that it can be maintained in the stomach environment for a period of time ensuring a constant flow of nutrients over this period to the intestine, but as there is limited chewing, it cannot be so well bound that it will not break down in the stomach.

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Figure B.2 Schematic representation of chronological development of manufactured feeds in the salmon industry The current semi-moist pellet used for crocodiles in Australia is produced with wheat gluten as the binder. A significant amount of trial and error has resulted in definition of the optimum balance between wheat gluten, oil and water so that pellet binding is optimal. Pellet sizes vary depending on the age of crocodiles being fed, ranging from a mince during the weaning period to pellets exceeding 5 cm in diameter for adult breeding crocodiles. The semi-moist pellets produced are generally sinking, but potential exists to produce floating pellets, but this may be at the expense of water stability in the first instance. Semi moist pellets are produced using a modified mixer/mincer with a range of tube die attachments. A gearbox allows the auger speed to be varied, so that the amount of mechanical energy imparted on the feed can be controlled. Ideally, if the temperature of the mix can reach approximately 52°C through friction, the semi-moist pellet binding will be optimal. INGREDIENT SELECTION: Ingredients used in manufactured diets for crocodiles must match the capacity of the crocodile to digest these components as well as being acceptable. While diets consumed by crocodiles in the wild are some guide to potential ingredients in manufactured feed, this is generally more likely to reflect the feed that is available rather than the most appropriate ingredients for use in the supply of nutrients in an intensive production system. Crocodiles have a high capacity to digest protein from both animal and vegetable sources (Coulson and Hernandez, 1983; Staton and Vernon, 1991; Manolis, 1993), and fats (Manolis, 1993). It also appears that crocodiles have some capacity to utilise carbohydrates as a source of dietary energy (Staton et al. 1990b). As a consequence, a wide range of feed ingredients can be utilised in manufactured crocodile feeds, resulting in significant flexibility in formulations, and potential to closely match diet specifications to the nutrient requirements of the crocodiles. While we have a basic understanding of the types of food constituents that can be digested by crocodiles, further refinement of manufactured feeds will be achieved through a better understanding of the enzymic and microbiological profiles in the gut, and how manufactured feeds can influence these factors. For example, while some enzymes have been identified (including pepsin, intestinal protease, trypsin, chymotrypsin, carboxypeptidase, aminopeptidase and α-amylase) their activity and relative proportions have not been identified. Digestive enzymes, whether of microbial or endogenous origin are the key to understanding digestive processes in the intestinal tract. However, not only is the total digestive profile important, it is also essential to understand the regional localisation of specific digestive enzymes so that the structure of feed pellets can be matched with digestive capabilities. In order to maximise the digestibility and absorption of nutrients, we need to understand which digestive enzymes have the greatest activities, where they are located and whether they change in level with changes in feed.

Salmon Aquaculture 1971

2001

Bait

Moist

Pellets/ Extruded

FCR2.5-3.5

0.9-1.2

Crocodile feeds

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In addition to enzyme activities and profiles, microbial populations also play an important role in the efficiency of digestion and manufactured feeds composition and form. Microorganisms play an important role in digestive processes in most terrestrial species, and there is good evidence that microorganisms also exist in the digestive tract of aquatic species and reptiles. However, it is not clear what role these bacteria play. They could play a passive role in restricting the establishment of significant numbers of pathogenic bacteria in the gut, or a more active role by promoting or enhancing digestion of the normal diet. When crocodiles are fed a manufactured diet containing components not normally found in the wild, the action of microbial populations may be essential in achieving optimal digestion and growth response. Integration of the microbial and enzyme components of the farmed crocodilian gut could provide useful information about the capability of crocodiles to digest manufactured feeds, and help in the structural and compositional design of a feed pellet. Manufactured diets for crocodiles that have been used to produce acceptable growth rates in crocodiles consist primarily of: Proteins including wheat gluten, meat and bone meal, feather meal, blood meal, poultry meal and fish meal;

• Water; • Oils or tallows; • Vitamins and minerals; • Preservatives and anti-microbials such as potassium sorbate, phosphoric acid and propylene

glycol to manipulate water activity and confer increased shelf life. • Anti-oxidants and mycotoxin binders.

While fresh meat is used as a basal diet in many crocodile production systems, it is not necessary to include it in manufactured diets to achieve acceptable intakes. Fresh meat is not currently used as a ingredient in manufactured diets in Australia and this does not effect the efficacy of the resulting diet. In addition, it appears that crocodiles do not have distinct preferences for particular ingredients in manufactured diets. Research undertaken by the Queensland Department of Primary Industries and Fisheries in Townsville compared the influence of fresh meat content (0 or 50%), fat level (10 or 15%), fat type (chicken tallow or canola oil) and an attractant (0 or added chicken flavour). All combinations of these factors were tested (Table B.1) through a total of 16 different diets, using paired preference tests to reveal no significant preference for any combination in crocodiles of different sizes. One characteristic of crocodile nutrition in the wild is the fact that they are known to consume a significant proportion of indigestible material and to retain proportions of this in the gut as gastroliths. This is similar to the consumption of shell grit by poultry to enhance the action of the gizzard. Previous research undertaken in Australia had demonstrated that diets with increasing levels of kaolin (a fine clay used in the production of porcelain) as an indigestible filler and decreasing levels of fat promoted superior performance in growing crocodiles (Figure B.3). It was hypothesized that the kaolin may be acting as a gastrolith and could be an important addition to manufactured feeds. Subsequent research however, demonstrated that addition of dietary kaolin has no influence on crocodile performance, nor do other clays such as bentonite (Figure B.4). DIET SPECIFICATIONS: There is limited information available on the nutrient requirements of crocodiles. Studies comparing the performance of crocodiles fed different forms of fresh meat clearly demonstrate that farmed crocodiles can respond to differences in nutrient intake, but they have done little to define the actual requirements of specific nutrients. Garnett (1985) made some progress in the assessment of fatty acid requirements showing that saturated fatty acids were apparently digested less efficiently by C. porosus than longer chain, unsaturated fatty acids and that C20:5 (eicosapentaenoic acid, EPA) and C22:6 (docosahexaenoic acid, DHA) are essential in diets for these crocodiles. Staton et al. (1990a) suggested that a dietary source of arachidonic acid may also be required for maximum growth of alligators (Alligator mississipiensis). Staton et al (1990b) went further and estimated optimum digestible energy:dietary protein ratios for young alligators to be

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1.96-2.60 KJ/g protein which was similar to the requirements of other aquatic ecotherms of equal size. Table B.1 Diet combinations used to test preference for fresh meat, fat level, fat type and attractant in manufactured semi-moist pellets.

Diet Fat content (%) Fresh meat (%)

Fat type Attractant

1 10 0 Chicken tallow No 2 15 0 Chicken tallow No 3 10 25 Chicken tallow No 4 15 25 Chicken tallow No 5 10 0 Canola oil No 6 15 0 Canola oil No 7 10 25 Canola oil No 8 15 25 Canola oil No 9 10 0 Chicken tallow Chicken flavour 10 15 0 Chicken tallow Chicken flavour 11 10 25 Chicken tallow Chicken flavour 12 15 25 Chicken tallow Chicken flavour 13 10 0 Canola oil Chicken flavour 14 15 0 Canola oil Chicken flavour 15 10 25 Canola oil Chicken flavour 16 15 25 Canola oil Chicken flavour Despite some basic information on the nutrient requirements of crocodiles, there are a large number of factors that influence our capacity to accurate define diet specifications. First and foremost is the fact that individual crocodiles farmed in groups have a large variation in daily intake and crocodiles also have a high capacity to vary their metabolic rate. In addition, it has been shown that environmental temperature can have a significant influence of the rate of intake and the feed conversion efficiency. As a consequence, definition of generic nutrient requirement information for crocodiles will be difficult and the need for highly specified diets is diminished. Instead, diet specifications based on broad parameters such as crude protein, crude fat and digestible energy are likely to be sufficient. Figure B.3 Response of growing crocodiles to increasing levels of dietary fat and dietary kaolin.

800900

100011001200130014001500160017001800

5 10 15

Dietary fat (%)

Tota

l gai

n (g

)

Small/medium Medium/large

2.52.72.93.13.33.53.73.94.14.3

5 10 15

Dietary fat (%)

Feed

con

vers

ion

ratio

Small/medium Medium/large

c

a

c

a

d

b

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Figure B.4. Influence of dietary kaolin on the growth and feed conversion efficiency of growing crocodiles. The composition of the first manufactured diets produced in Australia were based on available literature (eg. Staton and Vernon, 1991) and local experience, and resembled the following: Dry matter content: 60% Crude protein content (as fed) 35-40% Crude fat content (as fed) 16% Fresh product: 20-50% Binders: Nil Cost: $1.00-$1.50/kg. Subsequent research, such as that examining dietary fat content (Figure B.3), and growth studies has resulted in a significant revision of these specifications for growing crocodiles, with diet composition now reflecting the following: Dry matter content: 63% Crude protein content (as fed) 42% Crude fat content (as fed) 5% Fresh product 0% Binders: 0% Cost $0.70-$0.80/kg. Other additives: Shell grit Mycotoxin binders Acidifiers Vitamin D and Fe supplements FEEDING INITIATION: Manolis (1993) suggested that one of the most important steps in the development of manufactured feeds involved ensuring rapid initiation of feeding. Unless feeding initiation is completed rapidly, long-term performance of the crocodiles can be compromised. Manolis et al. (1989) also demonstrated that within the first month of life, C. porosus hatchlings showed clutch-specific preferences for certain foods, and in general, there was an avoidance of “smelly” foods. The primary drivers of initial preference are poorly understood, but it appears that weaning management rather than particular feeds has the greatest impact on the rate of acceptance and feeding initiation. Research undertaken by the Queensland Department of Primary Industries and Fisheries has assessed the use of a wide range of attractants for juvenile crocodiles. Some of the attractants assessed as part of a structured initiation trial include:

1300

1350

1400

1450

1500

1550

1600

1650

+ Kaolin + Water + Bentonite

Diet

Wei

ght g

ain

(g)

Small/Medium Medium/Large

0.00.51.01.52.02.53.03.54.04.5

+ Kaolin + Water + Bentonite

Diet

FCR

Small/Medium Medium/Large

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• Fresh meat; • Chicken head digest; • Fresh blood; • Liver digest; • A range of proprietary digests and attractants; • A variety of oil and meat combinations. These experiments revealed a high level of variability in response, and all demonstrated minimal “attractive properties”. Responses to these attractants was so poor that the experiment had to be prematurely terminated. In the absence of attractants, it appears that the best approach to ensuring a rapid initiation of feeding on manufactured feeds is via “staged weaning”. This process involved the gradual replacement of fresh meat with manufactured pellets. Staged weaning processes currently under investigation are aimed at reducing the time required to progress from fresh meat to 100% semi-moist pellets and resemble the following over a 6 week period:

90% meat : 10% pellets 7 days 80% meat : 20% pellets 7 days 70% meat : 30% pellets 7 days 60% meat : 40% pellets 7 days 50% meat : 50% pellets 7 days

100% manufactured pellets FEEDING FREQUENCY: Crocodiles are opportunistic feeders and as a consequence, their digestive systems and metabolism may be unsuited to frequent feeding. This is further supported by general observations of daily feed intake of farmed crocodiles (Figure B.5). It can be seen that feed intake drops after each large feed suggesting that daily feeding may not be required. Figure B.5 Variation in daily intake of manufactured feeds by growing C. porosus. Initial research has shown that there is significant improvement in feed conversion efficiency of crocodiles are not fed daily. Subsequent research using fifteen individually caged animals balanced across five clutches undertaken by the Queensland Department of Primary Industries and Fisheries examined feeding frequencies of 3, 4 or 5 days. It was demonstrated that feeding every five days had no negative influence on final body weight, and that there were numeric improvements in feed

0

100

200

300

400

500

600

700

800

Days Fed

Gra

ms

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conversion efficiency (Figure B.6). Further research is required to examine the effects of infrequent feeding on carcase quality. Figure B.6 Influence of feeding frequency of growth and feed conversion of C. porosus. OVERALL FEEDING MANAGEMENT: While significant progress has been made in the development of manufactured feeds for crocodiles in Australia, it is important to remember that the diet itself is only half of the equation. Using the “salmon model”, successful feeding of manufactured diets to crocodiles is going to depend equally on the feeding management. It is unlikely that a suitable manufactured diet will be developed that promotes acceptable levels of intake, growth and feed conversion in the absence of appropriate management. As diet development progresses, additional factors that need to be considered include the impact of manufactured diet use in crocodile health and overall production hygiene, the capacity of manufactured feeds to manipulate body composition and overall product quality, and the role of different diets for different crocodile production phases. CONCLUSIONS: In summary, crocodile nutrition research conducted to date has resulted in the development of manufactured diets with no fresh product needed that will support growth of crocodiles at an acceptable rate. A research program is now needed to capitalize on these initial investigation with a view to reducing the nutritional costs of producing crocodiles. REFERENCES: Coulson, RA & Hernandez, T 1983, ‘Alligator Metabolism Studies on Chemical Reactions in vivo’,

Pergamon Press: London. Garnett, S 1985, ‘Fatty acid nutrition of the estuarine crocodile Crocodylus porosus’, Comparative

Biochemistry and Physiology. 81B: 1033-1035. Jansen-van Vuuren, RA 1993, ‘Progress in the development of artificial crocodile diets in

Zimbabwe’, Journal of the Zimbabwe Society for Animal Production. 5: 87. Manolis, SC, Webb, GJW, Barker, SG & Lippai, C 1989, ‘Nutrition of crocodiles’, in Proceedings

of the Intensive Tropical Animal Production Seminar. Department of Primary Industries, Townsville.

7000

7500

8000

8500

9000

9500

10000

3 4 5

Feeding frequency

Bod

y w

eigh

t (g)

Initial Final

1.60

1.62

1.64

1.66

1.68

1.70

1.72

1.74

1.76

1.78

3 4 5

Feeding frequency

FCR

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Manolis, SC 1993, ‘Crocodile nutrition’, in Proceedings of the Second Regional East-Asia and Oceania Crocodile Conference.

Staton, MA, Edwards, HM, Brisbin, IL, Joanen, T and McNease, L 1990a, ‘Essential fatty acid

nutrition of the Amercian alligator (Alligator mississippiensis)’, Journal of Nutrition 120: 674-685.

Staton, MA, Edwards, HM, Brisbin, IL, Joanen, T & McNease, L 1990b, ‘Protein and energy

relationships in the diet of the American alligator (Alligator mississippiensis)’, Journal of Nutrition 120: 775-785.

Staton, MA & Vernon, BP 1991, ‘Formulated crocodile feeds’, in Proceedings of the Intensive

Tropical Animal Production Seminar. Department of Primary Industries, Townsville. pp 239-248.

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References Coulson, R.A. and Hernandez, T. (1983). Alligator metabolism, studies of chemical reactions in

vivo. Pergamon Press, New York. Garnett, S.T. and Murray. R.M. (1986) Parameters affecting the growth of the estuarine crocodile

Crocodylus porosus in captivity. Aust. Journal of Zoology. 34, 211-23 Jansen-van Vuuren, R.A. (1995). A review and update of the nutrition of Nile Crocodile

(Crocodylus niloticus) from birth toone year of age and the developmental of artificial-type diets for juvenile in Zimbabwe-1998 to 1994.

Kercheval, D.R. and Little, P.L. (1990). Comparative growth rates of young Alligators utilizing

rations of plant and/or animal origin. Volume 1 Proceeding of the 10th Working meeting of the Crocodile Specialist Group of the Species Survival Commission of the IUCN – The World Conservation Union. Gainesville, Florida. USA23rd-27th April 1990

Manolis, S.C., Webb, G.J.W., Barker, S.G. and Lippai, C. (1989), Nutrition of Crocodiles.

Proceedings of the Intensive Tropical Animal Production Seminar. Department of Primary Industries, Townsville.7th-8th August 1991.

Staton, M.A., Brisbin, I.L., and Pesti, G.M. (1986). Feed formulation for alligators: An overview

and initial studies. Proceeding of the 8th Working meeting of the Crocodile Specialist Group of the Species Survival Commission of the IUCN. Quito Ecuador. 13th-18th October 1986

Stanton, M.A. and Venon, B.P. (1991) Formulated Crocodile Feeds. Proceedings of the Intensive

Tropical Animal Production Seminar. Department of Primary Industries Townsville.7th-8th August 1991.

van Barneveld, R.J. (2003). Crocodile Nutrition Research Strategic Directions2002-2005. Webb,G.J.W, Hollis, G.J and Manolis, S.C. (1986). Feeding, Growth and Food Conversion Rates of

wild juvenile Saltwater crocodiles (Crocodylus porosus) Journal of Herpetology. Vol 25, No 4 pp 473-477.

Whitehead, P.J. (1990) Yolk depletion and metabolic rate of hatchling Crocodylus johnstoni. Copeia

1990(3) pp871-875