SID 5 Research Project Final Report -...

SID 5 (Rev. 3/06) of 26

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SID 5 Research Project Final Report

Note

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Project identification

1. Defra Project code AW0234

2. Project title

Detection, causation and potential alleviation of bone damage in laying hens housed in non-cage systems

3. Contractor organisation(s)

Department of Clinical Veterinary Science University of Bristol Langford Bristol BS40 5DU

54. Total Defra project costs £ 428,542

(agreed fixed price)

5. Project: start date ................ 01 September 2004

end date ................. 31 August 2008

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6. It is Defra’s intention to publish this form.

Please confirm your agreement to do so. ................................................................................... YES NO

(a) When preparing SID 5s contractors should bear in mind that Defra intends that they be made public. They should be written in a clear and concise manner and represent a full account of the research project which someone not closely associated with the project can follow.

Defra recognises that in a small minority of cases there may be information, such as intellectual property or commercially confidential data, used in or generated by the research project, which should not be disclosed. In these cases, such information should be detailed in a separate annex (not to be published) so that the SID 5 can be placed in the public domain. Where it is impossible to complete the Final Report without including references to any sensitive or confidential data, the information should be included and section (b) completed. NB: only in exceptional circumstances will Defra expect contractors to give a "No" answer.

In all cases, reasons for withholding information must be fully in line with exemptions under the Environmental Information Regulations or the Freedom of Information Act 2000.

(b) If you have answered NO, please explain why the Final report should not be released into public domain

Executive Summary

7. The executive summary must not exceed 2 sides in total of A4 and should be understandable to the intelligent non-scientist. It should cover the main objectives, methods and findings of the research, together with any other significant events and options for new work.

Recent media focus on issues of animal health and wellbeing, particularly national campaigns and television programmes aimed at the general public, have created a somewhat unfavourable impression of the poultry industry which may not be a fair reflection of the progress made in recent years. One consequence is that consumer choice is increasingly driven by perceived welfare issues. Such concerns have led to cage systems being phased out (by 2012), and although alternative housing confers many welfare benefits, such as freedom of movement, such gains are not without cost. A major threat to bird welfare in such systems is the susceptibility of hens to incurring fractures of the keel during the production cycle and, while the scale of the problem has not previously been systematically studied recent, limited evidence suggests that the situation is getting worse with very high levels of damage affecting up to 80% of birds within a single flock. Such fractures vary in severity and often result in gross skeletal damage and even death in some birds. It is likely that even minor skeletal damage may impact on aspects of bird behaviour and wellbeing. The main aims of the project were to: (1) provide an accurate assessment of the current levels of old breaks in end-of lay hens housed in a variety of system designs and identify the important risk factors. (2) determine when damage occurs during the normal laying cycle and identify critical time and control points. This was achieved using a prospective study of flocks housed in barn and two differing free range systems, enabling close observation of behaviour. (3) investigate the use of a new non-invasive method of monitoring bone breakage, metabolism and repair without the requirement for euthanasia, dissection or handling of live birds. A valid, non-invasive method would provide a useful audit tool. 4) evaluate intervention aimed at alleviating this welfare problem. An existing commercial population of birds, receiving dietary supplementation with omega-3 (n-3) poly unsaturated fatty acids (PUFA) to provide an altered human dietary intake, was examined and compared with birds housed in similar conditions.

A total of 67 flocks housed in eight broad categories of housing type were assessed at the end of the production period: Within each flock the presence of keel fractures was determined in 100 birds using a palpation technique and 20 birds dissected to permit validation of the palpation data and removal of the tibia, humerus and keel bones for measurement of breaking strength. Within the recent EU Directive and as a component of various assurance schemes there is a requirement for a designated, minimum amount of perch space per bird. However, differences in interpretation exist such that in Scotland only raised (aerial) perches are deemed to contribute to the total perch available. This has resulted in significant differences in internal design. In general terms, both the prevalence and severity of keel damage

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increased as the complexity of the environment increased. Of particular note was the increased damage (average of over 80% of birds affected) in flocks housed in systems equipped with multi level perches.

To determine when skeletal damage begins to accumulate, flocks housed in barn and free range systems, including those equipped with aerial perches, were assessed throughout the production period. In all three systems the onset of skeletal damage occurred at about 30 weeks of age and in the case of flocks housed with aerial perches, levels of damage were already high (30%). Thereafter there was a continuing accumulation of keel fractures. Analysis of bird activity identified some of the most important factors in determining the likelihood of fractures occurring (the height of the perches where available and the maximum height which birds could achieve within the house). Examination of the biochemistry and biomechanical properties of bones removed from the birds in this study confirmed that there is no decrease in ultimate breaking strength, indeed the ultimate stress increases with age of the bird. However there was a decrease in ultimate and yield strain and energy to fracture. The consequence is that although bones are not inherently weaker, they do become stiffer and less able to absorb energy, hence may be more likely to sustain fracture at a given load. The bone mineral density also increased with age, which may increase strength yet reduce compliance in line with the mechanical properties.

One reason why limited research has been carried out to identify the time of onset of fractures is because relevant information about fractures has had to be obtained by full dissection of birds or using palpation which still involves considerable disturbance to the birds. An additional technique was tested that would permit the detection of levels of damage in individuals, but without handling or capturing the individual birds. This involved measuring the presence and levels of the collagen cross-link, lysyl-pyridinoline (L-pyr) which is found only in mineralising tissue, principally bone. It’s presence in serum and urine of humans is a reliable indicator of bone turnover, repair and resorption. Faecal samples were collected to examine the relationship between the presence of broken bones and the excretion of lysyl pyridinoline. If proven to be an accurate indicator of bone breakage it was thought that such an assay might be developed as an auditing tool for farm assurance schemes, to ensure that farmers met their statutory obligations, or for use in further research. However age related changes confounded the interpretation and at present it is not considered a viable assessment method. Previous nutritional strategies to prevent osteoporosis in the laying hen have aimed to provide key nutrients important to bone development and maintenance, either by enhancing calcium absorption or retention, or by maximising peak bone mass. However, these interventions have met with limited success, principally because medullary and not structural bone is formed after the onset of lay. As an alternative approach we have examined whether dietary n-3 PUFA supplementation (already in use for production of ‘Columbus’ eggs) can influence the basic biochemical and biomechanical properties of bone and hence the incidence and severity of bone breakage in laying hens. Flocks were examined throughout the laying period and the flocks that were fed the n-3 supplemented diet presented substantially fewer broken keels in comparison to the free range flocks fed a standard ration. This reduction (up to 60% at 50 weeks assessed by dissection) was significant throughout production. Peak bone breaking strength, ultimate and yield stress, and energy to fracture were found to be increased in birds fed the n-3 supplemented diet and importantly, the decrease in energy to fracture observed in control birds with age was not observed, thus making them less liable to breakage. The bone mineral density and the structural bone volume were also increased with supplementation. Biochemical analysis further supported the hypothesis that the n-3 supplementation promoted bone formation (with an increase in bone turnover markers), thus enabling repair of accumulated microdamage and increasing the material strength of the bones, and the resistance to fracture. This strategy for reducing the overall prevalence of keel fractures will be investigated in more detail in a recently initiated BBSRC funded study: Production of welfare friendly eggs – improving bone health and reducing bone breakage in laying hens using omega-3 modified diets.

Project Report to Defra

8. As a guide this report should be no longer than 20 sides of A4. This report is to provide Defra with details of the outputs of the research project for internal purposes; to meet the terms of the contract; and to allow Defra to publish details of the outputs to meet Environmental Information Regulation or Freedom of Information obligations. This short report to Defra does not preclude contractors from also seeking to publish a full, formal scientific report/paper in an appropriate scientific or other journal/publication. Indeed, Defra actively encourages such publications as part of the contract terms. The report to Defra should include:

the scientific objectives as set out in the contract;

the extent to which the objectives set out in the contract have been met;

details of methods used and the results obtained, including statistical analysis (if appropriate);

a discussion of the results and their reliability;

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the main implications of the findings;

possible future work; and

any action resulting from the research (e.g. IP, Knowledge Transfer).

This project was started in November 2004 and had four primary objectives described below. During the second year of the project Defra requested an extension of the existing studies within Objective 01 and Objective 02 to permit assessment of flocks housed in free range systems in Scotland. Specifically, within the EU Directive there is a requirement for a designated, minimum amount of perch space per bird. However, differences in interpretation exist between England & Wales and Scotland such that in Scotland only raised (aerial) perches are deemed to contribute to the total perch available, whereas in England & Wales the use of a slatted floor is accepted as contributing to the perch requirement. Potentially this might mean that flocks housed in non-cage systems in Scotland have access to more included structures than their counterparts in other parts of the UK. To examine this, a further 13 flocks housed with aerial perches in Scotland were examined within Objective 01 and 7 free-range flocks with access to aerial perches were included in Objective 02. This necessarily extended the time course of the project.

Objective 01 To conduct a retrospective epidemiological survey to examine the associations and inter-relationships between breed, management, housing and nutritional factors on the current prevalence and severity of bone damage in end of lay hens housed in a range of alternative systems. To ensure a representative sample of flocks was assessed, the survey included farms from across both England & Wales and Scotland where there is the potential for differences in house design.

Objective 02 To investigate the time course of the accumulation of bone damage within the normal laying cycle in flocks housed in non-cage systems by conducting a prospective replicated study of flocks housed in alternative systems. This included both barn and free range units in England & Wales and free range units in Scotland. Associations between the accumulation of bone damage, bird behaviour, changes in management practice, and bone strength were examined. Objective 03 To develop a novel non-invasive monitoring system measuring levels of lysyl pyrodinoline in the faeces of laying hens and to apply the methodology to investigation of the time course of accumulation of bone damage. Objective 04 Evaluation of potential intervention strategies, particularly a promising dietary approach, compatible with current practices, aimed at reducing the problem of bone damage in laying hens Objective 01 Husbandry systems in current use encompass a wide range of design features and it was necessary to assess considerably more flocks than was originally intended to ensure that all the most common system designs were included in the survey. As a consequence to comprehensively assess the potential impact of house design on the cumulative incidence of fractures to the keel, a total of 67 flocks housed in the following broad categories were assessed at the end of the production period: furnished cage (FC), single tier barn (B), free range (FR), free range equipped with aerial (suspended) perches (FRAS), free range equipped with aerial (A-frame) perches (FRAA), organic static (OS), organic mobile (OM) and organic mobile equipped with fixed aerial perches (OMAF). The primary features of each system are described in the following tables.

Table 01: Summary of system designs - availability of perching space

System No flocks Available perch space

Perches 15cm/bird Aerial Available alternative perches Feed/drink Other*

Free range 12 6/12 - - 5/12 12/12

Free range A frame perches

7 7/7 7/7 7/7 0/12 7/7

Free range suspended perches

6 6/6 6/6 6/6 0/12 6/6

Organic mobile 8 0/8 - - 8/8 8/8

Organic mobile fixed perches

4 4/4 4/4 4/4 4/4 4/4

Organic static 11 9/11 - - 0/11 11/11

Barn 10 0/10 - - 4/12 10/10

Furnished cage 9 9/9 - - 0/9 9/9

(*other includes roof supports, stanchions, water supply pipes, ledges etc)

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The information provided in Tables 01 and 02 illustrates the wide variation in design and physical dimensions of internal structures between system types. There was in addition variation within these broad categories of system types.

Table 02: Summary of maximum heights of internal structures (mean per system, cm)

Upper nest box ledge

Slats above litter

Angle of ramp

Perches above litter

Perches above slats

Drinkers/feeders above litter

*Other above litter

FR 78 103 59 153 69 64 319

FR-AA 54 97 62 180 89 - 334

FR-AS 51 98 48 280 182 - 298

OM 60 70 14 - - 120 165

OM-AF 60 70 14 120 130 120 165

OS 23 88 40 152 65 - 301

B 51 86 47 - - 62 291

FC - - - - 16 - -

The information provided in Table 01 illustrates the wide variation in design of internal structures both within and between system types. Table 02 summarises the gross differences between system types in terms of the actual dimensions of internal structures but does not describe in detail the variation that exists within system types.

Within each flock 100 birds were palpated on the slaughter line following removal from a gas killing unit, for the presence of old breaks and 20 birds dissected to allow close inspection and validation of the palpation data and to permit removal of the tibia, humerus and keel bones for measurement of breaking strength using a three point bending test. Following removal from the bird the severity of damage to the keel was described using a five point photographic scale shown below. (Figure 01)

Figure 01. Severity of keel fractures scored 0 (normal) to 4 (very severe) from left to right

During dissection less than 1% of the birds were found to have incurred an old fracture of the furculum and of these only 8 birds had a broken furculum in the absence of damage to the keel. As a consequence these were removed from the analysis and figures given below for both palpation and dissection data relate solely to broken keels.

Background information describing the environment, production performance and management of each individual flock was collected from the individual farms and from records held by the production company. Information relating to the overall condition and disease status of each flock was obtained from the Meat Hygiene Service rejection data recorded at the slaughter plant. This data identified the number of birds deemed to be dead on arrival and the number of birds rejected due to trauma, contamination, disease, carcass damage, tumours etc. This post mortem data might provide an indication of the health status of the flock.

Results

The data were analysed using one way analysis of variance using Tukey post hoc tests to test for differences between pairs of system types and grouping into homogeneous subsets. The relationships between fracture rates, bone strength, individual components of system design, and a range of production variables were investigated using a generalized linear modeling approach.

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There was wide variation in the incidence of keel damage both between and within system types so that the overall range observed for flocks housed in non cage systems was 28 to 89% assessed using palpation and 30 to 95% when assessed by dissection. The corresponding ranges for flocks housed in furnished cages were 22 to 44% and 15 to 55% respectively. Nevertheless using palpation to identify the presence of old breaks, significant differences between system types were evident (P<0.001). Flocks from the free range system fitted with aerial perches had a significantly greater prevalence of keel damage than flocks from non perch systems (P<0.001) and the other two designs which incorporated different perch types (P<0.05).

Table 03: Prevalence of broken keels in flocks of hens housed in different systems (mean ± se)

n O/B palpation O/B dissection Average severity of

breaks

Free range 12 62 ± 3.1 67 ± 4.3 1.91 ± 0.07

Free range A-frame 7 68 ± 1.8 78 ± 3.4 2.15 ± 0.14

Free range suspended 6 87 ± 1.5 86 ± 1.5 2.59 ± 0.14

Organic mobile 8 39 ± 3.5 45 ± 3.1 1.61 ± 0.03

Organic mobile fixed 4 67 ± 6.7 84 ± 5.2 2.26 ± 0.02

Organic static 11 51 ± 4.5 59 ± 5.1 1.83 ± 0.08

Barn 10 53 ± 2.0 63 ± 2.7 1.80 ± 0.10

Furnished cages 9 32 ± 2.9 36 ± 4.6 1.45 ± 0.09

Similarly where dissection was used to identify old breaks significant differences between systems were also evident (P<0.001) and the ranking of systems was identical. There were in addition large differences between systems in terms of average severity of damage where a fracture has occurred and the systems associated with the highest mean severity score were those equipped with aerial perches (15cm/bird) regardless of design. Of particular note was the large increase in mean prevalence of damage recorded in birds housed in the organic mobile system where aerial perches had been fitted to four of the sheds (P<0.001) and an increase in severity of keel damage (P<0.01). Prior to incorporation of perches, flocks from this system were found to have the lowest mean prevalence of keel damage of all the non cage systems examined and the lowest mean severity score.

The validity of the palpation procedure can be described by reference to the true skeletal condition, confirmed by dissection, using standard measures of reliability (Armitage and Berry 1991). These measures are necessarily influenced by the prevalence of damage in the population as a whole as well as by the severity of the break identified by dissection. Not surprisingly where the break is relatively mild (eg furnished cages and organic mobile systems) identification of such breaks becomes more difficult. Nevertheless the combined results for the 67 flocks assessed indicated that diagnosing old breaks of the keel by palpation was 91% correct and the predictive value of such a diagnosis was 0.95 demonstrating the usefulness of this technique.

Based on the severity scores for each keel assessed using the photographic scale above, the distribution of scores using the applied descriptions shows marked differences between the system types. Presentation of the data using these three categories highlights the impact of all three designs of aerial perches encountered on the occurrence of severe keel damage.

Table 04: Assessment of severity of old breaks (%)

n None Minor Severe

score 0 & 1 score 2 score 3 & 4

Free range 240 57 27 16

Free range A-frame 140 46 29 25

Free range suspended 120 26 29 45

Organic mobile 160 81 13 6

Organic mobile fixed 80 29 37 34

Organic static 220 41 45 14

Barn 200 65 22 14

Furnished cages 180 87 10 3

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The project has identified large differences in design and dimensions of the internal structures commonly found within the laying environment and this necessarily creates considerable overlap between the eight system types we have described in terms of identifying the important factors influencing the prevalence of keel fractures. The following figures describe the relationship, on a flock basis, (excluding flocks housed in furnished cages) between the prevalence of keel fractures in end of lay birds and the overall height of a number of those structures which can be accessed in a single downward flight.

Figure 02a: Relationship between the maximum accessible perch height above slats and the prevalence of broken keels assessed by palpation (r² = 0.4136, P< 0.01)

Figure 02b: Relationship between the maximum accessible perch height above litter and the prevalence of broken keels assessed by palpation (r² = 0.5966, P< 0.001)

There was a significant association between perch height above both the slats and the litter and the prevalence of old breaks of the keel at the end of lay. Similarly both variables were also significantly correlated with average severity score for keel damage (r²=0.399, P<0.01 and r²=0.302, P<0.01 for perch height above slats and litter respectively). Interestingly, the relationship between maximum height available and the prevalence of keel damage was less obvious (Figure 3a and 3b). In a single model live weight was also found to be a significant factor such that increasing live weight was associated with a reduced prevalence of broken keels. The complexity of design of the internal environment means that simple relationships such as those above relating to the height of structures could be influenced by many contributory factors. These may be responsible for large variation

Figure 03a: Relationship between the maximum accessible height above slats and the prevalence of broken keels assessed by palpation (r² = 0.1129, P<0.05)

Figure 03b: Relationship between the maximum accessible height above litter and the prevalence of broken keels assessed by palpation (r² = 0.1715, P<0.01)

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Bone strength was measured as peak breaking force (kg) using a Stevens Compression tester as previously described and results are summarised below.

Table 05: Peak bone breaking strength (Kg)

System Tibia Humerus Keel A Keel B

mean se mean se mean se mean se

FR 22.9 0.30 18.7 0.39 31.4 1.06 13.9 0.37

FR-AF 20.8 0.34 19.1 0.23 25.1 0.74 10.9 0.39

FR-AS 23.6 0.73 21.9 0.55 27.6 0.88 11.5 0.66

OM 28.8 0.66 21.3 0.27 33.2 0.93 13.3 0.70

OM-A 24.3 0.85 23.4 0.89 29.4 1.52 12.7 0.48

OS 24.9 0.75 19.5 0.35 31.0 0.64 12.2 0.40

B 21.9 0.38 18.6 0.46 30.6 0.75 13.7 0.46

FC 19.1 0.61 14.3 0.51 23.6 0.93 9.7 0.48

As one might anticipate, housing system had a significant effect on breaking strength of all three bones measured (P<0.001). The system associated with the lowest bone breaking strength was the furnished cage, particularly for the humerus. Conversely birds from all the organic systems showed the highest breaking strength for tibia. It is not clear whether this is a dietary effect or possibly a consequence of a reduced stocking density which may encourage more overall activity. Given the importance of egg production efficiency to the successful operation of any design of housing, production records were obtained from all flocks assessed at the end of lay and key measures highlighted. The production industry uses a number of different measures including HHA (hen housed average, Table 06), and %production (Table 07). The former describes cumulative efficiency while the latter describes output at any given point within the production cycle and compensates for bird losses.

Table 06: Egg production as HHA (hen housed average

20 weeks 30 weeks 70 weeks

mean se mean se mean se

FR 2.75 0.52 63.17 0.97 265.58 3.87

FR-AA 2.29 0.64 60.57 2.49 243.87 3.33

FR-AS 2.00 0.45 62.83 1.38 295.83 5.56

OM 4.50 0.63 58.38 1.51 262.38 6.28

OM-AF 2.75 0.63 59.00 3.81 276.25 5.66

OS 2.60 0.67 58.40 2.11 244.90 2.23

B 2.00 0.54 60.00 1.76 263.30 8.87

FC 3.71 1.21 68.00 2.30 294.29 5.08

The data suggested differences between systems, with hen house average at 70 weeks of age being significantly increased in flocks housed in furnished cages and free range fitted with aerial perches type systems compared to flocks housed in organic mobile with aerial perches (P< 0.05) and all other system types (P<0.001). Nevertheless there was no significant correlation between HHA at either 50 or 70 weeks of age and the prevalence of broken keels analysed across system types.

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Table 07: Egg production as % Production

20 weeks 30 weeks 70 weeks

mean se mean se mean se

FR 32.98 5.98 89.46 1.07 63.12 2.62

FR-AA 28.97 8.93 82.66 1.53 69.47 3.88

FR-AS 25.87 5.26 91.15 0.73 74.50 2.65

OM 45.39 4.64 86.68 1.59 66.40 2.69

OM-AF 37.68 8.17 90.05 3.56 80.48 2.97

OS 31.41 7.17 82.23 2.09 63.80 2.77

B 25.81 6.49 86.47 2.03 58.37 3.89

FC 41.07 11.84 94.49 0.88 78.37 0.87

There were large, significant differences in production between systems at 20 weeks of age which reflect differences in the onset of lay but smaller though significant differences at 30 weeks of age around the time of peak lay, when demand for egg shell mineral might be at a peak. There was no significant correlation between production at either 50 or 70 weeks of age and the prevalence of broken keels or severity of keel damage across system types. Ongoing mortality levels necessarily affect measures of flock productivity (HHA) and might also give an indication of overall flock wellbeing. The two flocks which exhibited high figures for hen housed average, as well as those housed in free range fitted with ‘a-frame’ type perches, showed significantly lower levels of cumulative mortality at 70 weeks of age compared with flocks housed in all other systems.

Figure 04. Cumulative mortality during production (means per system)

During the early stages of production (up to 30 weeks) mortality in the three organic systems was significantly higher than for all the other system types and the high levels persisted in flocks housed in the two organic mobile systems. By 70 weeks of age there was large variation in total mortality levels between the system types and although one might anticipate low mortality in flocks housed in furnished cages, it is interesting to note that flocks from the two free range systems equipped with aerial perches, also showed low levels of mortality despite high levels of observed severe keel damage. Overall there was no significant correlation between cumulative mortality at all ages and the prevalence of broken keels. It was not within the scope of this study to determine the causes of mortality throughout production but carcase rejection data collected by the Meat hygiene Service during processing was collated since it might provide an indication of overall flock health at the end of lay.

0.0

2.0

4.0

6.0

8.0

10.0

12.0

14.0

16.0

18.0

20.0

20 30 40 50 60 70 80

FR

FR-AA

FR-AS

OM

OM-AF

OS

B

FC%

Age (weeks)

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Table 08: Carcase rejection data collected during processing (%)

System Live Weight (kg) Diseased ¹ DOA Total Rejects ²

Mean se Mean se Mean se Mean se

FR 1.70 0.04 2.75 0.37 0.33 0.12 3.56 0.58

FR-AA 1.80 0.02 1.80 0.18 0.18 0.05 2.18 0.20

FR-AS 1.84 0.04 1.73 0.37 0.10 0.12 2.26 0.58

OM 1.95 0.02 4.63 0.75 0.10 0.03 4.74 0.77

OM-AF 1.87 0.01 1.82 0.32 0.41 0.12 2.44 0.39

OS 1.82 0.04 3.36 0.45 0.12 0.02 3.67 0.51

B 1.64 0.06 2.55 0.25 0.14 0.04 2.99 0.30

FC 1.96 0.04 1.78 0.35 0.19 0.12 2.08 0.49

Includes: ¹ tumours, septicaemia, abnormal smell, emaciation, acites, peritonititis and salpingititis. Discussion of Objective 01

There was wide variation in design of internal structures both within and between system types such that the original five categories of system described in the proposal were inadequate to enable a comprehensive survey to be undertaken. As a consequence additional flocks representing broad system types were included in Objective 01. The overall prevalence of keel fractures, identified by dissection, in flocks housed in non-cage systems was found to range from 30 to 95%. System type was found to have a major effect on the prevalence of keel fractures in flocks assessed at the end of lay and in general terms both the prevalence and the severity of damage increased as the apparent complexity of the system increased. As an example the design of the free range system (below left) is relatively benign whereas that below right appears more complex and potentially more likely to result in bird damage. In particular where systems have been equipped with aerial perches which conform to the SEERAD interpretation of the EC requirement (and an increasing number of farm welfare assurance schemes) for provision of sufficient perch space per bird, the minimum levels of damage observed were greatly increased. In flocks from free range houses fitted with rows of suspended perches above the slatted area illustrated on the right, the average prevalence of broken keels was consistently high (on average 85% when assessed by palpation) and the average severity score was 2.59. Conversely in a single flock housed in a system similar to the one shown on the left the prevalence of broken keels at the end of lay was found to be 48% and the average severity 1.80.

However, where such perches are not provided birds will seek to access other structures within the house to satisfy their strong motivation to perch. This appears to be particularly important during the dark period and assessment of video recordings of flock behaviour and activity within Objective 02 of the project identified a substantial increase in flights to and from all available ‘perch structures’ during dimming of the lights and a corresponding increase in activity as lighting levels are increased at the start of the day. Nevertheless the relationship between maximum height available and the prevalence and severity of keel damage incurred was less pronounced than that observed for dedicated perch heights. This may reflect the large difference in usage of the two facilities since relatively few birds access other structures compared with perch use. Housing system had a significant effect on bone breaking strength and as anticipated flocks housed in the furnished cage had the lowest peak strength, particularly for the humerus, presumably as a consequence of

SID 5 (Rev. 3/06) of 26

reduced flapping and general activity. Conversely birds from all the organic systems tended towards greater peak breaking strengths for all the bones measured. It is not clear whether this is a dietary effect or possibly a consequence of a reduced stocking density which may encourage more overall activity. In the case of the organic mobile houses fitted with aerial perches this improved bone strength was not sufficient to prevent the high levels of keel damage observed. Conversely the relatively weak bones in birds housed in the furnished cages did not result in a high level of damage presumably because of lack of opportunity for injury. Historically there has thought to be an association between production (and demand for egg shell mineral) and bone quality which might render high production flocks susceptible to bone fragility. However although there were large differences between systems in terms of egg production these were not related to rates of fracture.

Objective 02

The results obtained from Objective 01 provided a comprehensive and accurate estimate of the overall prevalence of keel fractures in end of lay hens housed in a range of systems and permitted investigation of the associations between housing type and design, and the occurrence of such bone damage. However, it is also important to determine when skeletal damage begins to accumulate and to quantify the relative contributions of house design, bird behaviour, and intrinsic changes in bone strength and physiology throughout the laying period. The prospective study was carried out on 6 free range and 6 barn flocks situated within England and Wales and 7 free range flocks situated in Scotland. Flocks were visited once at their rearing farm just before point of lay (England & Wales only), and on four further occasions during the laying period (20, 30, 50 and 70 weeks). The first visit laying house was made within the first few weeks of placement to determine whether fractures occurred during the initial transport and placement in the laying house. On each visit, the house lights were turned down to minimise bird movement, and 100 birds sampled by palpation. At the rearing farm and during assessments at 20 and 30 weeks of age, a sub-sample of 10 birds were humanely killed and subject to full dissection, for continued monitoring of the accuracy of the palpation method and to provide material for biochemical and biomechanical testing. This minimised the requirement to sacrifice healthy birds at the start of their productive period and was an acceptable compromise to the farm managers who were reluctant to lose valuable birds at such an early age. At subsequent ages (50 and 70 weeks) 20 birds were similarly selected and dissected. At each age within the laying environment video surveillance equipment, utilising 4 cameras with wide-angle lenses were mounted in each house, approximately 3m above floor level, and calibrated for angle and field of view, so that bird distribution could be accurately mapped. Automatic timer switches enabled us to obtain recordings at 4 times throughout the day including dawn and dusk when activity was at a high level. Evaluation of these recordings enabled the incidence of wing flapping, flights and attempted flights, poor landings and other critical events to be made.

Within each house the design of the physical structure of the housing was described and measured and particular note made of any included structures. At the end of the laying period production records were collected from each house along with records of changes to working practices during the complete laying cycle. The main features of housing design are summarised for the 3 systems in the following tables. There was considerable variation between houses within each system type.


System No flocks Available perch space

Perches 15cm/bird Aerial Available alternative perches Feed/drink Other*

Barn 6 0/6 - - 2/6 6/6

Free range 6 4/6 - - 2/6 6/6

Free range with suspended perches

7 7/7 7/7 7/7 0/7 7/7

(*other includes roof supports, stanchions, water supply pipes, ledges etc)


Top nest box Slats above

litter Angle of

ramp Perches

above litter Perches

above slats Available

alternative perches

Barn 43 90 49 - - 275

Free range 70 78 44 168 104 325

Free range with suspended perches

45 78 45 259 179 311

SID 5 (Rev. 3/06) of 26

The major difference between the 3 systems was in the provision of dedicated perches which satisfied the Scottish interpretation of the EU Directive. In the Free range houses only limited (low level) perches were present and no perches were present in the barn houses. Additionally, in the houses equipped with suspended perches vertically displaced poles were installed in three rows above the slats only, since the Directive prohibits placement of perches above the litter to prevent excessive soiling and degradation which might result in an increase in potential foot health problems.

An example of a free range unit equipped with suspended perches is shown below.

Results

No keel damage was found in flocks assessed at the end of the rearing phase and while considerable bird activity was provoked by the catching operation and packing of birds into the transport modules, only two birds out of 160 were found to have incurred damage when flocks were assessed approximately one week after placement. The data were analysed using a generalized linear model and there was a significant effect of age on fracture incidence (P<0.001) for flocks housed in all three systems (Fig 05).

Figure 05 Prevalence of broken bones assessed by dissection throughout the production cycle described using a generalized model.

The accumulation of damage in flocks housed in the barn and free range systems follows a similar pattern while those housed in the free range with aerial perches system incur bone damage at an earlier age (P<0.001). There was considerable variation in prevalence within system, particularly for free range flocks (Fig 06b). Nevertheless at all ages, flocks housed in free range fitted with aerial suspended perches, had sustained significantly higher levels of damage than those housed in either standard free range or barn housing systems P<0.001 for 30 and 70 weeks and P<0.01 for 50 weeks of age. There was no difference between flocks housed in the barn or free range systems.

SID 5 (Rev. 3/06) of 26

Figure 06 Prevalence of broken bones assessed by palpation at different ages in flocks of hens housed in barn, free range and free range fitted with aerial suspended perches systems.

a) mean ± se b) mean ± range

The validity of the palpation procedure as applied to live birds within the production unit was assessed by reference to dissection in up to 20 birds per sample (total of 1105 birds):The percentage correct and predictive value of palpation was comparable to that observed in freshly killed birds in Objective 01 (91% and 0.93 respectively) but the sensitivity of the procedure was slightly reduced This is a consequence of the need to handle the live birds with due care and consideration during the palpation, so that minor damage is sometimes undetected.

The dissection procedure permitted evaluation of the severity of fracture to be noted and using the same categories as described for Objective 01, the relative proportion of birds in each category was calculated. The table below again highlights the increase in the proportion of birds at all ages with keel fractures assessed as being severe in flocks housed in the free range system where aerial suspended perches had been installed.

Table 11. Severity of keel damage (% frequency within system)

None Minor Severe

score 0 & 1 score 2 score 3 & 4

30 weeks Barn 93 5 2

F/R 98 2 0

F/R - Aerial 72 14 14


F/R 63 22 15

F/R - Aerial 35 26 39


F/R 68 19 13

F/R - Aerial 32 26 42

Biomechanics

Bone breaking strength was measured in those birds taken for dissection and the results are summarised below as treatment means.

Figure 07. Peak bone strength at different ages in flocks of hens housed in free range barn, free range and free range with aerial suspended perches systems (mean ± se)

a).Tibia b). Humerus

SID 5 (Rev. 3/06) of 26

c) Keel (below manubrial spine) d) Keel (lateral plate)

Using standard three point bending methodology, with no correction for bone size, there was no significant effect of age on peak bone breaking strength in either the humerus or the keel but there was a highly significant increase in tibia breaking strength from 20 weeks through to 70 weeks of age for flocks housed in barn (P<0.05), free range (P<0.001) and free range with aerial suspended perches (P<0.001). There was no significant effect of housing system on peak breaking strength of any of the bones assessed. A detailed examination of the biomechanical characteristics of the tibia enabled 5 measures of intrinsic (material) properties, calculated by taking into account cross sectional area and 6 extrinsic (structural) measures to be calculated from the ‘deformation – load’ curves obtained during detailed 3-point bending tests using an Instron biomechanical testing machine. An effective ‘treatment’ or change in management practice should improve extrinsic biomechanical properties of bone without impairing the intrinsic properties. A schematic representation of the mechanical properties is shown below. Figure 08: Bone mechanical properties

In line with the findings from the peak breaking strength tests, ultimate stress increased with age (Figure 09a). In addition the yield stress (fig. 09b) and Young's Modulus of Elasticity (fig 11a) also increased with age. Figure 09a: Ultimate Stress (mean ± sem) Figure 09b: Yield Stress (mean ± sem) Figure 4b: Yield Stress (mean ± sem)

0

50

100

150

200

16 18 30 50 70

Age (weeks)

Ulitm

ate

Str

ess M

Pa

Barn

FR

FR-AS

0

20

40

60

80

100

16 18 30 50 70

Age (weeks)

Yie

ld S

tress M

Pa

Barn

FR

FR-AS

0

20

40

60

80

100

16 18 30 50 70

Age (weeks)

Yie

ld S

tress M

Pa

Barn

FR

FR-AS

SID 5 (Rev. 3/06) of 26

Figure 10a Ultimate Strain (mean ± sem) Figure 10b Yield Strain (mean ± sem)

Figure 11a Young's Modulus of Elasticity (mean ± sem) Figure 11b Energy to Fracture (mean ± sem)

Although the overall strength of the tibia increased with age, the ultimate and yield strains (fig 10a and b) and the work (energy) to failure (fig 11b) decreased in all housing systems. The largest decrease in these measures of toughness was seen between 18 and 30 weeks, the age at which there was a significant increase in the incidence of fracture. Bone Mineral Density Dual energy x-ray absorbtiometry (DEXA) for determination of bone mineral density (BMD) was undertaken on tibias from all flocks analysed throughout the production cycle. There was a significant increase in BMD with age in all housing systems with little or no difference between systems (P<0.001). Figure 12 Bone mineral density in flocks of hens housed in free range, barn and free range with aerial suspension systems (mean ± sd)

Bone Calcium/ Phosphate Ratio

The bone calcium and phosphate levels were measured from ashed bone, and the ratio of these bone mineral ions calculated. Previous data suggests that the ratio increases as the bone mineral matures, either with age or decrease in bone turnover. However in this study there were no effects of either age or housing system on the Ca:P ratio with an average ratio of 1.8 (data not shown).

0

0.005

0.01

0.015

0.02

0.025

0.03

16 18 30 50 70Age (weeks)

Barn

FR

FR-AS

0.000

0.002

0.004

0.006

0.008

16 18 30 50 70

Age (weeks)

Barn

FR

FR-AS

0

5

10

15

20

16 18 30 50 70

Age (w eeks)

MP

a

Barn

FR

FR-AS

0.000

0.050

0.100

0.150

0.200

0.250

0.300

16 18 30 50 70

Age (w eeks)J

Barn

FR

FR-AS

0

0.05

0.1

0.15

0.2

0.25

0.3

0.35

0.4

0.45

18 20 30 50 70

Age (weeks)

BM

D (

g/s

qm

m)

Free Range

Barn

Free Range Aerial Suspension

SID 5 (Rev. 3/06) of 26

Histomorphorphometry

Figure 13a bone volume Figure 13b example of bone cross section

Material from only a limited number of flocks was analysed by the Roslin Institute and the remainder analysed at Bristol. Results indicate no difference between systems in TBV or trabecular width but reduced MBV in free range housed birds. Biochemistry The data revealed large variation not only between birds but also between flocks within the same management system giving rise to large standard deviations and limiting us to describing changes in trends between the systems we have studied. Hydroxyproline analysis of the tibiotarsus bones reveals a slight fall in bone collagen content (Figure 14a) with age which is in keeping with an expected increase in mineralization over the same period. It also reveals marginally more bone collagen in the barn flocks at all ages and considerably less bone collagen in the aerial perch flocks Figure 14a: Average % collagen (Hydroxyproline) of whole bone from all Objective 02 flocks (mean ± sd)

Figure 14b: Average moles pyrrole / mole collagen from all Objective 02 flocks (mean ± sd)

Analysis of the mature, pyrrolic collagen crosslink (Figure 14b), the major bone collagen crosslink, revealed an increase with age, particularly in the free range flocks. The increase was most pronounced at visits 30 and 50 weeks corresponding with the increase in bone strength reported previously.

0

5

10

15

20

25

20 30 50 70

% C

olla

gen

of

we

t w

eig

ht

Weeks of age

FR

Barn

FR AS

0.00

0.50

1.00

1.50

2.00

2.50

3.00

20 30 50 70

mo

les

of

pyr

role

/ m

ole

of

colla

gen

Weeks old

FR

Barn

FR AS

SID 5 (Rev. 3/06) of 26

Figure 15a: Non-pyrrolic crosslinking of tibiotarsal collagen from all Objective 02 flocks (mean ± sd)

Figure 15b: Distribution between intermediate and mature non-pyrrolic crosslinks in tibiotarsal collagen from Objective 02 flocks

Analysis of collagen non-pyrrolic crosslinking revealed the expected trends in bone collagen development (Fig 15a). The tibiotarsus from all birds is predominantly stabilised with the ketoimine intermediate crosslink (mauve) which diminishes with age to yield the mature ketoimine products, in bone, of lysyl and hydroxylysyl pyridinoline (green and brown) If this data is combined with the aldimine intermediate and mature crosslinking (Fig 15b) then differences between the flocks become apparent. Free range flocks showed a less pronounced transition from intermediate to mature crosslinks in comparison with barn or FR AS flocks and may indicate a more immature collagen. Bone Enzyme Activity Bone alkaline phosphatase (BAP) activity is a marker of osteoblast number and activity and gives an indication of bone turnover. The BAP activity measured was low compared to other species and generally decreased with age as expected in avian bone, which is thought to undergo very little turnover once maturity is reached. The presence of medullary bone in the tibia resulted in high variability in the data and as a result no significant difference in activity between housing systems was found (data not shown). Matrix metalloproteinase (MMP) activity, which gives additional information on bone turnover (formation), was quantified in the bone extracts and was found not to vary significantly either between housing systems or with age. Again the medullary bone in the tibia samples was thought to be responsible for the high variability encountered. Behaviour analyses. During each visit a series of CCTV cameras, linked to a video recorder, was set up in each system to observe the flock and bird behaviour over 4 x 2-hour recording periods to cover a typical 24-hour period. On each occasion the flock was assessed for 1) the estimation of the numbers birds per view; 2) numbers of flights; 3) bad landings; and 4) numbers of wing flaps. For ease of presentation in the present report activity recorded from all camera views and time slots within each 24 hour recording period has been combined and the data analysed using a generalized linear modelling approach. Figure 16: Summary of activity of birds in flocks housed in barn, free range and free range with aerial suspended perches corrected for numbers of birds in each view a) Flights per bird

b) Probability of flight ending in a bad landing

0

0.1

0.2

0.3

0.4

0.5

0.6

0.7

0.8

0.9

20 30 50 70

mo

les

/ m

ole

of

colla

gen

Weeks of age

0.00

0.10

0.20

0.30

0.40

0.50

0.60

0.70

0.80

20 30 50 70m

ole

s /

mo

le o

f co

llage

nWeeks of age

SID 5 (Rev. 3/06) of 26

There was an overall significant effect of age on total number of flights per bird (P<0.01) with a small but significant decrease in the total number of flights per bird at 30 and 50 weeks of age compared to flight activity at 18 weeks in flocks housed in all 3 systems. In addition total flights per bird were significantly different between the 3 systems (P<0.001). This total included flights to and from dedicated perches where present, alternative structures available for perching, nest boxes and between the slats and the litter. Nevertheless the presence of aerial perches resulted in an increase in total flight numbers, a high proportion of which were likely to be over relatively long distances. The probability of a flight ending in a bad landing and thus increasing the likelihood of damage to the keel was found to decrease with age in flocks housed in all 3 systems followed by an increase at age 70 weeks in flocks housed in the 2 free range systems (Figure 16b).

There was a positive relationship when the data was analysed using a generalized linear model, between the number of flights per bird and the prevalence of fractures assessed by dissection when analysed across all systems, which was significant (P<0.05) for a one sided test. Similar analysis found no relationship between the proportion of bad landings observed and the fracture incidence.

Discussion/principle findings of Objective 02

The onset of keel damage was found to occur at some point between 18 and 30 weeks, depending on system design, with a further dramatic increase between 30 and 50 weeks of age. Where free range houses had been equipped with aerial suspended perches the onset of damage was earlier and overall both the level and severity of damage was found to be significantly greater than that sustained by flocks in either conventional free range or barn systems. Intuitively one might anticipate a relationship between activity and the prevalence of keel damage. However although differences between systems in total bird activity were identified there was no clear association between observed bad landings (which might be responsible for the occurrence of fractures) and the levels of keel damage assessed throughout the laying period. Interestingly there was a nearly significant reduction in fracture rate associated with increased flapping even where no flights had taken place. Such activity may be associated with maintenance of bone structure and strength.

There was no decrease in peak bone breaking strength with age, indeed tibia strength showed a significant increase in flocks housed in all systems. Detailed examination of other components of fracture mechanics, showed a decrease in ultimate and yield strains and the work (energy) to failure with age. The largest decrease in these measures of toughness was seen between 18 and 30 weeks, the age at which there was a significant increase in the incidence of fracture. These biomechanical properties determine how much energy is required before the bone will fracture, which could also be an indication of how much microdamage the bone has sustained. An accumulation of microdamage would increase the propagation of fractures and decrease the overall strength of the bone. These factors are also influenced at a molecular level, by the quality of the collagenous (and mineral) matrix- i.e. the bone becomes less compliant, which may affect the ability of the bone to withstand trauma, and thus be more susceptible to fracture. A reduction in collagen content, revealed by hydroxyproline analysis, concurred with the observed increase in bone mineral density revealed by DEXA analysis. This increase in BMD together with the increased mature collagen cross-linking, accounts for the observed increase in stiffness (Young’s Modulus) of the bone revealed by mechanical testing. Together these determinations support the biomechanical data that the bones become stronger and stiffer with age which affects the ability of the bone to withstand trauma in a reversible fashion, and thus be more susceptible to fracture. Overall, the detailed investigation of bone collagen biochemistry has revealed a trend of increased bone collagen crosslinking with age (particularly mature collagen crosslinking) that could explain the increased brittleness observed during mechanical testing. The large variability in the data, caused by variability between individual birds, as well as between flocks within a single management system, limits our ability to assign significance to specific areas of data. However the observed trends do provide a clear guidance of where future work in this important area should focus its efforts. Closely controlling housing variables, as well as those already controlled within this study, would greatly reduce data variability and empower the conclusions reached.

Objective 03.

Faecal samples from birds assessed within the project were collected to corroborate the preliminary data relating broken bones to the excretion of lysyl pyridinoline, a marker of bone collagen resorption. Lysyl pyridinoline is a bone collagen specific crosslink,and therefore an increased level of lysyl pyridinoline in faecal matter provides an indicator of increased bone collagen turnover, which is certain to occur following bone breakage

The overall trend observed was as expected, with greater excretion of lysyl pyridinoline being associated with greater bone metabolism. However, as Figures 17 and 18 reveal, when the lysyl pyridinoline data for young birds from the early visits of Objective 02 is compared with keel score the upward trend is reversed as bone collagen turnover is very much higher in these younger birds, despite there being no keel damage, than in older birds with broken bones.

SID 5 (Rev. 3/06) of 26

Figure 17: Relationship between keel score and picomoles of lysyl pyridinoline / mg faecal matter for barn (4), free range (4), free range aerial suspension flocks (2) and PUFA flocks (2) (mean ± sd)

Figure 18: Relationship between lysyl pyridinoline and the age of birds from barn (4), free range (4), free range aerial suspension (2) and PUFA flocks (2) (mean ± sd)

Analysis of faecal material recovered from those birds euthanazed for dissection revealed considerable variation in collagen content between birds at any time point and an overall downward trend with age in most flocks suggesting that general collagen metabolic rates are lower in older birds. However it is interesting to note that lysyl pyridinoline levels are appreciably higher in the PUFA flocks at visit 3 (70 weeks) which may indicate that in these birds, bone collagen metabolism is up-regulated and represents a larger proportion of general collagen turnover, in keeping with the hypothesis that n-3 fatty acids stimulate bone collagen metabolism. However in the present trial the time between bone breakage/repair and sampling was unknown and not controllable. As a consequence the lysyl pyridinoline may not accurately reflect the severity of the breakage. Having studied material collected at each visit from up to 4 flocks from each of the 4 systems assessed within the project, further analysis was curtailed.

Objective 04.

To determine whether dietary n-3 PUFA supplementation can influence the incidence and severity of osteoporosis and bone breakage in laying hens, in a commercial environment, birds from six supplemented flocks were examined at 30, 50 and 70 weeks of age by palpation. As described in Objective 02, at 30 weeks of age 10 birds and at 50 and 70 weeks of age, 20 birds were euthanased to validate the palpation and provide bone material for biochemical and biomechanical analysis. The 70 week assessments were an additional sampling point which was included based on results obtained in Objective 02 which indicated that there was a significant increase in bone damage between 50 and 70 weeks of age in some flocks, depending on system design. Non-supplemented flocks (controls) for comparison were those free range flocks examined in Objective 02. At each visit (30, 50 and 70 weeks of age) 100 birds were palpated and a subsample humanely killed and dissected as previously described. The volatility of the market for the Columbus eggs produced meant that flocks could be changed on to and off the supplemented diet as demand for the product varied. As a consequence one of the selected flocks had to be discarded at 50 weeks and a replacement flock started. The primary features of each system are described in the following tables.


No flocks Available perch space

Perches 15cm/bird Aerial

Available alternative perches

Feed/drink Other

Free range (Obj 02) 6 4/6 - - 2/6 6/6

Free range with n-3 supplementation

6 1/6 - - 2/6 6/6


Top

nest box Slats above

litter Perches

above litter Perches

above slats

Available alternativeperches

Free range Obj 02) 70 78 168 104 325

Free range with n-3 45 78 259 179 311

R2 = 0.0419

-0.010

0.040

0.090

0.140

0.190

0.240

0.290

0.340

0.390

0 1 2 3 4

Keel Score

Ly

sy

lpy

rid

ino

lin

e (

pm

/mg

)

0.000

0.020

0.040

0.060

0.080

0.100

0.120

0.140

0.160

0.180

0.200

18 20 30 50 70

Age (weeks)

lysy

lpy

rid

ino

lin

e (

pm

/mg

)

Free range

Barn

Free Range aerial suspension

PUFA

SID 5 (Rev. 3/06) of 26

As with the housing described in Objective 02, considerable variation in design was evident between the production units housing flocks fed the n-3 supplemented ration. Only one of the n-3 supplemented flocks had access to dedicated perches compared with four of the control (free range) flocks. However the maximum height of alternative structures which permitted perching was similar between the supplemented and control groups. Results. The prevalence of keel damage showed a reduction in the numbers of birds with broken keels throughout the production cycle in hens fed the n-3 supplemented diet, when birds were assessed by both palpation and dissection in comparison to the free range flocks fed a standard ration (Objective 02). This reduction was significant at both 50 weeks of age when assessed by palpation and dissection (P<0.01) and at 70 weeks of age when assessed by dissection (P<0.01). Figure 19: Incidence of broken bones in free range hens fed either standard or Columbus rations (mean ± se) a) Broken keels assessed by palpation

b) Broken keels assessed by dissection

The observed decrease in overall prevalence was accompanied by a similar reduction in the proportion of birds found to have sustained a severe (score 3 & 4) fracture of the keel when assessed at 50 weeks of age (15% of birds affected in flocks fed the standard ration compared to only 1.7% of birds affected from flocks fed the supplemented ration). A similar reduction was observed at 70 weeks of age, 13% vs 3.3%. This decrease in severity necessarily affected the resultant validation of palpation estimates, such that the sensitivity of assessment, ie the proportion of individuals with old breaks diagnosed by palpation, was reduced to 0.66 since mild breaks are obviously much more difficult to accurately detect and classify. Biomechanics Peak bone breaking strength showed a small but consistent increase in birds fed the n-3 supplemented diet in comparison to those fed a normal ration and this was significant for the Humerus and Keel bones at 70 weeks of age (P<0.01 and 0.05 respectively).

Table 14. Peak bone breaking strength

System Age Tibia Humerus Keel A Keel B

mean se mean se mean se mean se

Free range 30 20.6 0.39 20.0 0.66 29.1 1.07 14.1 0.55

50 22.3 0.54 18.7 0.78 29.6 1.14 13.6 0.74

70 23.9 0.75 19.3 0.49 31.4 0.58 13.4 0.70

Free range

with n-3

30 22.7 0.59 22.7 0.39 32.6 1.11 15.1 0.65

50 23.1 1.10 21.9 0.50 31.4 1.78 13.4 0.45

70 25.7 1.36 21.8 0.32 36.4 1.21 15.2 0.55

Examination of the stress- strain curves obtained for tibia bones showed the ultimate (fig 20a) and yield stress (not shown), and Young's Modulus of Elasticity (fig 21a) of the n-3 supplemented birds were consistently higher than the FR at all ages examined. There was little difference in the ultimate and yield strains (data not shown), but the energy to fracture was significantly higher (fig 22a), again at all ages, in the n-3 supplemented bones compared to the FR, with less of the negative trend with age in the n-3 flocks.

SID 5 (Rev. 3/06) of 26

Figure 20a Tibia Ultimate Stress (mean ± sem) Figure 20b Humerus Ultimate Stress (mean ± sem)

Figure 21a Tibia Young's Modulus of Elasticity (mean ± sem)

Figure 21b Humerus Young's Modulus of Elasticity (mean ± sem)

Figure 22a Tibia Energy to Fracture (mean ± sem) Figure 22b Humerus Energy to Fracture (mean ± sem)

The humerus of a selection of standard FR and n-3 supplemented birds also underwent detailed biomechanical analysis, and confirmed a significant increase in ultimate stress (P<0.01; fig. 20b), Young's Modulus (P<0.001; fig 21b), and energy to fracture (P<0.001; fig. 22b) with n-3 supplementation. Of interest, the humerus showed significant changes in biomechanics earlier and to a greater extent than the tibiotarsus. Bone Mineral Density The flocks fed the PUFA supplemented diet had consistently higher levels of mineral in both tibia and humerus bones than the control free range flocks. This increase was significant at both 30 weeks (P<0.05) and 70 weeks (P<0.001) weeks of age in the humerus, due to the absence of the medullary bone reducing the variation as described in Objective 02. These data suggests that the n-3 diet may be improving bone strength by promoting mineralization as outlined above.

0

50

100

150

200

30 50 70

Age (w eeks)

MP

a

FR

FR-N3

0

50

100

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30 70Age (w eeks)

MP

a

FR

FR-N3

0

5

10

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Age (w eeks)

GP

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FR

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a

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J

FR

FR-N3

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0.3

30 70

Age (weeks)

J

FR

FR-N3

SID 5 (Rev. 3/06) of 26

Figure 23: Bone mineral density in flocks of hens housed in free range (6) and PUFA (6) flocks (mean ± sd a. Tibia

b. humerus

Calcium Phosphate Analysis Calcium and phosphate analysis of the tibia showed no significant change with diet, although at 30 weeks in both tibiatarsus and humerus there was a lower Ca:P ratio in the n-3 supplemented birds (fig 24), suggesting a less mature mineral matrix, possibly due to an increase in bone turnover in response to the n-3 diet.

Figure 24 Calcium: Phosphate Ratio (mean ± sem)

Biochemistry The bone alkaline phosphatase activity of the tibia was again complicated by the variable presence of the metabolically active medullary bone (data not shown). However quantification of BAP in the humerus showed an increase in the n-3 supplemented flocks (P<0.05, figure 25a), and also an increase in tartrate resistant acid phosphatase (TRAP), which is a marker of osteoclast activity, (fig 25b) at 30 weeks of age (P<0.01) and end-of-lay (P<0.05), which supports the suggested theory of a stimulation of bone turnover by n-3. This bone turnover would result in the replacement of any damaged (through microfracture) structural bone, slowing the accumulation of damage, so increasing bone strength. Figure 25a. Humerus BAP activity Figure 25b. Humerus TRAP activity

0

0.05

0.1

0.15

0.2

0.25

0.3

0.35

0.4

0.45

0.5

30 50 70

Age (weeks)

BM

D (

g/s

qm

m)

Free Range

Free Range PUFA

0

2

4

6

8

30 70 Age (weeks)

0

0.4

0.8

1.2

1.6

2

30 70 Age (weeks)

30 50 70

1.4

1.5

1.6

1.7

1.8

1.9

FR

Columbus

Age (weeks)

0

50

100

150

200

250

30 70 Age (weeks)

* ***

SID 5 (Rev. 3/06) of 26

Matrix metalloproteinase activity (MMP-2) quantification in the tibiotarsus showed increased levels of total MMP-2 activity in birds fed the high n-3 diet compared to those on the standard diet (figure 26). MMP-2 has been associated with osteoblast activity and bone turnover. This finding supports our hypothesis of n-3 stimulation of bone turnover/repair in supplemented birds.

Figure 26. Total MMP-2 activity

Figure 27a: Average % collagen (Hydroxyproline) of whole bone from all objective 4 flocks (mean ± sd)

Figure 27b: Average moles pyrrole / mole collagen from all objective 4 flocks (mean ± sd)

Figure 27a reveals reduced levels of bone collagen in the PUFA flocks compared with the free range control flocks, which may be in keeping with the increased bone metabolism evidence in figures 25 and 26, moreover levels of pyrrole (Figure 27b) a mature bone collagen crosslink are also low in keeping with the premise of elevated levels of bone collagen metabolism. Figures 28a and 28b confirm the above finding of reduced bone collagen maturation in the PUFA flocks as the level of mature, non-pyrrolic crosslinking, is also markedly reduced in the PUFA flocks at all ages.

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Figure 28a: Non-pyrrolic crosslinking of tibiotarsal collagen from all Objective 04 flocks (mean ± sd)

Figure 28b: Distribution between intermediate and mature non-pyrrolic crosslinks in tibiotarsal collagen from all Objective 04 flocks.

Histomorphometry Histomorphometric analysis of the tibial metaphyses of four supplemented flocks showed an increase in both trabecular and medullary bone volumes when compared to the four flocks fed the standard diet assessed as part of Objective 02. There appeared to be little difference in trabecular width. The data indicates that the Columbus diet is either promoting both medullary and structural bone formation, and / or slowing resorption of the structural bone, both of which would have positive effects on the bone material properties

Figure 29: Bone volume of hens fed supplemented ration (mean ± sd)

Investigations of the enzymic activity (TRAP, ALP, MMP2) suggest that the significantly reduced breakage and improved mechanical properties in n-3 supplemented birds results from increased metabolic activity (turnover rate) of the structural bone. This enables repair of microfractures, rather than their accumulation, and maintained mechanical integrity compared with birds on standard diet with a lower turnover of structural bone. Discussion of Objective 04 Dietary supplementation with omega-3 fatty acids substantially reduced (by up to 60%) the levels of keel breakage in laying hens throughout the production cycle. However the influence of housing design on fracture incidence was evident from analysis of Objective 01 and 02 data and there was a significant difference in perch provision between the supplemented and control groups such that dedicated perches were only provided in one of the N-3 fed flocks compared to four of the control fed flocks. Alternative structures were available in all houses and the mean maximum heights were similar. Nevertheless both the tibia and humerus of birds fed the supplemented diet were significantly stronger and presumably more able to withstand fracture (by an improved ability to store energy before failure) than those fed a standard diet in similar housing. N-3 supplementation is known to promote bone formation and mineral absorption, both of which would improve bone material properties. In the case of laying hens the apparent lack of turnover after bone growth has ended may result in an accumulation of microdamage, a decrease in ability to absorb energy, and an increase in stiffness. Stimulation of bone turnover by n-3 supplementation may allow this microdamage to be repaired. The basis for this hypothesis was investigated by detailed biochemical assessments of bone material collected throughout the production period. Quantification of bone alkaline phosphatase activity BAP showed an increase in the n-3 supplemented flocks and also an increase in tartrate resistant acid phosphatase (TRAP), which is a

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marker of osteoclast activity. These findings support the suggested theory of a stimulation of bone turnover by n-3. This bone turnover would result in the replacement of any damaged (through microfracture) structural bone, slowing the accumulation of damage, so increasing bone strength. In addition the reduced amount of pyrrole and other mature cross links in bone collagen extracted from birds from the PUFA flocks, further supports the hypothesis that collagen turnover is being stimulated by the n-3 fatty acids thereby reducing the level of mature collagen crosslinking. The overall outcome is therefore an increase in the energy to fracture helping to protect the bird from bone breakage. Overall conclusions of the study Levels of bone breakage in UK flocks can be as high as 90% at the end of lay and housing design is the major determinant of breakage levels. In general terms, both the prevalence and the severity of keel damage increased as the complexity of the environment increased. Of particular note was the increased damage in flocks housed in systems equipped with multi level perches although these were very much restricted to a limited number of farms.

To determine when skeletal damage begins to accumulate, flocks housed in barn and free range systems, including those equipped with aerial perches, were assessed throughout the production period. In all three systems the onset of skeletal damage occurred at about 30 weeks of age and in the case of flocks housed with aerial perches, levels of damage were already high (30%). Thereafter there was a continuing accumulation of keel fractures. Analysis of bird activity identified some of the most important factors in determining the likelihood of fractures occurring and the height of the perches where available was found to be significantly related to fracture prevalence. Other structures may also be responsible but their overall usage is much reduced in comparison. Examination of the biochemistry and biomechanical properties of bones removed from the birds in this study confirmed that there is no decrease in ultimate breaking strength, indeed the ultimate stress increases with age of the bird. However there was a decrease in ultimate and yield strain and energy to fracture. The consequence is that although bones are not inherently weaker, they do become stiffer and less able to absorb energy, hence may be more likely to sustain fracture at a given load. The bone mineral density also increased with age, which may increase strength yet reduce compliance in line with the mechanical properties. Bone strength was influenced by system design and as expected birds housed in furnished cages had the weakest bones while those housed in all organic systems tended to have the strongest. However it is clear that bone strength and system design both contribute to the overall prevalence since flocks from furnished cages which had the lowest bone strength also had the lowest levels of damage.

One reason why limited research has been carried out to identify the time of onset of fractures is because relevant information about fractures has had to be obtained by full dissection of birds or using palpation which still involves considerable disturbance to the birds. An additional technique was tested that would permit the detection of levels of damage in individuals, but without handling or capturing the individual birds. This involved measuring the presence and levels of the collagen cross-link, lysyl-pyridinoline (L-pyr) which is found only in mineralising tissue, principally bone. It’s presence in serum and urine of humans is a reliable indicator of bone turnover, repair and resorption. Faecal samples were collected to examine the relationship between the presence of broken bones and the excretion of lysyl pyridinoline. If proven to be an accurate indicator of bone breakage it was thought that such an assay might be developed as an auditing tool for farm assurance schemes, to ensure that farmers met their statutory obligations, or for use in further research. However age related changes confounded the interpretation and at present it is not considered a viable assessment method. Previous nutritional strategies to prevent osteoporosis in the laying hen have aimed to provide key nutrients important to bone development and maintenance, either by enhancing calcium absorption or retention, or by maximising peak bone mass. However, these interventions have met with limited success, principally because medullary and not structural bone is formed after the onset of lay. As an alternative approach we have examined whether dietary n-3 PUFA supplementation (already in use for production of ‘Columbus’ eggs) can influence the basic biochemical and biomechanical properties of bone and hence the incidence and severity of bone breakage in laying hens. Flocks were examined throughout the laying period and the flocks that were fed the n-3 supplemented diet presented substantially fewer broken keels in comparison to the free range flocks fed a standard ration. This reduction (up to 60% at 50 weeks assessed by dissection) was significant throughout production. Peak bone breaking strength, ultimate and yield stress, and energy to fracture were found to be increased in birds fed the n-3 supplemented diet and importantly, the decrease in energy to fracture observed in control birds with age was not observed, thus making them less liable to breakage. The bone mineral density and the structural bone volume were also increased with supplementation. Biochemical analysis further supported the hypothesis that the n-3 supplementation promoted bone formation (with an increase in bone turnover markers), thus enabling repair of accumulated microdamage and increasing the material strength of the bones, and the resistance to fracture. This strategy for reducing the overall prevalence of keel fractures will be investigated in more detail in a recently initiated BBSRC funded study: Production of welfare friendly eggs – improving bone health and reducing bone breakage in laying hens using omega-3 modified diets, while the impact of keel fractures on individual birds is being studied in a current Defra funded study: The impact of keel fractures on the welfare of laying hens

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References to published material

9. This section should be used to record links (hypertext links where possible) or references to other published material generated by, or relating to this project.

The primary findings of this project will be described in three or four, peer-reviewed, research papers based on the project objectives. The first paper describing the overall situation in the UK will shortly be sent to British Poultry Science for publication.

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