Duteau 2015. Fish Silage Project Final Report

Yukon Research Centre 1 / 52

Michel Duteau, Cold Climate Innovation Centre

Yukon College | 500 College Drive, PO Box 2799, Whitehorse, Yukon Y1A 5K4

Fish Silage Project: Experimental protocol and Annotated Bibliography

MICHEL DUTEAU Yukon Research Centre, Yukon College, 500 College Drive, Whitehorse YT Y1A 5K4 Phone: (867) 689-8490, Fax: (867) 456-8672, email: [email protected]

Oct 27, 2015

Yukon Cold Climate Innovation Centre at Yukon College

Michel Duteau, Cold Climate Innovation Centre


Michel Duteau and Amlie Janin, NSERC Industrial Research Chair in Mine Life Cycle


PROJECT INVESTIGATORS AND PARTNERS

This project was prepared in agreement with and in partnership with:

_________________________________ _______________________________

Michel Duteau Ziad Sahid

Yukon Research Centre Yukon Research Center

_________________________________

Jonathan Lucas

Grizzly Pigs Farm

Yukon Cold Climate Innovation Centre is providing the funding for this project, through its funding partnerships and agreements.



INTRODUCTION

Fish silage definition

Fish silage can be defined as a liquid product made from whole fish or parts of fish that are liquefied by the action of enzymes in the fish in the presence of an added acid (Tatterson and Windsor, 2001).

Current situation

It is estimated that fish processing for human consumption yields around 40% of edible meat, while the remnant 60% composed of bones, skin, head, viscera, meat scraps and scales, is fishery by-products (Gildberg 1993 in Ramirez, 2013).

The technology required to produce fish silage is much simpler than that needed for fish meal. Fish silage thus has a net advantage in areas where the tonnage of waste material is insufficient to justify the production of fish meal and it is estimated that fish silage is most likely to be successful in areas where fish offal or waste fish is regularly available, but the cost of sending it to the nearest meal plant is prohibitive, and where there are farms, particularly pig farms, close by (Tatterson and Windsor, 2001).

According to estimates by Bimbo (2012), cold crude fish silage sold for USD 70-173/metric ton on the Alaska market during 1998-2007. For the purpose of the present analysis, a conservative estimate would be that farmers would have to pay CAD 200/metric ton for fish silage on the Yukon market in 2015.

Aim and objectives

The objectives of fish silage production in the Yukon are to:

- lower the cost of animal production in the Yukon (pigs, broiler chickens and laying hens),

through the production of a local feed option

- unlock the value of fish waste (Icy Waters Ltd. fish offal and casualties)

- make use of available fish resource (chum salmon)

The vision is that fish silage can be manufactured at a commercial scale and distributed as a wet mash to animal husbandry operations in the Yukon

An introductory experiment was conducted during summer 2014 at Icy Waters Ltd., where it was established that it is possible to transformed fish offal into fish silage using formic acid.

With this project, we intend in developing guidelines as to fish silage production in the Yukon from two main sources: Icy Waters fish waste, and chum salmon. We also want to assess the bioequivalence of fish silage when compared to feed stuff that is conventionally used in the Yukon i.e. we want to prove that replacing conventional (imported) protein with locally-sourced fish silage does not have a negative impact on animal productions. Thus, a comparative experiment is designed to test the fish silage diet on a sample of pigs, broiler chickens and laying hens, and infer conclusions onto all such animals in Yukon conditions.



EXPERIMENTAL PROTOCOL

Setup

The feeding trials were designed to be conducted in the fall (October-December) of 2015 at Grizzly Pigs farm, situated North of Whitehorse Yukon, on the Mayo Road. Grizzly Pigs Farm produced pig, broiler chickens, and eggs. Following dismantlement of Grizzly Pigs Farm in the fall of 2015, the feeding trials can be conducted where the animals now are hosted, contingent on conditions suitability.

Grizzly Pigs Farm rears two kinds of pig, with hybrids and back crossings: English Large Black (Figure 3) and Landrac-Durac (pink; Figure 4 ). The pigs range in outdoor wind-protected (low bush) paddocks, and have access to sheltered wood hutches. All piglets are weaned (separated from the mother) at 1 month. Male piglets are castrated at 4-5 days (barrows). All pigs are vaccinated for Parvovirus and Legionella twice a year. The pigs are butchered (market weight) at 100 kg (220 lb), which is attained at approximately 4 months of age.

In the fall (Sept-Oct) of 2015, 6 litters are expected, with 4-6 piglets each. 3 pink and 1 black sows were bred with a same pink boar. 1 black and 1 hybrid black/pink sows were bred with a same black boar. All in all, this is 3 litters of pure pink, 1 hybrid, 1 pure black, and 1 hybrid backcrossing to a black. Overall, 24 piglets are expected. According to the owners experience, these piglets in all likelihood should be similar enough for the purpose of this experiment.

Water is provided 1-2 times a day in one bucket per paddock. Feed is provided once a day in individual bowls for each pig (Figure 4). Pigs at Grizzly Pigs Farm are usually fed a commercial pig grower in the form of pellets containing oat, barley, wheat, corn, protein supplement, and canola oil (manufactured by Federated Co-operatives Ltd, Saskatoon, SK; Figure 5). This grower is certified FeedAssure, a feed safety management and certification program developed by the Animal Nutrition Association of Canada (ANAC). This grower is imported from Southern Canada and is bought from C&D Feed (Whitehorse, YT). Yukon Grain Farm (Steve Mackenzie-Grieve) also has pig grower available, which would likely not be as standardized and slightly more expensive (approximately 6$ extra per bag).

The broiler chickens stay in a heated building (garage). In the fall of 2015, 30 chicks of a .. mix are expected, which should all be similar enough for the purpose of this experiment. Broiler chickens are usually fed a commercial feed also available at C&D Feed.

The laying hens are housed in two heated temporary buildings (tarp sheds). One is insulated with straw, and the other one is insulated with foam. One is facing South, and the other one is facing North. The individual space area is approximately 1 sq ft, which amounts to 25 animals per building. In the fall of 2015, a new hatch is expected. Water is provided once a day. The water troughs are cleaned every 2-3 days. Feed is provided ad libitum. Light is provided 6 am to 10 pm. The hens usually lay eggs for 10 weeks. They will have been laying for 2 weeks prior to the experiment. The hens are expected to start laying mid-September. Two types of laying hens are available: Brown Hybrid Leghorn and Columbine Rock. Typical productivity at Grizzly Pigs Farm is 2 eggs per 3 days and 1 egg per 2 days for Brown Hybrid Leghorn and Columbine Rock, respectively. Typical overall productivity at Grizzly Pigs Farm is 5-6 eggs per week per hen for the first 8 months. Potentially, the buildings could be inverted mid-experiment, so as to account for the difference in living conditions. Another way to circumvent this would be to assign



the hens to cages and feed them accordingly, providing for a Complete Block Design. Laying hens are usually fed a commercial feed also available at C&D Feed.

The animals are cared for according to guidelines of the Canadian Council on Animal Care (1993).

Experimental plan

Testing

The objective of this experiment is to test the bioequivalence of a recommended fish silage dosage (test diet) as an alternative to conventional feed (control diet) for pigs, broiler chickens and laying hens. For more complex trials (e.g. comparison of different levels of inclusion of fish silage), more sophisticated experimental setup would be necessary, along with finer statistical tools.

This experiment is thus designed as a simple comparison of the means of the two diets for a series of response variables. Bioequivalence is granted if no statistically significant difference is found between the means. The hypothesis of there being a difference between the group means is tested with a series of univariate t-tests, and a Bonferroni correction (Kuehl, 2001) is applied to adjust () and minimize the experiment-wise error rate (i.e. take into account the fact that a series of statistical tests are performed on the same individuals). The results depend on the size of the difference between the means, divided by the standard error of the difference. Alternatively, a multivariate analysis could be performed, considering all response variables at once.

The null hypothesis is stated as H0: d d0, where d0 is the minimum difference between the groups that is to be detected. The alternate hypothesis is: H1: d > d0.

Experimental design

Ideally, all individuals are the same (age, sex, ancestry), and all conditions are the same (environmental exposure -wind, sun, snow, rain, temperature-, floor space per individual, type of watering system, type of feeding through, type of faeces management system). When such an ideal situation is unattainable, the differences become nuisance factors, and can potentially become sources of variability. Randomization is essential to reduce the contaminating effect of nuisance factors (e.g. sex and ancestry) and reduce variability: subjects are randomly assigned to one treatment or the other (control diet vs. test diet). When needed, blocking can be used to reduce the effect of a specific nuisance factor (e.g. exposure to wind): creating homogeneous blocks in which the nuisance factors are held constant. However, a simple t-test will not suffice in analyzing results for a Randomized Block Design; an ANOVA would be called for and the F-test would become the initial statistic of importance.

Confidence interval

In this experiment, bioequivalence is granted with a confidence interval of 95%, i.e. the null hypothesis of no difference between the means is rejected when p < 0.05. The probability of making a Type I error (rejecting the null hypothesis when it should not be rejected) is thus 5%.

Power of the test

A test's power is the probability of rejecting the null hypothesis when it should be rejected. The power of a statistical test is calculated as 1-, where is the probability of making a Type II error (accepting a



null hypothesis when it should be rejected). A test's power is influenced by the choice of confidence interval (1- ), and depends on the sample size and the magnitude of the effect (the degree of departure in the population from the null hypothesis). It is conventional to set 80% as the target value for statistical power. This convention implies a four-to-one trade off between -risk and -risk. ( is the probability of a Type II error; is the probability of a Type I error, 0.2 and 0.05 are conventional values for and ). When a test shows that a significant difference is present, then usually there is no need to further consider the statistical power of the study. However, if no significant differences are detected, then questions may arise as to whether detection of significant differences in the means would have been made had there been more replications in the experiment. In a bioequivalence experiment, it is thus paramount to make sure that the power of the test be sufficient (80%) and thus insure validity of the conclusions, by using the proper minimum amount of replicates.

Number of replicates

It is tempting to declare that for a specific experiment, a critical minimum quantity of replicates (n) is necessary to detect a statistically valid difference (assess bioequivalence); however, the interaction of specific breed, type of feed, environmental conditions etc. makes it impossible to make concrete declarations of sample size or levels of significance (Roush, 2004). In order to approximate the minimum number of replicates, it is helpful to conduct an a priori power analysis. The website of R. V. Lenth (www.stat.uiowa.edu/rlenth/Power/) provides links to several power analysis calculators. When the coefficient of variation (CV) and the magnitude of the effect is known for a specific trait, a table such as that presented in Roush (2004) or Reese (2010) can be used to estimate the minimum number of replicates. A trait with a small CV needs fewer samples for detection than a trait with a large CV. In the same way, a trait with a large magnitude of the effect (e.g. 0.8) needs fewer samples for detection than a trait with a medium (e.g. 0.4) or small (e.g. 0.1) magnitude of the effect.

Because the total number of replicates that are required depends both on the variability of the trait (response variable) under scrutiny and the magnitude of the effect that is expected, the number of replicates is specific to each trait. In order for the power to reach 80% throughout all the traits for a specific animal (pigs, broiler chickens, laying hens), it is important to calculate n using the trait that has the highest CV, and lowest magnitude of the effect.

As a guidance, Reese (2010) determined that for two-sample diet experiments with pigs, 4 pens per diet is necessary to detect a potential difference of 15% or higher, assuming a CV of 5% - hence a total of 8 pens is necessary. In this case, the pen (group of pigs) is the replicate (hence, 3 degrees of freedom). The number of pigs per pen should be as high as possible (in order to have averages with minimum standard error, and to be able to account for dead pigs), taking into account the comfort of the animals and the total number of pigs available for the experiment. Based on Reese (2010)s recommendations, three pigs per pen should be used for this experiment, for a total number of 24 animals. Floor space should be equal for every individual.

For two-sample diet experiments with broiler chickens and laying hens, MacMillan suggested that 60 individuals be used per diet. In this case, the individual is the replicate.



Time frame

For pigs, the experiment takes place over the growing-finishing cycle. During this period of approximatively 3 months, the pigs grow from 25 kg (55 lb) to 100 kg (220 lb, market weight). Alternatively, the experiment could take place during the growing period only (25 kg to 60 kg liveweight). Additionally, 7 days should be allocated for adaptation to the feed, and 10 days for adaptation to the cage.

The feeding trial on broiler chickens should be carried out through a normal production range (6-8 weeks).

For laying hens, the experiment should take place through the first half of the laying period (point of lay to 22 weeks).

Fish silage production

Fat in the silage may increase poly-unsaturated fatty acids (PUFA) content, including the long-chain omega-3 fatty acids C22:6 (DHA), C22:5 (DPA) and C20:5 (EPA). This may be beneficial for human nutrition, since the consumption of long-chain omega-3 fatty acid may strength immune and nervous systems, as well as prevention of the cardiovascular diseases and some types of cancer (Ramirez, 2013). However, this may have an adverse effect on the sensory quality of meat, leading to the development of a rancid or fishy taste (Raa and Gildberg 1982; Krogdahl 1985). Hence, fish silage fed to pigs, broiler chickens or laying hens needs to be defatted.

In this experiment, defatted fish silage is produced following Jangaard (1987; Figure 1 and Figure 2):

- The raw material is first minced; suitably small particles can be obtained by using a hammer mill grinder fitted with a screen containing 10 mm diameter holes (Tatterson and Windsor, 2001).

- Immediately after mincing, formic acid is added at a level of 1525 g kg1 (1.5-2.5 %) wet weight depending on the ash content in the raw. The more bone the higher rate of acid is required to bring pH down -high calcium content will neutralize the acid and therefore the product requirement will be higher. When making large batches, acidity should be monitored and adjusted empirically to stay within the 3.6-4 range; if it is above 4 more acid should be added; if it is below 3.8 less acid could probably have been used, with a saving in cost. It is important to mix thoroughly so that all the fish comes into contact with acid, because pockets of untreated material will putrefy.

- Ethoxyquin is added as an anti-oxidant at 200300 ppm wet weight (200-300 mg/kg wet weight) - The fish silage is let to cure for liquefaction to operate, and occasional stirring helps to ensure

uniformity. The rate of liquefaction highly depends on the temperature of the process. For instance, white fish offal can take about two days to liquefy at 20C, but takes 5-10 days at 10C, and much longer at lower temperatures. Thus in winter it would be necessary to heat the mixture initially, or to keep it in a warm area until liquid (Tatterson and Windsor, 2001).

- In a subsequent step, fish silage is heated to 95C, and passed through a decanter and a centrifuge to separate the fat from the rest. Fish fat could potentially be valued through routes such as energy production (e.g. biodiesel), compost, dog food, etc.



Fish silage of the correct acidity keeps at room temperature for at least two years without putrefaction. The protein becomes more soluble, and the amount of free fatty acid increases in any fish oil present during storage, but these changes are unlikely to be significant nutritionally (Tatterson and Windsor, 2001).

According to Tatterson and Windsor (2001), the fish silage can be blended with cereals to make a semidry feed or wet mash.

Pre-experiment measurements

Variability of farm-specific production performance

Variability of production performance is measured before the experiment, in order to determine the minimum number of replicates needed to assess bioequivalence of the test diet when compared to the control diet. Variability is specific to the farm where the experiment takes place. Variability expresses chance variation i.e. the difference that exists between individuals, despite the best effort to feed and treat a group alike. For instance, variability of weight gain is a measure of weight gain difference that exists between individuals because of factors that cannot be explained or anticipated. Metrics used to assess production performance are detailed in the Response Variables section. Variability should be assessed over a whole production cycle (e.g. weaning to slaughter for pigs). Variability is expressed in terms of Coefficient of Variation: CV = SD/X * 100% where CV = Coefficient of Variation SD = Standard Deviation X = Treatment Mean

Weight at time zero of the experiment

Weight of each individual animal used in the experiment is measured at the time of inception of the experiment.

Pig weight is determined using heart girth as a proxy (Groesbeck et al., 2002): Pig weight (lb) = 10.1709 heart girth (in) - 205.7492

Quality of the feed

Quality of the feed ingredients and quality of the feed is determined prior to the experiment (Table 1). Dry matter content should equate to the addition of Protein, Fat, and Ash content. Similar to Kjos (2001, 2000, 1999), all analyses are conducted according to standard procedures described by the Association of Official Analytical Chemists (1990). Protein content is calculated as the Nitrogen content (Kjedahl) multiplied by 6.25. Fat is measured as HCl-ether extract; fatty acid composition is analyzed by GLC procedures. Metabolizable energy is determined according to procedures described in Krogdahl (1985), following Just (1982)s method and using the Rostock equation described by Schiemann et al. (1971). Quality is determined for the crude fish matter, crude fish silage, fresh de-fatted fish silage, aged (e.g. 3 months) de-fatted fish silage, the wet mash, and the control feed.



pH

Dry matter content (% of diet)

Protein content (g kg Dry Matter-1)

Fat content (g kg Dry Matter-1)

Ash content (g kg Dry Matter-1)

Crude fiber (g kg Dry Matter-1)

Nitrogen free extracts (g kg Dry Matter-1)

Fatty acid composition (g kg Dry Matter-1)

Calcium (g kg Dry Matter-1)

Phosphorus (g kg Dry Matter-1)

Magnesium (g kg Dry Matter-1)

Metabolizable energy (MJ kg Dry Matter-1)

Table 1: Quality parameters for the feed and feed ingredients

Feeding and Diets

If not otherwise stated, feeding and diets follow Kjos (2001, 2000 and 1999)s recommendations. In order to be able to draw valid conclusions on the bioequivalence of fish silage as a protein source, the test and control diets are isoenergetic, i.e. balanced on a metabolizable energy basis.

Feeding scheme

Contingent on the farm habits, pig rations are provided once or twice a day. The pigs are fed individually. Feed quantity is adjusted daily following a standard feeding/growth chart (e.g. Thomke et al., 1995). From Kjos (1999)s observations, the average daily feed intake can be assumed to be approximately 1.89-1.99 kg day-1. Following Kjos (1999)s recommendation for the prevention of adverse effect on sensory quality of the meat, the experimental diet is fed until slaughter only if the de-fatted fish silages fat level is lower than 3.4 g kg1 DM; if the fat level is up to 5.7 g kg1 DM, the experimental diet can be fed until 60 kg liveweight, and control feed is fed for the remainder of the finishing period (until 100 kg).

For broiler chickens, feed and water are provided ad libitum. From Kjos (2000)s observations, the average net feed intake can be assumed to be approximatively 79.1-82.9 g kg-1. Following Kjos (2000)s recommendation for the prevention of adverse effect on sensory quality of the meat, the experimental diet can be fed until slaughter if the de-fatted fish silages fat level is lower than 10 g kg-1 DM;

For laying hens, feed and water are provided ad libitum.

Test diet

Test diet compositions for pigs, broiler chickens and laying hens are presented in Table 2. All test diets are a compound feed based on a non-protein commercial mix (e.g. barley, oat, wheat, corn, canola), and protein is supplied by fish silage and soybean meal; fish silage is supplied at the maximum proportion recommended in the literature to prevent adverse effect on production performance, and the remainder of necessary protein supply is provided by soybean meal. Rendered animal fat is used to adjust the metabolizable energy with that of the control diet; for instance, Kjos (2001, 2000, 1999)



utilized rendered fat consisting of approximately 70% lard and 30% tallow. Vitamin E is added to prevent lipid oxidation in meat tissues and prevent adverse effect on sensory quality of the meat. Lysine, methionine and tryptophan are added in order to meet or exceed the National Research Council requirements for amino acids for poultry (1994) and for swine (1998). In the same way, vitamins are added in order to supply surplus amounts according to requirements (ref), and to equalize diets. Diet compositions are provided here on a relative basis, and final individual ingredient weights will need to be determined from the fish silage quality data (starting with protein content).

For pigs, the fish silage is provided in a proportion of 9% of the total dietary protein; in Kjos(1999)s experiment, for instance, 9% of the total dietary protein content corresponded to 50 g fish silage / kg diet (circa 5% of the total diet on a weight basis). The remainder of the protein need is supplied by soybean meal; in Kjos(1999)s experiment, for instance, 162 g kg-1 was necessary to complete protein requirements. From Kjos (1999)s experiment, it can be approximated that 25% of the soybean meal that would be necessary to complete dietary protein requirements (circa 210 g kg-1) can be replaced by fish silage. For illustrative purpose, the metabolizable energy level in Kjos (1999)s diets was 14.4-14.8 MJ kg-1 DM).

For broiler chickens, the fish silage is provided in a proportion of 21% of the total dietary protein; in Kjos (2000)s experiment, for instance, 21% of the total dietary protein content corresponded to 100 g fish silage / kg diet (circa 10% of the total diet on a weight basis). For illustrative purpose, the metabolizable energy level in Kjos (2000)s diets was 11.32-11.77 MJ kg-1 DM).

For laying hens, the fish silage is provided in a proportion of 12% of the total dietary protein; in Kjos (2001)s experiment, for instance, 12% of the total dietary protein content corresponded to 50 g fish silage / kg diet (circa 5% of the total diet on a weight basis). For illustrative purpose, the metabolizable energy level in Kjos (2001)s diets was 10.6-10.7 MJ kg-1 DM).

Pigs Broiler Chickens Laying Hens

Commercial non-protein feed Basis Basis Basis

De-fatted fish silage 9% of the total dietary protein

content

21% of the total dietary protein

content

12% of the total dietary protein

content

Soybean meal To complete protein needs

To complete protein needs

To complete protein needs

Rendered Animal fat To adjust metabolizable

energy

To adjust metabolizable

energy


energy

Vitamin E Yes ? ?

Lysine Yes ? ?

Methionine Yes ? ?



Tryptophan ? ? ?

Vitamin premix Yes1 Yes2 Yes3

Table 2: Test diet composition for pigs, broiler chickens, and laying hens. The relative proportion of each ingredient is indicated in the column for the specific animal production.

Control diet

Control diet compositions for pigs, broiler chickens and laying hens are presented in Table 3. All control diets are a compound feed based on a non-protein commercial mix (e.g. barley, oat, wheat, corn, canola), and protein needs are supplied by soybean meal entirely. Rendered animal fat is used to adjust the metabolizable energy with that of the test diets. Vitamin E, essential amino acids, and Vitamin premix are also added, in the same way as for the test diets. Control diets compositions are provided here on a relative basis, and final individual ingredient weights will need to be determined from the ingredients quality data.


Commercial non-protein feed Basis Basis Basis

De-fatted fish silage None None None

Soybean meal Entire protein needs

Entire protein needs

Entire protein needs

Rendered Animal fat To adjust metabolizable

energy


energy


energy

Vitamin E Yes ? ?

Lysine Yes ? ?

Methionine Yes ? ?

Tryptophan ? ? ?

Vitamin premix Yes4 Yes5 Yes6

Table 3: Control diet composition for pigs, broiler chickens, and laying hens. The relative proportion of each ingredient is indicated in the column for the specific animal production.

1 Trace elements and vitamins included to provide the following amounts per kg of diet: 70 mg of Zn; 50 mg of Fe; 40 mg of Mn; 10 mg of Cu; 0.5 mg of I; 0.2 mg of Se; 6000 IU of vitamin A; 400 IU of cholecalciferol; 40 mg of dl- -tocopheryl acetate; 3 mg of riboflavin; 10 mg of d-pantothenic acid; 20 g of cyanocobolamine; 20 mg of niacin; 0.2 mg of biotin; 1.5 mg of folic acid; 2 mg of thiamin; 3 mg of pyridoxine. 2 Trace elements and vitamins provide the following amounts per kg diet: 70 mg of Zn; 50 mg of Fe; 40 mg of Mn; 10 mg of Cu; 0.5 mg of I; 0.2 mg of Se;6000 IU of vitamin A; 400 IU of cholecalciferol; 40 mg of d1-a-tocopheryl acetate; 8 mg of riboflavin; 15 mg of d-pantothenic acid; 20 mg of cyanocobolamine; 60 mg of nicacin; 0.2 mg of biotin; 2 mg of folic acid; 4 mg of thiamin; 6 mg of pyridoxine. 3 Trace elements and vitamins to provide the following amounts per kg of diet: 60 mg of Zn; 25 mg of Fe; 100 mg of Mn; 5 mg of Cu; 0.5 mg of I; 0.2 mg of Se; 12,000 IU of vitamin A; 3000 IU of cholecalciferol; 40 mg of d1-a-tocopheryl acetate; 8 mg of riboflavin; 15 mg of d-pantothenic acid; 30 mg of cyanocobalamine; 40 mg of niacin; 0.1 mg of biotin; 1 mg of folic acid; 4 mg of thiamin; 6 mg of pyridoxine 4 Same as in experimental diet 5 idem 6 idem



Response variables

Measurements are taken throughout the experiment to assess bioequivalence of the experimental diet and the control diet. Response variables can be categorized in terms of production performance, economics, metabolism data, physical characteristics of the end product, sensory quality of the end product, and nutritive quality of the end product. If not indicated otherwise, all response variables are measured same as in Kjos (2001, 2000 and 1999). According to budget, logistics, and technical feasibility, measurements of some response variables might be prioritized, modified, or eliminated.

Production performance

Production performance metrics for pigs, broiler chickens, and laying hens are presented in Table 4. Weight of each individual is measured at the beginning and at the end of the experiment, and net weight gain is calculated from these observations. The number of days necessary to fatten up to market weight (circa 100 kg for pig and circa 2.5 kg for broiler chickens) is recorded, and average daily gain is calculated from these observations. For pigs, weight is also recorded every 14th day; for broiler chickens, weight is also recorded every 7th day. For pigs, feed intake is recorded daily; if any feed is rejected, it is measured and subtracted from the ration weight. For broiler chickens and laying hens, feed consumption is recorded every 7th day. Net feed intake is calculated using this observation and the average daily feed intake is calculated, integrating the number of days the experiment unfolded. Feed efficiency equates to a feed-to-gain ratio, and is calculated as net feed intake over net weight gain. Net energy intake and average daily energy intake are calculated by integrating the metabolizable energy value of the feed. Energy efficiency (energy-to-gain ratio) is calculated as energy intake over weight gain. For laying hens, eggs are collected and counted daily, and an average daily egg quantity and egg weight production is calculated once a week. Hen-day production represents the average quantity of eggs that is produced per hen per day and is calculated as:




Initial weight (kg) X X X

Final weight (kg) X X X

Net Weight gain (kg) X X X

Number of days to market X X

Average daily gain (kg day-1) X X X

Net feed intake (kg) X X X

Average daily feed intake (kg day-1) X X X

Feed efficiency (kg kg-1 gain) X X X

Average daily energy intake (MJ day-1) X X X

Energy efficiency (MJ kg-1 gain) X X X

Average daily egg production (quantity day-1) X

Average daily egg weight production (g day-1)

X

Average hen-day egg production (%) X

Table 4: Production performance metrics for pigs, broiler chickens, and laying hens. Those metrics marked with an X in the column are suggested for the specific animal production.

Economics

Economical metrics for pigs, broiler chickens, and laying hens are presented in Table 5. All economical metrics are calculated considering a fish silage cost of CAD 200/metric ton.

Economical metric Pigs Broiler Chickens Laying Hens

Cost per weight gain (S/kg) X X X

Cost per egg produced ($/egg) X

Cost per energy intake ($/MJ) X X X

Return on investment ($/$) X X X

Table 5: Economical metrics for pigs, broiler chickens, and laying hens. Those metrics marked with an X in the column are suggested for the specific animal production.

Metabolism data

Metabolism metrics for pigs and broiler chickens are presented in Table 6 (no metabolism data for laying hens). For pigs, blood samples are taken at start of the experiment (circa 25 kg), at 60 kg live weight, and immediately before slaughter (circa 100 kg); the blood samples are taken from the jugular vein, approximately 1 h after the morning feeding, using heparinized vacutainers for the plasma samples and polyethylene tubes (TT tubes) for whole blood. For broiler chickens, blood samples are taken from the jugular vein of all chicks immediately after slaughter, using heparinized vacutainers for the plasma samples and polyethylene tubes (TT tubes) for the whole blood samples. Vitamin E is determined in blood plasma using the method of McMurray and Rice (1982) with modifications indicated in Kjos (2000



and 1999). Ceruloplasmin is determined in blood plasma according to Schosinsky et al. (1974). Glutathione peroxidase is analyzed in whole blood following the method of Paglia and Valentine (1967).

Pigs Broiler Chickens

Vitamin E X X

Ceruloplasmin X X

Glutathione peroxidase X X

Table 6: Metabolism metrics for pigs and broiler chickens. Those metrics marked with an X in the column are suggested for the specific animal production. No metabolism metrics are suggested for laying hens.

Physical characteristics of the end product

Physical characteristic metrics for pigs, broiler chickens and laying hens are presented in Table 7. For pigs, carcass characteristics are measured 1 d after slaughter. Lean percentage is determined using a GP2Q pistol (Hennessy System), measuring the diameter of the loin muscle (longissimus thoracis et lumborum) and backfat thickness at two sites (between the last 3rd and 4th rib, 6 cm from the midline, and behind the last rib, 8 cm from the midline). A tracing of a cross section of the cutlet, behind the last rib, is made using tracer paper. Meat area in the cutlet is determined with a planimeter (Coradi AG, Zrich, Switzerland). The P2 backfat thickness is measured 8 cm from the midline behind the last rib using tracer paper and a ruler. Subjective evaluation of subcutaneous fat firmness using a scale from 1 to 15, in which 15 is the firmest score. Subjective evaluation of fat colour using a scale from 1 to 15, in which 15 is the most favorable colour. For broiler chickens, carcass weight and weight of the abdominal fat pad are registered at the time of slaughter. For laying hens, egg characteristics are taken on all eggs from two randomly chosen days within the first and the second half of the experimental period, respectively. The eggs are stored at 4oC and the analyses must take place within 10 days. Thickness of albumen is determined on cracked eggs using a micrometer. Haugh unit is calculated on the basis of thickness of albumen and egg weight. Yolk color index is evaluated by Roche Yolk Colour Fan (F. Hoffmann La Roche Ltd., Basel, Switzerland), on a scale of 114 (1 = very pale yellow; 14 = very dark orange).




Slaughter weight (kg) X X

Carcass weight (kg) X X

Dressing percentage (%) X X

Lean (%) X

Meat area in the cutlet (cm2) X

P2 backfat thickness, P2 (mm) X

Subcutaneous fat firmness (1-15) X

Fat colour (1-15) X

Weight of the abdominal fat pad X

Thickness of albumen X

Yolk color index X

Table 7: Physical characteristics of the end product for pigs, broiler chickens, and laying hens. Those metrics marked with an X in the column are suggested for the specific animal production.

Sensory/organoleptic quality

Organoleptic quality (meat and egg acceptability) metrics for pigs, broiler chickens, and laying hens are presented in Table 8. For pigs, sensory quality analysis is conducted on samples of loin, flank, and belly that have been stored in a freezer at 16C, for 1 mo (short time storage) or for 6 mo (long time storage). The samples of belly are processed (cured and smoked) to make bacon. Meat for sensory analysis is vacuum-packaged prior to storage. The sensory analysis is conducted according to international standards (ISO 3972 Sensory analysis Methodology Method of investigating sensitivity of taste); a trained panel of eight members evaluate the samples, using a scale from 1 to 9, where 1 is the lowest and 9 the highest intensity, for all parameters. The sensory analysis can be conducted at the Norwegian Meat Research Laboratory, Oslo, Norway.

For broiler chickens, sensory quality is analyzed on thigh meat taken from 15 chicks of each dietary treatment, randomly chosen from each of the replicate pens. The samples are taken 1 h post-mortem, and are frozen immediately. Sensory quality analysis is conducted on pieces of meat that have been frozen for 1 mo and 6 mo and with the same method as described for pigs (see hereinabove).

For laying hens, sensory analysis is conducted on two sets of 12 eggs, collected from two randomly chosen days. The eggs are stored at 4oC and analyzed for sensory evaluation after 7 days and after 35 days, respectively. The sensory analysis is conducted according to international standards (ISO 3972 Sensory analysis Methodology Method of investigating sensitivity of taste). Similar to Kjos (2001), sensory analysis can be conducted at the Norwegian Food Research Institute, s, Norway, using a computerized system for recording of data (Compusense Five, Compusense, Guelph, ON). The eggs are boiled for 10 min and then cooled in cold water for 5 s before sensory evaluation. A trained panel of 11 members evaluate both albumen and yolk for the parameters odor, off-odor, taste, off-taste, whiteness and hardness. Each assessor evaluates the samples on the computerized system, using a continuous



scale. The computer translates the responses into numbers between 1 to 9, where 1 equals no intensity and 9 equals high intensity of the parameter.


Loin [odour, off-odour, taste, off-taste, juiciness, tenderness] (1-9)

X

Flank [odour, off-odour, taste, off-taste] (1-9)

X

Belly [odour, off-odour, smoke odour, taste, off-taste, smoke taste, salt taste] (1-9)

X

Thigh meat [odour, off-odour, taste, off-taste, rancid taste, juiciness, tenderness] (1-9)

X

Albumen [odor, off-odor, taste, off-taste, whiteness, hardness]

X

Yolk [odor, off-odor, taste, off-taste, yellowness, hardness]

X

Table 8: Sensory quality of the end product for pigs, broiler chickens, and laying hens. Those metrics marked with an X in the column are suggested for the specific animal production.

Nutritive quality of the end product -contents of fatty acids

Nutritive quality metrics for pigs, broiler chickens, and laying hens are presented in Table 9. Fatty acid results are presented as relative distribution of the individual fatty acids (g 100 g1 of total fatty acids). Total poly unsaturated fatty acids (PUFA) include the long-chain omega-3 fatty acids C22:6 (DHA), C22:5 (DPA) and C20:5 (EPA). Fatty acids composition is analyzed by GLC procedures according to the methods described by Ulbreth and Henninger (1992) for extracted/methylated samples. The fatty acid methyl esters are determined on a Perkin Elmer Autosystem gas chromatograph (Perkin Elmer Corp., Norwalk, CT) with a SGE capillary column no. 5QC3/bpx70, 0.25, 25 + 25 m (SGE International Pty. LTD, Ringwood, Victoria, Australia). For pigs, the content of fatty acids is measured in subcutaneous fat. For broiler chickens, fatty acids composition is analyzed on (samples of) the abdominal fat pad of all the chicks and (of) the breast meat of five chicks of each treatment randomly chosen. For laying hens, nutritive quality (cholesterol and fatty acid content) is measured on 4 eggs collected randomly throughout the experiment period. The eggs are stored at 4oC and analyzed within 10 days. Cholesterol in egg yolk is determined spectrophotometrically in Encore Chemistry System (Baker Instruments, UK), using Cholesterol Enzumatique PAP 100, kit. Ref. 61 224 from bioMeriedux (France).




Proportion of individual fatty acids (in meat/in yolk) (g 100 g total fatty acids-1) C14:0 C16:0 C16:1 C18:0 C18:1 C18:2 (n-6) C18:3 (n-3) C20:1 C20:4 C20:5 (n-3) C22-1 C22:5 (n-3) C22:6 (n-3)

X X X

Proportion of poly unsaturated fatty acids (PUFA) (g 100 g total fatty acids-1)

X X X

Cholesterol in egg yolk X

Table 9: Nutritive quality of the end product for pigs, broiler chickens, and laying hens. Those metrics marked with an X in the column are suggested for the specific animal production.



PHOTOS AND FIGURES

Figure 1: Typical fish silage installation (adapted from Jangaard, 2007)

Figure 2: Processing method for concentrated, defatted fish silage (adapted from Kjos, 1999).



Figure 3: English Large Black sow and her 6 piglets at Grizzly Pigs Farm (July 2015)

Figure 4: Pink sow at Grizzly Pigs Farm (July 2015)



Figure 3: Example of a small pig hutch at Grizzly Pigs Farm (July 2015

)

Figure 4: Feed bowls for individual pigs

Figure 5: Commercial Grower Feed utilized for pigs, broiler chickens and laying hens at Grizzly Pigs Farm



ANNOTATED BIBLIOGRAPHY

Addcon, 2015. The principle of making fish silage to preserve by-products for the feed industry. Webpage.

Notes This document presents how to use ENSILOXR, a product that can be used for making fish silage. This product consists of formic acid and an antioxidant, the latter helping in protecting the oil content. Addcon is based in Germany.

Al-Marzooqi, W., Al-Farsi, M., Kadim, I., Mahgoub, O., Goddard, J., 2010. The effect of feeding different levels of sardine fish silage on broiler performance, meat quality and sensory characteristics under closed and open-sided housing systems. Asian-Australasian Journal of Animal Sciences. 23 (12), 1614-1625.

Abstract Two experiments were conducted to evaluate the use of fish silage prepared from Indian oil sardines, Sardinella longiceps, as partial replacement of soybean meal as a sole source of protein for growing broiler chickens. The main objective of Experiment 1, an ileal digestibility assay, was to assess the nutritional value of fish silage compared with soybean meal for feeding broiler chickens. The two test ingredients, soybean meal and dried fish silage, were incorporated into semi-synthetic diets, as the only component containing protein. The ileal digestibility coefficients of amino acids of fish silage were considerably higher than those of soybean meal (p



Association of Official Analytical Chemists 1990. Official methods of analysis. 15th ed. AOAC, Washington, DC.

Methods for analysis the quality of the fish silage can be found in there (Kjos, 1999).

Balios, J., 2003. Nutritional value of fish by-products, and their utilization as fish silage in the nutrition of poultry. Proceedings of the 8th International Conference on Environmental Science and Technology8-10.

Abstract It can be concluded from the experiments that fish silage is a very good alternative source of protein when partly replacing other more expensive sources of protein. In our days, with consumers being more and more sensitive in matter related to the pollution of the environment, fish silage provides the means of utilizing fish waste from the canning industry, instead of being thrown away. Among the advantages of making fish silage are: Fairly low capital cost, can be made by unskilled workers, there is no smell of the final product and can be stored, under favourably conditions, for up to two years. Disadvantages are, the high transportation cost and also that high inclusions in the diet of the farm animals can affect negatively the flavor of meat and eggs (fishy taint).

Bimbo, A., 2012. Alaska seafood byproducts: potential products, markets and competing products. Anchorage, Alaska: Alaska Fisheries Development Foundation. 277.

Summary

Composts, hydrolyzates, digests and silage must be market driven since they are either very high in water content (silage) or bulky thus making transportation costs a key factor. There is a tendency to interchange hydrolyzates, silage and digest nomenclature. For our purposes, fish solubles is the concentrated stickwater from fishmeal production. Silage is the autolysate or fish digest using the internal enzymes in the fish plus acid for stability. The acid inhibits and destroys the bacteria allowing the internal fish enzymes to digest the fish mass. Cold silage is the product that represents the fish material in liquid form without removal of water or oil. Hot or concentrated or advanced silage involves oil and water removal and evaporation and results in a more concentrated product. If the raw material is low in fat, no oil removal is needed. Fish solubles are sometimes marketed as hydrolyzates or something similar. []A recent headline from Alaska indicates that fertilizers are in short supply and the prices have increased 400% so there could be a market for fish waste in agriculture now. Liquid silages, fish solubles etc. are used as organic fertilizers and have found niche markets for golf courses, the growing of cranberries etc. [] (Table 103-11- present the composition of branded fish silages (e.g. ash content, proteins, energy, amino acidsetc.)) []About 40,000 tons of raw silage is processed with finished products shipped to Norway, Finland, Denmark, France and Holland. A similar co-op set up could be put in place in Alaska as well but this must be market driven. [] SCANBIO SCOTLAND LTD ENSILER EQUIPMENT: manufactures off the shelf silage plants of all sizes and shapes that can be moved from place to place. [] As already mentioned, there is a shortage of fertilizer in Alaska so perhaps silage production could fill that need. []There is very little information available on the price structure for Fish Silage, Hydrolyzates And Fish Solubles. The only information is on the internet and this only reflects retail sales of products in pint and quart

http://seafood.oregonstate.edu/.pdf%20Links/Alaska-Seafood-By-Products-Potential-Products-Markets-and-Competing-Products.pdfhttp://www.norfab.co.uk/enciliers.asphttp://www.norfab.co.uk/enciliers.asp



bottles and 5 gallon pails. Some of the hydrolyzates from France have sold in the US$900+/ton range for early weaned pig and milk replacer diets. However, the early weaned pig market is only 8 weeks out of the life of the pig. Omega Protein is the only company that sells fish solubles as a separate product. Based on their SEC filing, during the period 1998-2007 as shown in the following figure, fish solubles sold in the US$175 $432/metric ton over that period. If we assume that the fish solubles are 50% solids and that conventional cold silage is 20% solids and that the nutrient composition is comparable, we could estimate that over that same period of time, cold crude fish silage would have sold in the $70 - $173/metric ton. []

Coates, J.W., Holbek, N.E., Beames, R.M., Puls, R., O'Brien, W.P., 1998. Gastric ulceration and suspected vitamin A toxicosis in grower pigs fed fish silage. The Canadian Veterinary Journal. 39 (3), 167.

Abstract In 3 feeding trials, gastric ulceration was diagnosed in 2 of 12 lame and recumbent grower pigs fed a diet of 50% fish silage produced from the offal of farmed Atlantic salmon. Premature femoral physeal closure and elevated serum retinyl palmitate levels, features of vitamin A toxicosis, were also observed.

Cameron, C. D. T. Acid fish offal silage as a source of protein in growing and finishing rations for bacon pigs. Canadian Journal of Animal Science 42.1 (1962): 41-48. Abstract

Three factorially designed experiments, involving 136 growing-finishing Yorkshire pigs, were carried out to determine the feeding value of acid-ensiled cod and haddock offal. Rate of gain, feed efficiency and carcass characteristics indicated that this product was a satisfactory source of supplementary protein. However, a moderate off-flavor was detected in the meat from pigs fedfish silage to market weight. The intensity of the off-flavor was not affected by removal of fish silage from the ration of pigs at approximately 170 pounds body weight when slaughtered at 200 pounds. The results from discontinuing the feeding of fish silage when the pigs reached body weights of 100 and 150 pounds on off-flavor in the meat were not conclusive.

Canadian Council on Animal Care 1993. Guide to the care and use of experimental animals. Vol. 1, 2nd ed. Canadian Council on Animal Care, Ottawa, ON.

Collazos, H., Guio, C., 2007. The effects of dietary biological fish silage on performance and egg quality of laying Japanese quails (Coturnix coturnix japonica). World Poultry Science Association, Proceedings of the 16th European Symposium on Poultry Nutrition, Strasbourg, France, 26-30 August, 2007: World's Poultry Science Association (WPSA). 37-40.

Abstract An 8 week experiment was conducted to evaluate the effects of biological fish silage supplementation in laying Japanese quails diets on performance and egg quality. A total of 120, 60 d-old laying japanese quails were allotted in a randomized experimental design with four treatments (Controls, 2, 4 and 6% of biological fish silage), with five replicates and 6 birds per replicate. Diets were formulated to meet or exceed NRC recommendations. Feed and water were supplied ad libitum and light was scheduled for 16 hours of light and 8 hours of dark each day. Feed consumption was measured weekly and feed conversion was calculated. Laying percentage,

http://www.ncbi.nlm.nih.gov/pmc/articles/PMC1539927/pdf/canvetj00149-0041.pdfhttp://www.ncbi.nlm.nih.gov/pmc/articles/PMC1539927/pdf/canvetj00149-0041.pdfhttp://pubs.aic.ca/doi/pdf/10.4141/cjas62-006http://pubs.aic.ca/doi/pdf/10.4141/cjas62-006http://www.cabi.org/Uploads/animal-science/worlds-poultry-science-association/WPSA-france-2007/82.pdfhttp://www.cabi.org/Uploads/animal-science/worlds-poultry-science-association/WPSA-france-2007/82.pdf



egg weight, and egg mass were recorded daily during 8 to 16 wk of age. Random samples of 8 eggs from each treatment were collected weekly to measure egg quality: such as, eggshell thickness, Haugh units, egg specific gravity, and yolk percentage. Productive parameters such as feed intake, egg weight, feed efficiency, body weight variation, and egg mass were not affected (P>0.05), only laying percentage was affected (P0.05) by dietary treatments. Results obtained indicate that biological fish silage can be included in laying diets of Japanese quails up to 6% without adverse effects. Introduction A particular problem in animal nutrition is the lack of quality protein sources with a good amino acid profile, due to availability and relative high cost. Objective Determine the effects of (biological) fish silage supplementation in laying Japanese quails diets on performance and egg quality when supplemented over standard corn and soymeal diet. Materials and Methods Experiment with biological fish silage. Experiment performed on Japanese qualis. Completely Randomized experimental design. 4 treatment groups. 120 quails used. 6 quails per (replicate) cage. 5 (replicate) cages. Each cage was the experimental unit. Metallic cages were used. Initial age of 60 days. The (biologica) fish silage was prepared following FAO procedures (FAO, 1992), from slaughter by-products (heads, guts, remains after deboning) of tilapia (oreochromis spp). Wastes were washed, ,cooked for 15 minutes to reach 91oC, in order to avoid contamination problem, drained and fine grounded (2mm), and added 15% molasses. The fish silage was preserved by mean of lactic acid bacteria (Lactobacillus bulgaricus and Streptococcus thermophilus), the microbial culture was previously prepared, to be added to the substract and molass (5% W:W).The culture microorganisms concentration was of 10 x 108 cfu. The mixture was placed in a incubator at 40oC for 96 hours, in anaerobic conditions. The biological fish silage had 29.10% crude protein and 48.90% Dry Matter. Base ration was based on corn and soybean meal as main ingredients. Diets were formulated to meet or exceed NRC recommendations (NRC 1994) and contained 20% of Crude protein and 2605 Kcal/kg of ME. Control diet (CO): No fish silage Diet 1: 2% biological fish silage Diet 2: 4% biological fish silage Diet 3: 6% biological fish silage Metrics (Performance): Weight gain (Average daily gain); Feed intake (Average ME intake); Feed efficiency (Egg Production): Laying percentage (hen-day egg production (%)?); Egg weight; Egg mass (Egg Characteristics): Eggshell thickness; Haugh units; Egg specific gravity; Yolk percentage



Results (Performance and Egg production): Productive parameters such as feed intake, egg weight, feed efficiency, body weight variation, and egg mass were not affected (P>0.05). The level of fish silage affected egg production (laying percentage), which was higher (77.03%) in controls, and lower in T2 (67.84%). Egg weight, Weight gain, and eggshell thickness, and yolk percentage increased linearly as level of silage increased. (Egg Characteristics): Egg quality parameters were no affected (P>0.05) by dietary treatments Conclusion The results obtained in this experiment showed that biological fish silage supplementation to the diet tended to improve egg weight, weight gain, eggshell thickness, and yolk percentage. Fish silage can be included in laying diets of Japanese quails up to 6% without adverse effects.

National Research Council, 1994. Nutrient requirements of poultry. National Research Council. National Academy Press Washington USA. National Research Council, 1998. Nutrient requirements of swine. National Academic Press, Washington, DC. Dapkevicius, M.L.E., Nout, M.R., Rombouts, F.M., Houben, J.H., Wymenga, W., 2000. Biogenic amine formation and degradation by potential fish silage starter microorganisms. Int. J. Food Microbiol. 57 (1), 107-114. Abstract

Fish waste can be advantageously upgraded into animal feed by fermentation with lactic acid bacteria (LAB). This procedure is safe, economically advantageous and environment friendly. The pH value of the fish pastes decreases to below 4.5 during ensilage. This pH decrease is partly responsible for preservation. Decreased pH values and relatively low oxygen concentrations within the silage facilitate decarboxylase activity. Biogenic amines may constitute a potential risk in this kind of product since their precursor amino acids are present in fish silage. It is of great importance to ensure that the LAB strains chosen for starters do not produce biogenic amines. Some bacteria, among which some LAB species, are able to degrade these metabolites by means of amino oxidases. This could be of interest for fish silage production, to control biogenic amine build-up in this product. Seventy-seven LAB cultures isolated from fish pastes submitted to natural fermentation at two temperatures (15 and 22C) and selected combinations of these isolates were examined for histamine, tyramine, cadaverine and putrescine production. Of the isolates tested, 17% were found to produce one or more of these biogenic amines. The behaviour of diamine oxidase was tested under the conditions present in fish silage. Addition of 12% sucrose or 2% NaCl did not affect histamine degradation. Addition of 0.05% cysteine decreased histamine degradation. Degradation occurred at all temperatures tested (15, 22 and 30C), but not at pH 4.5. Forty-eight potential fish silage starters were tested for histamine degradation in MRS broth containing 0.005 g l1 histamine and incubated at 30C. Indications were found that five of these isolates could degrade as much as 2056% of the histamine added to the medium within 30 h, when used as pure cultures. No histamine degradation was observed with combinations of cultures. Histamine degradation (5054%) by two of these isolates was also observed in ensiled fish slurry.

http://www.researchgate.net/profile/Maria_Dapkevicius/publication/40137267_Biogenic_amine_formation_and_degradation_by_potential_fish_starter_microorganisms/links/0f317533fe186cb58a000000.pdfhttp://www.researchgate.net/profile/Maria_Dapkevicius/publication/40137267_Biogenic_amine_formation_and_degradation_by_potential_fish_starter_microorganisms/links/0f317533fe186cb58a000000.pdf



de Lurdes, M., Dapkeviius, E., Batista, I., Nout, M.R., Rombouts, F.M., Houben, J.H., 1998. Lipid and protein changes during the ensilage of blue whiting (Micromesistius poutassou Risso) by acid and biological methods. Food Chemistry. 63 (1), 97-102. Abstract

Fish waste is a potential source of protein for animal nutrition. Ensilage could provide an advantageous means of upgrading these residues. Careful control of the degree of proteolysis and lipid oxidation is required to produce silages of high nutritional value. This paper studies the changes in lipids and protein during storage (15 days) of acid silages (with 0, 0.25 and 0.43%, w/w, of formaldehyde) and biological silages (with 10 and 20% molasses or dehydrated whey) prepared from blue whiting. A remarkable reduction in protein solubilisation values was achieved by adding formaldehyde. However, formaldehyde addition led to an increase in the peroxide value of the oil extracted from the silages. Ensiling by biological methods seems promising. It yielded both a considerable reduction in protein solubilisation and in basic volatile nitrogen when compared with acid ensilage. In addition, the oil from biological silages had lower peroxide values than the oil from acid silages with added formaldehyde.

DFO-MPO, 1987. Fish Silage Workshop. in: DFO-MPO, ed. Atlantic Fisheries Development. Universit Sainte-Anne, Church Point, Nova Scotia 103. Abstract

This publication contains the proceedings of the Fish Silage Workshop held at Church Point, Nova Scotia, June 16-17, 1987. The workshop was sponsored by the Canadian Department of Fisheries and Oceans under the Fisheries Development Program and attracted about 130 participants. The proceedings contain fourteen papers presented or distributed at the workshop. Included is a review of recent developments in the production and use of fish silage concentrate especially in Norway and two papers by manufacturers of silage processing equipment. Several papers describe feeding trials with trout and salmon, and several domestic animals. Other papers give details of recent activities in Canada's Atlantic provinces, including various pilot plant studies and trials with the use of fish silage for fertilizer. The workshop was designed as an information workshop and no recommendations for future development were formulated.

Enes Dapkevicius, M.L., Nout, M.R., Rombouts, F.M., Houben, J.H., 2007. Preservation of Blue-Jack Mackerel (Trachurus Picturatus Bowdich) silage by chemical and fermentative acidification. Journal of food processing and preservation. 31 (4), 454-468.

Abstract We compared acidified and lactic acid fermented silage approaches for the preservation of blue-jack mackerel. Silages acidified with formic and propionic acids had stable pH (3.8) and low (19 mg/g N) levels of volatile nitrogen compounds (total volatile basic nitrogen, TVBN), but relatively high (82 g/100 g) final non-protein-nitrogen (NPN) values. The silage was fermented with Lactobacillus plantarum LU853, a homofermentative lactic acid bacterium with a high growth (0.51/h) and acidification rate at 37C (optimum temperature), able to grow in the presence of 40 g/L NaCl and to ferment sucrose and lactose. The silages at 37C reached safe pH < 4.5 values within 4872 h, either (F2a) or not (F0), in combination with 20 g/kg salt addition; F2a acidified more rapidly, which may be an advantage for its microbiological stability. Proteolysis resulting in 5359 g NPN/100 g N was lower in fermented than in acidified silages; however, in fermented silages, the levels of TVBN were much higher (5080 mg TVBN/g N) than generally considered acceptable.

http://www.researchgate.net/profile/Maria_Dapkevicius/publication/235635415_Lipid_and_protein_changes_during_the_ensilage_of_blue_whiting_(Micromesistius_poutassou_Risso)_by_acid_and_biological_methods/links/54f5db100cf2ca5efefd3ada.pdfhttp://www.researchgate.net/profile/Maria_Dapkevicius/publication/235635415_Lipid_and_protein_changes_during_the_ensilage_of_blue_whiting_(Micromesistius_poutassou_Risso)_by_acid_and_biological_methods/links/54f5db100cf2ca5efefd3ada.pdfhttp://www.researchgate.net/profile/Maria_Dapkevicius/publication/235635415_Lipid_and_protein_changes_during_the_ensilage_of_blue_whiting_(Micromesistius_poutassou_Risso)_by_acid_and_biological_methods/links/54f5db100cf2ca5efefd3ada.pdfhttp://www.dfo-mpo.gc.ca/Library/103595.pdfhttp://www.researchgate.net/profile/Maria_Dapkevicius/publication/227714675_Preservation_of_blue-jack_mackerel_(Trachurus_picturatus_BOWDICH)_silage_by_chemical_and_fermentative_acidification/links/02e7e517f9c9061d80000000.pdfhttp://www.researchgate.net/profile/Maria_Dapkevicius/publication/227714675_Preservation_of_blue-jack_mackerel_(Trachurus_picturatus_BOWDICH)_silage_by_chemical_and_fermentative_acidification/links/02e7e517f9c9061d80000000.pdf



Groesbeck, C. N., 2003 Use heart girth to estimate the weight of finishing pigs, Kansas State University

Cooperative Extension Service Swine Update Newsletter Spring, 2003.

Haskell, S. R., et al. Flavour studies on pork from hogs fed fish silage. Canadian Journal of Animal Science 39.2 (1959): 235-239.

Jangaard, P., 1987. Fish silage: A review and some recent developments. In Proceedings of Fish Silage Worksho p 8-33. DFO-MPO, ed. Atlantic Fisheries Development. Universit Sainte-Anne, Church Point, Nova Scotia. 103 p. Abstract

A brief summary of various enzymatic processes for preserving fish is given with emphasis on Canadian contributions. Recent developments in Norway are described in some detail as a result of a visit to that country in March 1987. These include research and development work on acid fish silage and silage concentrate and their use as a feed especially for salmon and fur animals.

Summary Introduction

Capital costs for (fish silage production) equipment are considerably lower than for a comparable fish meal plant and there are no odor problems. One disadvantage is that transportation of silage involves large quantities of water, and users should therefore be located as close as possible to the plant. Historical A better word to describe fish silage would perhaps be liquid fish, liquefied fish protein or when more concentrated, protein concentrate. In this report, fish silage is defined as silage produced by adding inorganic and/or organic acids to lower the pH sufficiently to prevent bacterial spoilage. The fish silage becomes liquid because the tissue structures are degraded by a process called autolysis by enzymes naturally present in the flesh. Lactic acid fermentation One reason fish spoils more quickly than flesh or warm blooded animals is that tissues become less acid post mortem in contrast to mammalian tissues. By encouraging the growth of lactic acid bacteria, the spoilagerprocesses leading to the reduction of trimethylamine oxide to trimethylamine and the degradation of amino acids to ammonia by spoilage bacteria are suppressed. Lactic acid bacteria are well-known in dairy products such as yogurt. Although these bacteria are natural inhabitants of fish, they are present in low numbers. Fish also contains only small amounts of free sugar which is the essential substrate for growth of such bacteria (Raa et al., 1983; Mackie et al., 1971). Therefore, to preserve fish or animal waste products by fermentation, it is essential to add a sugar source, preferably with a starter culture of proper lactic acid bacteria which, by rapid conversion of the sugar to acid, preserves the whole mass. A considerable amount of fermentable sugar must be added to obtain a stable silage with a pH around 4; for example, 20 kg of a dry mixture of malt and oatmeal was required for 100 kg of fresh herring (Nelson and Rydin, 1963), or more than 10% molasses (Roa, 1965). [] Both spoilage bacteria and lactic acid bacteria will contribute to the initial acid production because the conditions are anaerobic and sugars are available, but growth of the lactic acid bacteria will be favored as the silage becomes more acidic. If the pH falls to below 4, lactobacilli will become the predominant organism present and harmful bacteria (coliforms, enterococci, typhoid

http://pubs.aic.ca/doi/pdf/10.4141/cjas59-031



bacteria and even spores of Clostridium botulinum) are destroyed in such a silage (Raa et al., 1983). If oxygen is admitted to any extent, then aerobic microorganisms such as yeasts may develop. Yeasts are capable of growth at relatively low pH and utilize carbohydrate and protein. Mold spoilage may also be a problem, especially if any drying occurs, for instance at exposed surfaces (Mackie et al., 1971). A company in Troms, Norway, called BIOTEC Ltd. has developed a protein concentrate aimed at the fur animal market []. The fish is heat treated first to deactivate thiaminase and other enzymes and to stop microbial activity; fat can be removed by a decanter and formic acid, molasses and antioxidants added. When cooled, the lactic acid bacteria and finally a binder meal are added to give the product its desired consistency. It is claimed that the product can be stored for several months, that the lactic acid bacteria also acts as an antioxidant and that the flavor is superior to the bitter taste of acid silage. Acid silage

In 1936, experiments were started in Sweden with the A.I. Virtanen (AIV) process for preservation of fish and fish offal intended for use as animal feeds. Results of the trials were published by Edin (1940) and Olsson (1942). The Swedish experiments included, besides the AIV process (Hydrochloric + sulphuric acids) two other acid preservation methods: the Sulfuric Acid/Molasses Method and the Formic Acid Method (H. Peterson, 1953). The chief advantage of AIV acid over organic acids is its low cost, but this is probably outweighed by the disadvantage in that it is a highly corrosive liquid producing a corrosive product which requires neutralization. Olsson found that formic acid limited the growth of bacteria at a relatively high pH (4.0) as compared to mineral acids like sulphuric acid (pH 2) and that no neutralization was necessary before feeding the silage to animals. Backhoff (1976) found that the enzymes mainly responsible for the liquefaction of fish were those of the gut, skin and other parts of the fish, tather than those of the flesh. Work in Canada on acid fish silage was carried out at the Halifax Technological Station of the Fisheries Research Board of Canada by Freeman and Hooglan (1956, a,b ). It was found that the rate of autolysis increased with temperatures from 15oC to 37oC and reached a maximum after three days at 37oC. Researchers from the Vancouver Technological Station of the Fisheries Research Board of Canada found that liquefaction of the fish in an acid medium was achieved in 72 hours at 37oC. A study by Strasdine and Jones (1983) carried out at the British Columbia Research Council Laboratory on silage from dogfish wastes found established that by adding 1.5% formic acid and heating to 45oC, almost complete liquefaction was achieved in 24 hours. In the 1980s, the Province of Nova Scotia Department of Fisheries supported the construction and operation of a small fish silage plant at Casey Fisheries in Victoria Beach, Nova Scotia. Silage from this plant has been used for feeding trials with pigs at the Agriculture Canada Research Station in Nappan, Nova Scotia.

Acid silage production

Plant and Equipment

It is important for a plant of any size to have at least automated acid addition with a pH meter downline controlling the rate. It is claimed that it is better to stop autolysis soon after liquefaction to cut down on bitter flavors (peptides), fat autolysis (free fatty acids) and complete protein autolysis to amino acids. It might therefore be desirable to have a heat exchanger and holding cell to be able to heat the silage to 85oC or so and hold it to inactivate enzymes. The next step would be to add a decanter/separator to remove the oil from the silage. The last



scenario, and of course the most expensive, would be to have an evaporator to produce silage concentrate. A simplified sketch of a basic silage plant is shown in Figure 4. The raw material is brought to a feeding hopper with a screw feeder on the bottom. Formic acid (and antioxidant) held in tan acid tank of suitable resistant material (fiberglass or plastic coated, etc.) is fed to the fish with a metering pump before the grinder, thereby ensuring good mixing of acid with the fish. The fish should be ground so that no pieces are larger than 3-4 mm in diameter (Tatterson, 1976). The mass is then pumped in a Progressive Cavity Pump (Mono pump), where acid and fish are further mixed. A pH meter in the line adjusts the addition of acid automatically, or stops the plant, if the pH is not in the desired range (3.8 - 4). Example of addition of an evaporation step is at the Royal Seafood Ltd. plant in Bjugn, Norway, the silage is first heated to 95C, passed through a decanter and centrifuge to separate oil and sludge (Sobstad, 1987). The water phase is passed through a flash evaporator at 55C where it cools to 35C, is reheated and flash evaporated again until the solid content reaches 50-55%. A second effect evaporator operating at 35 and lower vacuum makes the system more energy efficient.

The process

The enzymes of importance in silage are various proteinases that break down proteins into peptides and individual amino acids and lipases that break down fats into free fatty acids and glycerol.

Protein changes

A silage gradually liquefies as connective protein tissues are broken down (into peptides and individual amino acids) by enzymes in the fish and become water soluble. This self-digestion is called autolysis and the rate is dependent on the activity of digestive enzymes in the raw material, the physiological condition of the fish when caught, the pH, the temperature and the preservative acids. The enzymes mainly responsible for liquefaction are from the viscera, skin and other parts of the fish other than flesh. The rate of autolysis is temperature dependant, and is quite low at temperatures below 10oC (Figure 15). As autolysis progresses, oil will be liberated and float to the top and bone fragments and undissolved tissues go to the bottom. It is important that a means of stirring the silage in the tank is provided for. There will always remain a fraction of the protein which is resistant to enzymatic digestion, for reasons not completely known. One drawback of acid fish silage is that the product often has a bitter flavour that could have an effect on animal acceptability of the product. Several authors have linked the bitter flavours to certain types of polypeptides formed as the protein molecules are broken down in the autolysis.

Lipid (Fat) changes

Free Fatty Acids (FFA) increase with the storage period, and with the temperature. An antioxidant should be added to the fish silage, in order to limit oxidation of the fat. Common practice in Norway is to add the antioxidant to the formic acid (200 ppm ethoxyquin). An inert gas (C02, N2) could also be used over the silage in the storage tanks. Other antioxidants possible are gallates, hydroxyquinone, BHA, BHT and anisole. If the silage is to be used to feed livestock, it is better to remove the oil as soon as it is feasible and store it separately.



The product

Quality and Analytical methods

For formic acid silage, the pH is recommended to be between 4.0 and 4.3. If pH is above 4.5, there is the danger of bacterial activity, decomposition and perhaps formation of toxic compounds. The pH value is not constant and should be checked regularly, especially when the silage is freshly produced, the temperature is high or the ash or bone content is high. Silage from fish and fish products has a certain buffering action. This means that relatively large quantities of formic acid can be added without the pH dropping correspondingly. It is therefore of interest for the user to find out how many kilos have been used per tonne raw material. When formic acid alone is used, the pH should not be below 3.8. The lower the pH, the better the storage ability. The higher the pH, the less acidic the finished feed will be. There has to be a balance between the two (Pedersen, 1987).

A working group was formed in Norway to establish quality standards for fish silage. The group has recommended that in addition to protein and fat, both ash and dry matter (not fat-free solids) be given. The reason for this is to be able to have a certain control over how well fat and protein analyses were carried out by the laboratories. Since % protein + % fat + % ash = % dry matter, it is then possible to double check if the protein, and especially the fat analyses, are correct. The value for ash will indicate if the silage was made from whole fish, mostly viscera or bony offal. The ash content of whole fish is usually in the 2-3% range.

The concept of the term quality is difficult to define. In commercial terms, it is often limited in the case of fish silage to pH and the contents of protein, fat, dry matter and ash. Total volatile nitrogen (Tot. Vol N) also often is cited, as well as the Trimethylamine nitrogen (TMA-N) and Trimethylamine oxide nitrogen (TMAO-N) content.

Just, A. 1982. The net energy value of balanced diets for growing pigs. Livest. Prod. Sci. 8: 541555.

Kjos, N., Herstad, O., Skrede, A., verland, M., 2001. Effects of dietary fish silage and fish fat on performance and egg quality of laying hens. Canadian Journal of Animal Science. 81 (2), 245-251.

Abstract A total of 45 laying hens were fed a control diet, or one of four diets containing 50 g kg1 fish silage and different levels of fish fat (1.8, 8.8, 16.8 or 24.8 g kg1), to determine the effect of fish silage and fish fat in the diet on performance and egg quality. Fish silage did not affect feed intake, egg production, fatty acid composition of yolk, yolk color or sensory quality of eggs, compared with the control. The diets with 16.8 or 24.8 g kg1 fish fat decreased feed intake (P < 0.001), egg production (P < 0.001), and hen-day egg production (P < 0.04), and increased yolk color index (P < 0.003). The proportions of the fatty acid C22:1 (P < 0.001), and PUFA as the sum of C18:2 n-6, C20:5 n-3, C22:5 n-3 and C22:6 n-3 (P < 0.02) in egg yolk were highest for the fish silage diets with 24.8, 16.8 or 8.8 g kg1 fish fat, and lowest for the diet with 1.8 g kg1 fish fat. Proportions of C18:1 (P < 0.001) and C20:1 (P < 0.001) were lowest for the diets with 16.8 or 24.8 g kg1 fish fat. Egg yolk cholesterol did not differ among treatments. The diet with 16.8 g kg1

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fish fat resulted in a more intense egg albumen whiteness as measured by the sensory study, compared with the other diets (P < 0.05). There was a linear relationship between dietary fish fat level and increased off-taste intensity of egg yolk (P < 0.03). Introduction Krogdahl (1985) reported that hens performed well on diets containing fish silage, without affecting the quality of eggs. Experiments with fish oils in diets for laying hens have shown that the levels of polyunsaturated fatty acids (PUFA) in egg yolk is positively related to the level of fish oil in diets (Van Elswyk et al. 1994; Herstad et al. 2000; Meluzzi et al. 2000). Fish silage contains 35% fat with a high level of PUFA, including the long-chain n-3 fatty acids C22:6 (DHA), C22:5 (DPA) and C20:5 (EPA). Fish silage may, therefore, increase the content of these long-chain PUFA in eggs, and this may affect sensory quality of eggs. However, n-3 enriched eggs may serve as a good source of these fatty acids in human nutrition. It is reported that consuming n-3 fatty acid enriched eggs affects human plasma lipids, thus such eggs may improve human health by reducing the risk of cardiovascular diseases (Hargis et al. 1991; Leskanich and Noble 1997). Objective Determine the effect of (defatted) fish silage and fish fat on performance and egg quality when compared to fish meal. Materials and Methods Experiment with formic acid fish silage, defatted, and fish fat. Experiment performed on laying hens. RCBD experimental design. 5 treatment groups. 45 hens used. 9 (replicate) hen per treatment group. Each hen was the experimental unit. Individual wire cages of 47 X 22.5 cm2 were used. Initial age of 22 weeks. Experiment conducted over two consecutive periods of 28 days (56 days total). The fish silage was prepared same as in Kjos (1999) (from slaughter by-product of farmed Atlantic salmon), except that Ethoxyquin was added as an antioxidant at 250 ppm wet weight. Crude fat as HCl-ether extract was analysed in fish silage and diets according to standard procedures described by the Association of Official Analytical Chemists (1990). Metabolizable energy of the diets was determined according to procedures described by Krogdahl (1985). Thickness of albumen was determined on cracked eggs using a micrometer, and Haugh unit was calculated on the basis of thickness of albumen and egg weight. Yolk color index was evaluated by Roche Yolk Colour Fan, (F. Hoffmann La Roche Ltd., Basel, Switzerland). Cholesterol in egg yolk was determined spectrophotometrically in Encore Chemistry System (Baker Instruments, UK), using Cholesterol Enzumatique PAP 100, kit. Ref. 61 224 from bioMeriedux (France). Sensory analysis was conducted according to international standards (ISO 3972 Sensory analysis Methodology Method of investigating sensitivity of taste).]), using a computerized system for recording of data (Compusense Five, Compusense, Guelph, ON). Base ration was based on barley, oats, maize gluten meal and soybean meal as main ingredients (and fish meal). The diets were designed to meet or exceed the National Research Council requirements for amino acids (NRC 1994). Rendered fat consisting of approximately 70% lard and 30% tallow was used to balance the level of metabolizable energy (ME) in all diets (11.8 MJ ME



kg DM1). Protein from fish silage accounted for 12% of total protein in these treatment diets. Defatted fish silage used contained 36 g kg1 crude fat. Control diet (CO): No fish silage; No fish fat Diet 1: 50 g kg-1 defatted fish silage + 1.8 g kg-1 fish fat (residual) Diet 2: 50 g kg-1 defatted fish silage + 8.8 g kg-1 fish fat Diet 3: 50 g kg-1 defatted fish silage + 16.8 g kg-1 fish fat Diet 4: 50 g kg-1 defatted fish silage + 24.8 g kg-1 fish fat Metrics (Performance): Weight gain (Average daily gain); Feed intake (Average ME intake); Feed-to-gain ratio (feed efficiency measured as kFUp kg1 of gain) (Egg Production): Egg production (g day-1); hen-day egg production (%) (Egg Characteristics): Albumen height; Yolk colour; Cholesterol; Fatty acid composition (Sensory Quality of Eggs): Odour, off-odour, taste, off-taste after 35 days and 7 days refrigerated storage. Results (Performance): Fish silage did not affect feed intake, egg production, fatty acid composition of yolk, yolk color or sensory quality of eggs, compared with the control. Feed intake was highest for diets CO and A, and lowest for diet C (16.8 g fish fat kg1) and diet D (24.8 g fish fat kg1). (Egg Production): In the present study, an inclusion level of 50 g kg1 diet, supplementing 12% of the total protein, had no negative effects on egg production. Egg production and egg weight were highest for diets CO and A, and were significantly depressed when the contents of fish fat were 8.8 g kg1 or higher (diets B, C and D) - high levels of fish fat negatively influence egg production. (Egg Characteristics): The diets with 16.8 or 24.8 g kg1 fish fat increased yolk color index (P < 0.003). The proportions of the fatty acid C22:1 (P < 0.001), and PUFA as the sum of C18:2 n-6, C20:5 n-3, C22:5 n-3 and C22:6 n-3 (P < 0.02) in egg yolk were highest for the fish silage diets with 24.8, 16.8 or 8.8 g kg1 fish fat, and lowest for the diet with 1.8 g kg1 fish fat. Proportions of C18:1 (P < 0.001) and C20:1 (P < 0.001) were lowest for the diets with 16.8 or 24.8 g kg1 fish fat. Egg yolk cholesterol did not differ among treatments. Adding up to 24.8 g kg1 fish fat to the diet causes only minor changes in fatty acid composition of egg yolk when compared with a fish meal based control. No difference was found in egg yolk cholesterol among diets. (Sensory Quality of Eggs): There was a linear relationship between dietary fish fat level and increased off-taste intensity of egg yolk (P < 0.03). To avoid reduced sensory quality of eggs, the fish fat level should be kept below 24.8 g kg1. The diet with 16.8 g kg1 fish fat resulted in a more intense egg albumen whiteness, compared with the other diets (P < 0.05). There were no significant differences in any of the sensory traits between eggs from period 1 (stored at 4C for 35 d) and period 2 (stored at 4C for 7 d). (Overall): High levels of fish fat in the diet caused reduced egg production and egg weight, and tended to cause a modest increase in the level of polyunsaturated omega-3 fatty acids in egg yolk. The reduction in egg production and egg weight observed for the two highest levels of dietary fish fat indicate that fish fat in diets for laying hens should be kept below 17 g kg1. Discussion



(Performance): Krogdahl (1985) found no differences in feed intake, weight gain and egg production of laying hens when herring meal was replaced with fish silage (7.4 or 14.2% fish silage in the diet, respectively). The highest level of dietary fish fat of 17.7 g kg1 tested by Krogdahl (1985) did not influence egg production. Hargis et al. (1991) and Meluzzi et al. (2000) reported that 30 g kg1 dietary menhaden oil did not affect egg production or egg weight. Baucells et al. (2000) found no reduction on performance of laying hens when feeding up to 40 g kg1 of fish oil. Whitehead et al. (1993) observed that egg weight was depressed when fish oil was fed in excess of 20 g kg1, and that feed intake and hen-day egg production (%) was depressed at 60 g dietary fish oil kg1. Van Elswyk at al. (1994) found significant differences in yolk and egg weight when feeding menhaden oil at 30 g kg1. Scheideler and Fr

Duteau 2015. Fish Silage Project Final Report

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