WO1999066876A2

WO1999066876A2 - Dietary manipulation to increase freezability and hypothermic storage of sperm and eggs

Info

Publication number: WO1999066876A2
Application number: PCT/IL1999/000351
Authority: WO
Inventors: Amir Arav
Original assignee: Agricultural Research Organization
Priority date: 1998-06-25
Filing date: 1999-06-24
Publication date: 1999-12-29
Also published as: IL125096A; AU4530199A; WO1999066876A3

Abstract

The present invention provides a method for increasing freezability and hypothermic storage of a gamete of an animal, comprising the step of feeding the animal material comprising an element from the group consisting of a source of omega 3, 18:3 omega 3 (LNA), 20:5 omega 3 (EPA), 22:5 omega 3 (DPA) and 22:6 omega 3 (DHA) fatty acids and mixtures thereof, in an amount effective to increase freezability of sperm and oocytes or eggs, by substantially maintaining at least one fertility factor. Furthermore, the present invention provides a dietary supplement to increase freezability and hypothermic storage of animal sperm and oocytes or eggs, comprising an element from the group consisting of a source of omega 3, 18:3 omega 3 (LNA), 20:5 omega 3 (EPA), 22:5 omega 3 (DPA) and 22:6 omega 3 (DHA) fatty acids and mixtures thereof.

Description

DIETARY MANIPULATION TO INCREASE FREEZABILITY AND HYPOTHERMIC STORAGE OF SPERM AND EGGS

FIELD OF THE INVENTION A natural, inexpensive feed supplement that increases freezability and hypothermic storage of sperm and oocytes.

BACKGROUND OF THE INVENTION The influence of dietary fat on membrane lipid composition has been widely investigated in recent years. It has been demonstrated that (i) membrane lipid composition can rapidly and profoundly be modified by the diet and that (ii) many membrane- associated functions can be altered by diet-induced changes in lipid composition (McMurchie, 1988, see Appendix for full references). The phospholipids of spermatozoa are characterised by extremely high proportions of long-chain, highly polyunsaturated fatty acids. In mammals polyunsaturates of the ω-3 series are the major acyl components of these cells, whereas in birds fatty acids of the ω-6 series predominate. There is considerable evidence that such fatty acids play an important, although as yet unspecified role in sperm function since cases of impaired fertility in both mammals and birds have been associated with reduced amounts of these polunsaturates in spermatozoa (Surai et al., 1997).

The composition of phospholipids and phospholipid bound fatty acid esters and aldehydes in ram, bull, boar, human and rabbit spermatozoa has been established. The spermatozoa of these species can be allocated into two groups on the basis of the ratio of the phospholipid-bound polyunsaturated:saturated fatty acids, which can be correlated to cold shock. The ratio for the spermatozoa of ram, bull and boar, which are known to be very susceptible to cold shock is approximately three times the ratio found for the less sensitive spermatozoa of the rabbit and human (Darin-Bennet et al., 1973). The results of experiments on dog and fowl spermatozoa by Darin-Bennet et al., 1974 support their earlier findings that the ratio of polyunsaturated:saturated phospholipid fatty acids can be linked to the susceptibility of spermatozoa to cold-shock.

Cold shock is defined as an irreversible damage expressed shortly after exposure to low, but not freezing temperatures. The primary target of cold shock is thought to be the plasma membrane.

Comparative studies into methods of semen preservation by chilling and freezing in the presence of cryoprotective substances have shown that the success of these procedures is markedly species dependent. As most sperm lipids are incorporated into various types of membrane structures, differences in membrane lipid composition between species are probably responsible for the differential labilities during storage. Holt et al., 1985, disclose results of examination of thermal phase-transition behaviour. There was an indication that the lipids in the immediate vicinity of the calcium stimulated ATPase molecule shows a major thermal phase transition, evidenced by a change in the activation energy of the enzyme reaction, in the region of 23-24°C. Ayala et al., 1977, disclose the effect of 22:6 ω3 provided by dietary fish oil on the development of germinal tissue of rat testes and fatty acid composition of lipids. It was shown that at 7 and 9 weeks of age, development of germinal tissue in rats, which were fed fish oil was normal. The fatty acid composition showed a decrease in 22:5 ω6 acid content and an increase in 22:6 ω3 acid in triacylglycerol, phosphatidylcholine and phosphatidylethanolamine.

Coull et al., 1998, disclose the lipid and fatty acid composition of zona intact sheep oocytes. More than 50% of lipid in the oocytes was phospholipid or triglyceride. Long chain polyunsaturated fatty acids occurred infrequently.

The background art discloses species differences in the freezability and chilling injury resistance of sperm. Freezability of sperm, is defined as the effect of freezing sperm on sperm properties, such as sperm count, motility, viability and fertility. Increasing freezability of sperm, is defined as limiting a negative change due to freezing, on sperm properties, such as sperm count , motility, viability and fertility. Chilling injury resistance of sperm can be defined as the resistance to damage, which occurs medically upon exposure to low non-freezing temperatures. In particular there is a problem with freezing and chilling poultry sperm.

The prior art does not provide an effective method of increasing freezability or chilling injury resistance of sperm, eggs or oocytes, more specifically turkey sperm. There is therefore a need for such a method, which is disclosed herein as the present invention.

SUMMARY OF THE INVENTION The present invention provides a natural, safe, inexpensive feed supplement, which affects fatty acid composition of sperm, and egg membranes and increases the hypothermic storage and freezability of sperm in turkeys. Additionally, the present invention increases sperm and egg hypothermic storage and freezability in all types of poultry, other farm animals and in humans. Moreover, the present invention provides a feed supplement that is easy to use. In a first embodiment, the present invention provides a method for increasing freezability of a gamete of an animal, comprising the step of feeding the animal a ω-3 fatty acid-containing component, in an amount effective to increase freezability of sperm, by substantially maintaining at least one fertility factor.

In a preferred embodiment of a method for increasing freezability of a gamete of an animal the fertility factor is sperm viability.

In a preferred embodiment of a method for increasing freezability of a gamete of an animal the fertility factor is sperm motility.

In a preferred embodiment of a method for increasing freezability of a gamete of an animal the fertility factor is sperm concentration. In a preferred embodiment of a method for increasing freezability of a gamete of an animal the fertility factor is ability to fertilise a gamete from the opposite gender.

In a preferred embodiment of a method for increasing freezability of a gamete of an animal the animal includes an agriculturally useful animal.

In a preferred embodiment of a method for increasing freezability of a gamete of an animal the animal includes poultry. In a preferred embodiment of a method for increasing freezability of a gamete of an animal the animal includes a turkey.

In a preferred embodiment of a method for increasing freezability of a gamete of an animal the animal includes a lower mammal. In a preferred embodiment of a method for increasing freezability of a gamete of an animal the lower mammal is from the ovine species.

In a preferred embodiment of a method for increasing freezability of a gamete of an animal the lower mammal is from the bovine species.

In a preferred embodiment of a method for increasing freezability of a gamete of an animal the animal includes a non-mammalian aquatic animal.

In a preferred embodiment of a method for increasing freezability of a gamete of an animal the animal includes a human.

In a preferred embodiment of a method for increasing freezability of a gamete of an animal the gamete is a sperm. In a preferred embodiment of a method for increasing freezability of a gamete of an animal the gamete is an oocyte.

In a preferred embodiment of a method for increasing freezability of a gamete of an animal the ω-3 fatty acid-containing component is selected from the group consisting of a source of omega 3, 18:3 omega 3 (LNA), 20:5 omega 3 (EPA), 22: 5 omega 3 (DP A) and 22: 6 omega 3 (DHA) fatty acids and mixtures thereof.

In a preferred embodiment of a method for increasing freezability of a gamete of an animal the ω-3 fatty acid-containing component is of plant origin.

In a preferred embodiment of a method for increasing freezability of a gamete of an animal the ω-3 fatty acid-containing component is of animal origin. In a preferred embodiment of a method for increasing freezability of a gamete of an animal the ω-3 fatty acid-containing component is of single cell oil origin.

In a preferred embodiment of a method for increasing freezability of a gamete of an animal the plant source is selected from the group consisting of linseeds and purslane. In a preferred embodiment of a method for increasing freezability of a gamete of an animal the animal source is selected from the group consisting of fish oil and fish meal.

In a second embodiment the present invention provides a method for increasing freezability of an oocyte of an animal, comprising the step of feeding the animal a ω-3 fatty acid-containing component, in an amount effective to increase freezability of the oocyte, by substantially maintaining at least one fertility factor.

In a preferred embodiment of a method for increasing freezability of an oocyte of an animal, the fertility factor is decreased embryo mortality. In a preferred embodiment of a method for increasing freezability of an oocyte of an animal, the fertility factor is oocyte fertility.

In a preferred embodiment of a method for increasing freezability of an oocyte of an animal, the fertility factor is number of eggs.

In a preferred embodiment of a method for increasing freezability of an oocyte of an animal, the fertility factor is quality of eggs.

In a preferred embodiment of a method for increasing freezability of an oocyte of an animal, the fertility factor is hatchability.

In a preferred embodiment of a method for increasing freezability of an oocyte of an animal, the animal includes an agriculturally useful animal. In a preferred embodiment of a method for increasing freezability of an oocyte of an animal, the animal includes poultry.

In a preferred embodiment of a method for increasing freezability of an oocyte of an animal, the animal includes a turkey.

In a preferred embodiment of a method for increasing freezability of an oocyte of an animal, the animal includes a lower mammal.

In a preferred embodiment of a method for increasing freezability of an oocyte of an animal, the lower mammal is selected from the group consisting of animals from the ovine and bovine species.

In a preferred embodiment of a method for increasing freezability of an oocyte of an animal, the animal includes a non-mammalian aquatic animal. In a preferred embodiment of a method for increasing freezability of an oocyte of an animal, the animal includes a human.

In a preferred embodiment of a method for increasing freezability of an oocyte of an animal, the ω-3 fatty acid-containing component is selected from the group consisting of a source of omega 3, 18:3 omega 3 (LNA), 20:5 omega 3 (EPA), 22: 5 omega 3 (DP A) and 22: 6 omega 3 (DHA) fatty acids and mixtures thereof.

In a preferred embodiment of a method for increasing freezability of an oocyte of an animal, the ω-3 fatty acid-containing component is of plant origin.

In a preferred embodiment of a method for increasing freezability of an oocyte of an animal, the ω-3 fatty acid-containing component is of animal origin.

In a preferred embodiment of a method for increasing freezability of an oocyte of an animal, the ω-3 fatty acid-containing component is of single cell oil origin.

In a preferred embodiment of a method for increasing freezability of an oocyte of an animal, the plant source is selected from the group consisting of linseeds and purslane. In a preferred embodiment of a method for increasing freezability of an oocyte of an animal, the animal source is selected from the group consisting of fish oil and fish meal.

In a third embodiment the present invention provides a dietary supplement to increase freezability of animal sperm, comprising an element from the group consisting of a source of omega 3, 18:3 omega 3 (LNA), 20:5 omega 3 (EPA), 22: 5 omega 3 (DP A) and 22: 6 omega 3 (DHA) fatty acids and mixtures thereof.

In a preferred embodiment of a dietary supplement to increase freezability of animal sperm, the animal includes an agriculturally useful animal.

In a preferred embodiment of a dietary supplement to increase freezability of animal sperm, the animal includes poultry.

In a preferred embodiment of a dietary supplement to increase freezability of animal sperm, the animal includes a turkey.

In a preferred embodiment of a dietary supplement to increase freezability of animal sperm, the animal includes a lower mammal. In a preferred embodiment of a dietary supplement to increase freezability of animal sperm, the animal includes a non-mammalian aquatic animal.

In a preferred embodiment of a dietary supplement to increase freezability of animal sperm, the animal includes a human. In a preferred embodiment of a dietary supplement to increase freezability of animal sperm, the source of omega 3 is a plant source.

In a preferred embodiment of a dietary supplement to increase freezability of animal sperm, the source of omega 3 is an animal source.

In a preferred embodiment of a dietary supplement to increase freezability of animal sperm, the source of omega 3 is a single cell oil source.

In a preferred embodiment of a dietary supplement to increase freezability of animal sperm, the plant source is selected from the group consisting of linseeds and purslane.

In a preferred embodiment of a dietary supplement to increase freezability of animal sperm, the animal source is selected from the group consisting of fish oil and fish meal.

In a fourth embodiment the present invention provides a dietary supplement to increase freezability of animal oocytes, comprising an element from the group consisting of a source of omega 3, 18:3 omega 3 (LNA), 20:5 omega 3 (EPA), 22: 5 omega 3 (DP A) and 22: 6 omega 3 (DHA) fatty acids and mixtures thereof.

In a fifth embodiment the present invention provides a method for increasing hypothermic storage of a gamete of an animal, comprising the step of feeding the animal a ω-3 fatty acid-containing component, in an amount effective to increase hypothermic storage of sperm, by substantially maintaining at least one fertility factor. In a preferred embodiment of a method for increasing hypothermic storage of a gamete of an animal, the fertility factor is sperm viability.

In a preferred embodiment of a method for increasing hypothermic storage of a gamete of an animal, the fertility factor is sperm motility. In a preferred embodiment of a method for increasing hypothermic storage of a gamete of an animal, the fertility factor is ability to fertilise a gamete from the opposite gender.

In a preferred embodiment of a method for increasing hypothermic storage of a gamete of an animal, the animal includes an agriculturally useful animal.

In a preferred embodiment of a method for increasing hypothermic storage of a gamete of an animal, the animal includes poultry.

In a preferred embodiment of a method for increasing hypothermic storage of a gamete of an animal, the animal includes a turkey. In a preferred embodiment of a method for increasing hypothermic storage of a gamete of an animal, the animal includes a lower mammal.

In a preferred embodiment of a method for increasing hypothermic storage of a gamete of an animal, the animal includes a non-mammalian aquatic animal.

In a preferred embodiment of a method for increasing hypothermic storage of a gamete of an animal, the animal includes a human.

In a preferred embodiment of a method for increasing hypothermic storage of a gamete of an animal, the gamete is a sperm.

In a preferred embodiment of a method for increasing hypothermic storage of a gamete of an animal, the gamete is an oocyte. In a preferred embodiment of a method for increasing hypothermic storage of a gamete of an animal, the hypothermic storage includes chilling injury resistance.

In a preferred embodiment of a method for increasing hypothermic storage of a gamete of an animal, the ω-3 fatty acid-containing component is selected from the group consisting of a source of omega 3, 18:3 omega 3 (LNA), 20:5 omega 3 (EPA), 22: 5 omega 3 (DP A) and 22: 6 omega 3 (DHA) fatty acids and mixtures thereof.

In a preferred embodiment of a method for increasing hypothermic storage of a gamete of an animal, the ω-3 fatty acid-containing component is a plant source.

In a preferred embodiment of a method for increasing hypothermic storage of a gamete of an animal, the ω-3 fatty acid-containing component is an animal source. In a preferred embodiment of a method for increasing hypothermic storage of a gamete of an animal, the ω-3 fatty acid-containing component is a single cell oil source.

In a preferred embodiment of a method for increasing hypothermic storage of a gamete of an animal, the plant source is selected from the group consisting of linseeds and purslane.

In a preferred embodiment of a method for increasing hypothermic storage of a gamete of an animal, the animal source is selected from the group consisting of fish oil and fish meal.

In a sixth embodiment the present invention provides a method for increasing hypothermic storage of an oocyte of an animal, comprising the step of feeding the animal a ω-3 fatty acid-containing component, in an amount effective to increase hypothermic storage of the oocyte, by substantially maintaining at least one fertility factor.

In a seventh embodiment, the present invention provides a dietary supplement to increase hypothermic storage of animal sperm, comprising an element from the group consisting of a source of omega 3, 18:3 omega 3 (LNA), 20:5 omega 3 (EPA), 22: 5 omega 3 (DP A) and 22: 6 omega 3 (DHA) fatty acids and mixtures thereof.

In a preferred embodiment of the dietary supplement to increase hypothermic storage of animal sperm, the animal includes an agriculturally useful animal.

In a preferred embodiment of the dietary supplement to increase hypothermic storage of animal sperm, the animal includes poultry.

In a preferred embodiment of the dietary supplement to increase hypothermic storage of animal sperm, the animal includes a turkey.

In a preferred embodiment of the dietary supplement to increase hypothermic storage of animal sperm, the animal includes a lower mammal. In a preferred embodiment of the dietary supplement to increase hypothermic storage of animal sperm, the animal includes a non-mammalian aquatic animal.

In a preferred embodiment of the dietary supplement to increase hypothermic storage of animal sperm, the animal includes a human. In a preferred embodiment of the dietary supplement to increase hypothermic storage of animal sperm, the hypothermic storage includes chilling injury resistance.

The term 'hypothermic stability' is defined as the resistance to temperatures lower than physiological temperatures preferably between 0°C and 30°C. A 'lower mammal' can be defined as any non-human mammal. The term 'fertility factors' can be defined as gamete concentration, viability and motility in males and increased hatchability, increased fertility, decreased embryo mortality, increased numberof eggs and increased quality of eggs in females. The term 'a gamete' hereinafter includes both male and female gametes, a sperm and an oocyte or egg respectively. The present invention can also be applied to oocytes or eggs. Throughout the specification where the term sperm is used, oocyte or egg can equally apply. Throughout the specification where the term 'oocyte' is used egg can equally apply. The term 'a source of omega 3' as used herein refers to any animal, plant single cell oil, or synthetic source of omega 3.

BRIEF DESCRIPTION OF THE DRAWINGS The invention is herein described, by way of example only, with reference to the accompanying drawings, wherein:

FIG 1 shows the a graph of the effect of diet composition on chilling sensitivity of torn turkeys' sperm;

FIG 2 shows a graph of the effect of diet composition on the viability of torn turkeys' sperm after freezing and thawing;

FIG 3 shows the effect of fish oil on phase transition of ewe GV oocytes; and FIG 4 shows the effect of fish oil on membrane integrity in semen of bulls.

DETAILED DESCRIPTION OF THE PRESENT INVENTION The present invention provides a dietary supplement, which increases sperm and egg freezability. Without wishing to be bound by a single mechanism, the fatty acids in the composition of the present invention may be absorbed through ingestion as part of the diet and may exert their effect by being incorporated into the sperm or egg membrane and affecting fatty acid composition of the sperm or egg membrane. Sperm or egg concentration may be increased by metabolic or endocrine modification. This incorporation may lead to increased sperm concentration, motility, viability and fertility in males and increased hatchability, increased fertility, decreased embryo mortality, increased number of eggs and increased quality of eggs in females. The fatty acids in the composition may also affect, polyunsaturated fatty acid (PUFA) and long chain fatty acid concentration and the ratio of phospholipids to cholesterol in the plasma membrane.

The dietary supplement is at least one of the following or a mixture thereof of a source of omega 3, 18:3 omega 3 (LNA), 20:5 omega 3 (EPA), 22: 5 omega 3 (DP A) and 22: 6 omega 3 (DHA) fatty acids contained in ω-3 fatty acids. A source of omega 3 is preferably a plant, animal, or single cell oil source. More preferably a source of omega 3 is fish oil, fish meal, linseed and purslane. The composition of the present invention is envisioned as being added to the regular diet of agricultural animals, such as fowl, chicken, turkey, geese, mule ducks, ostrich and other poultry or even lower mammals, non-mammalian aquatic animals and humans. The exact route of administration may depend on the animal e.g. for animals the supplement can be given as part of a feed, for humans the supplement can be given in tablet form (see below for more detailed description of routes of administration) .

Example 1 : Experiment to investigate the effect of ω-3 supplement on chilling sensitivity of torn turkeys' sperm

Materials and Methods

Two groups of torn turkeys were maintained on a commercial diet containing 2830 kcal ME/kg, 9.5% protein, 2.95% fat (mostly from the ingredients and only 0.5% added poultry oil), and semen was collected every three days. The semen was evaluated for motility, viability and membrane integrity. The effect of chilling was tested by exposing the semen to different temperatures between 0 °C and 30 °C. After chilling for fifteen minutes at this temperature, the sperm was rewarmed and evaluated for motility, viability and membrane integrity. The two groups of torn turkeys were then fed a dietary supplement. One group's feed was supplemented with soybean oil (2.5 - 4.0 %), a common fat source in poultry feeds. The feed of the second group was enriched by ingredients supposed to affect the physical characteristics of the cell membranes (2.5 - 4.0 % fish oil, experimental diet). After a week on this diet, sperm was collected every three days for three months and was evaluated. The semen was evaluated for motility, viability and membrane integrity. The effect of chilling was tested by exposing the semen to different temperatures between 0 °C and 30 °C . After chilling for fifteen minutes at this temperat re, the sperm was rewarmed and evaluated for motility, viability and membrane integrity. Sperm concentration was counted using a Mekler chamber and computerised system. Motility was assayed three hours after collection, diluted 1 : 1 with a commercial diluter (GOMET, 1 1) and kept at a temperature between 16 and 22°C. The evaluation was done using a microscope or computerised system. Membrane integrity was determined using fluorescent staining (7.5μg/ml carboxy fluorescent diacetate (CFDA)) and a fluorescent microscope. The stained fluorescent cells were counted and divided by the non-stained cells. The computerised system for evaluating sperm motility (SMI-sperm motility index) and the results from the fluorescent intensity were expressed as: (Post thaw motility / Prefreezing motility) 100. Results The effect of cooling to various temperatures torn turkeys' sperm is shown in figure 1. The two groups (n=15) which were maintained on a commercial diet showed a high chilling sensitivity. After exposure to 16 °C, 35-45% of the sperm maintained membrane integrity (viability). When the torn turkeys were fed soybean oil, the sperm viability factors were slightly improved after chilling. Sperm from torn turkeys fed the ω- 3 dietary supplement did not show any chilling sensitivity and their sperm remained about 90% intact with exposure to any of the temperatures between 0 - 30 °C. Example 2: Experiment to investigate the effect of ω-3 supplement on the freezability of torn turkeys' sperm

Young torn turkeys were fed a basal diet containing 2830 kcal ME/kg, 9.5% protein, 2.95% fat (mostly from the ingredients and only 0.5% added poultry oil). When they entered maturity, three groups, of 15 birds each, were assigned to the experimental groups, while the rest continued on the basal diet (Control). The feed of group 2 was supplemented with soybean oil (2.5 - 4.0 %), a common fat source in poultry feeds. The feed of group 3 was enriched by ingredients supposed to affect the physical characteristics of the cell membranes (2.5 - 4.0 % fish oil, experimental diet). Semen from the three groups were diluted using GOMET solution containing 10% egg yolk and 7% glycerol. The semen were loaded into 5 ml straws (Minitub (Germany)) and frozen using an I.M.T (Alon 1000, IMT, Israel) freezing device at a rate of 300°C/min., to a temperature of -50°C and stored for 3-18 weeks in liquid nitrogen. Thawing was carried out by placing the straws in a water bath at 75°C for 20 seconds and then 30 seconds at 38°C.

Sperm concentration was counted using a Mekler chamber and computerised system. Motility was assayed three hours after collection, diluted 1 : 1 with a commercial diluter (GOMET, 11) and kept at a temperature between 22 and 30°C. The evaluation was done using a microscope or computerised system. Membrane integrity was determined using fluorescent staining (7.5μg/ml carboxy fluorescent diacetate (CFD A)) and a fluorescent microscope. The stained fluorescent cells were counted and divided by the non-stained cells. The computerised system for evaluating sperm motility (SMI-sperm motility index) and the results from the fluorescent intensity were expressed as: (Post thaw motility / Prefreezing motility) 100. Results

Post thaw viability was expressed using three different tests: (1) Motility using a microscope; (2) Sperm motility index (SMI) using a computerised system; and (3) fluorescence retainment by the intact membranes. Very few sperm cells survived freezing and thawing from the control and soybean fed groups. In contrast more than 80% expressed post thaw viability when the sperm was enriched with ω-3 fatty acids from the dietary supplement of the Experimental diet. (Figure 2).

Example 3: Experiment to investigate increasing freezability and cold storage capacity of sperm and eggs in avian species by dietary supplements of ω-3.

Turkey toms: A control group of about 170 young torn turkeys were fed a basal diet containing 2830 kcal ME/kg, 9.5% protein, 2.95% fat (mostly from the feed and only 0.5% added poultry oil). Fifteen toms (treatment group) were fed for 10 weeks with a diet supplemented by 2% fish oil (ω-3 supplement), 4% linseeds and 75 mg/kg vitamin E. The diet was formulated to give an energy: protein ratio similar to the control group. Semen was evaluated for motility after storage at 16-22°C for 8 hours.

Turkey hens: A control group of about 7000 female turkeys were fed a basal diet containing 2830 kcal ME/kg, 9.5% protein, 2.95% fat (mostly from the feed and only 0.5% added poultry oil). One hundred female turkeys were also fed for ten weeks (between week 15 and 25 of lay) with the same ω-3 supplement as described above for the toms (treatment group). Fertility and embryo mortality (on day ten of incubation) was measured daily. Storage of eggs was done at days 4, 17 and 28 at 4°C. The results are shown in Table 1. Results Turkey toms: The lipid profile of semen (data not shown) showed increasing DHA with 0.2 in the control group compared to 4.5% in the treatment group. Motility in sperm collected from the control toms declined by 20% after 8 hours at 16-22°C, while in the sperm from the ω-3 group no decrease in motility was observed (data not shown). The same increase in DHA and sperm motility was seen in the treatment group after 24 hours and after 48 hours at 4°C.

Turkey hens: From Table 1 it can be seen that female mortality was higher by 4% after 28 days of cold storage in the control group. Hatchability was higher in the ω-3 group by 4% compared to the control group for eggs which were stored for 17 days at 4°C. Table 1 :

storage capacity of sperm and eggs in male and female turkeys respectively.

Example 4: Experiment to investigate increasing freezability and cold storage capacity of sperm and eggs in ovine species by dietary supplements of ω-3.

Twenty female lambs aged approximately one month were fed with 4% protected fish oil and vitamin E. An additional eight female lambs were used as a control. Four months later the animals were slaughtered and the ovaries were evaluated for oocyte fluidity and chilling sensitivity. Fluidity was measured by FTIR and cold sensitivity was evaluated using CFDA staining after exposure to 16°C for 15 minutes.

Results

The lipid phase transition of lambs' oocytes showed differences between the control group and treatment group (Figure 3). In figure 3, it can be seen that the lipid phase transition between fluid (2852 cm^" ) to solid state (2851 cm^" ) is much more evident in the control group. The oocytes from the treated group had a flat phase transition at these temperatures, which suggest that they maintained lipid fluidity at these temperatures. Moreover, the oocytes from the treatment group were less cold sensitive than the oocytes from the control group (data not shown). Example 5: Experiment to investigate increasing freezability and cold storage capacity of sperm in bovine species by dietary supplements of ω-3

Four young bulls with low fertility were fed for four months with 4% fish oil and 50 U/kg vitamin E, which were protected using the Ca salt. An additional four bulls with high fertility and two bulls with low fertility were taken as the control group. Blood and semen samples were taken weekly and the lipid profile of the semen was evaluated. Chilling sensitivity of sperm was evaluated using CFDA staining after exposure to 16°C for 15 minutes.

Results The lipid profile showed increasing concentrations of DHA in the semen of the treated bulls. As can be seen from figure 4, the membrane integrity after chilling increased after almost two months of diet with protected fish oil. Full membrane protection was seen by ω-3 after two months of diet while the control bulls remained with low membrane integrity when no supplement was given. It was therefore shown that ω-3 supplements increased freezability and cold storage capacity of sperm in bulls.

Example 6: Possible methods of use

Examples 1 and 2 illustrate a specific method of use. A general method of use is now described.

18:3 omega 3 (LNA), 20:5 omega 3 (EPA), 22: 5 omega 3 (DP A) and 22: 6 omega 3 (DHA) fatty acids contained in ω-3 fatty acids can be administered to a subject in a number of ways, which are well known in the art. Hereinafter, the term 'subject' refers to the turkey or other poultry, lower mammal or human to whom 18:3 omega 3 (LNA), 20:5 omega 3 (EPA), 22: 5 omega 3 (DP A) and 22: 6 omega 3 (DHA) fatty acids contained in ω-3 fatty acids were administered. For example administration may be done orally, topically or parentally.

Compositions for oral administration, which is the preferred route of administration, is in a form that can be added to feed during feeding or before and include powders or granules, suspensions or solutions, in non-aqueous media, sachets, capsules or tablets, ω-3 fatty acids can be used in a protective form to prevent degradation, such as in a protective fat. Thickeners, diluents, flavourings, vitamins, dispersing aids, emulsifiers or binders may be desirable. Dosing is dependent on the responsiveness of the subject to 18:3 omega 3 (LNA),

20:5 omega 3 (EPA), 22: 5 omega 3 (DP A) and 22: 6 omega 3 (DHA) fatty acids contained in ω-3 fatty acids. The amount received by the subject is preferably controlled. For example as part of a diet it would be administered at the time the subject ate, or alternatively as a pill. The amount of the dose and frequency of dosing would be dependent on the responsiveness of the subject. Persons of ordinary skill in the art can easily determine optimum dosages, dosing methodologies and repetition rates.

Example 7: General method of increasing freezability of sperm or oocytes

The following example illustrates a method of increasing sperm or oocyte freezability, characterised by the effect of 18:3 omega 3 (LNA), 20:5 omega 3 (EPA), 22: 5 omega 3 (DP A) and 22: 6 omega 3 (DHA) fatty acids contained in ω-3 fatty acids on the fatty acid composition of sperm or oocyte membranes and is not intended to be limiting.

The method includes the step of adding 18:3 omega 3 (LNA), 20:5 omega 3 (EPA), 22: 5 omega 3 (DP A) and 22: 6 omega 3 (DHA) fatty acids contained in ω-3 fatty acids in an acceptable form to the feed of the subject e.g., by adding directly to the feed of the subject. 18:3 omega 3 (LNA), 20:5 omega 3 (EPA), 22: 5 omega 3 (DP A) and 22: 6 omega 3 (DHA) fatty acids are administered according to an effective dosing methadology. Semen is diluted using a suitable solution. The semen is frozen using a suitable freezing device to a suitable temperature and stored in liquid nitrogen. Thawing is carried out in a suitable device using established rates of thawing, such as in a water bath. Alternatively, eggs or oocytes are put in a suitable medium. The eggs or oocytes are frozen using a suitable freezing device to a suitable temperature and stored in liquid nitrogen. Thawing is carried out in a suitable device using established rates of thawing, such as in a water bath.

Fertility factors, such as sperm concentration, viability, motility and fertility for males or increased hatchability, increased fertility, decreased embryo mortality, increased number of eggs and increased quality of eggs in females can be assayed, using established procedures.

Example 8: General method of increasing chilling stability of sperm

The following example illustrates a method of increasing chilling stability of sperm, characterised by the effect of 18:3 omega 3 (LNA), 20:5 omega 3 (EPA), 22: 5 omega 3 (DP A) and 22: 6 omega 3 (DHA) fatty acids contained in ω-3 fatty acids on the fatty acid composition of sperm membranes and is not intended to be limiting.

The method includes the step of adding 18:3 omega 3 (LNA), 20:5 omega 3 (EPA), 22: 5 omega 3 (DP A) and 22: 6 omega 3 (DHA) fatty acids contained in ω-3 fatty acids in an acceptable form to the feed of the subject e.g., by adding directly to the feed of the subject. 18:3 omega 3 (LNA), 20:5 omega 3 (EPA), 22: 5 omega 3 (DP A) and 22: 6 omega 3 (DHA) fatty acids are administered according to an effective dosing methadology.

Semen is diluted using a suitable solution. The semen is chilled and then rewarmed and evaluated. Fertility factors, such as sperm concentration, viability, motility and fertility can be assayed, using established procedures e.g., such as those described above.

Alternatively in females, eggs or oocytes are placed in a suitable medium. The eggs or oocytes are chilled and then rewarmed and evaluated. Fertility factors, such as increased hatchability, increased fertility, decreased embryo mortality, increased number of eggs and increased quality of eggs can be assayed, using established procedures.

While the invention has been described with respect to a limited number of embodiments, it will be appreciated that many variations and other applications of the invention may be made. APPENDIX

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G.D. Coull, B.K. Speake, M.E. Staines, P.J. Broadbent and T.G. McEvoy,

Theriogenology, Lipids, (1998), Vol. 12, No.12.

W.V. Holt and R.D. North. (1985) J. Reprod. Fert. 73, 285-294.

E.J. McMurchie, (1988) in Advances in Membrane Fluidity, 189-237, Alan R. Liss Inc., New York, NY.

P.F. Surai, E. Kutz, G.J. Wishart, R.C. Noble and B.K. Speake. Journal of Reproduction and Fertility ( 1997) 110, 47-51.

Claims

What is claimed is:

1. A method for increasing freezability of a gamete of an animal, comprising the step of feeding said animal a ╧ë-3 fatty acid-containing component, in an amount effective to increase freezability of sperm, by substantially maintaining at least one fertility factor.

2. The method of claim 1, wherein said fertility factor is sperm viability.

3. The method of claim 1, wherein said fertility factor is sperm motility.

4. The method of claim 1 , wherein said fertility factor is sperm concentration.

5. The method of claim 1 , wherein said fertility factor is ability to fertilise a gamete from the opposite gender.

6. The method of claim 1, wherein said animal includes an agriculturally useful animal.

7. The method of claim 1, wherein said animal includes poultry.

8. The method of claim 1 , wherein said animal includes a turkey.

9. The method of claim 1 , wherein said animal includes a lower mammal.

10. The method of claim 9, wherein said lower mammal is from the ovine species.

11. The method of claim 9, wherein said lower mammal is from the bovine species.

12. The method of claim 1, wherein said animal includes a non-mammalian aquatic animal.

13. The method of claim 1, wherein said animal includes a human.

14. The method of claim 1 , wherein said gamete is a sperm.

15. The method of claim 1 , wherein said gamete is an oocyte.

16. The method of claim 1, wherein said ╧ë-3 fatty acid-containing component is selected from the group consisting of a source of omega 3, 18:3 omega 3 (LNA), 20:5 omega 3 (EPA), 22: 5 omega 3 (DP A) and 22: 6 omega 3 (DHA) fatty acids and mixtures thereof.

17. The method of claim 1, wherein said ╧ë-3 fatty acid-containing component is of plant origin.

18. The method of claim 1, wherein said ╧ë-3 fatty acid-containing component is of animal origin.

19. The method of claim 1, wherein said ╧ë-3 fatty acid-containing component is of single cell oil origin.

20. The method of claim 17, wherein said plant source is selected from the group consisting of linseeds and purslane.

21. The method of claim 18, wherein said animal source is selected from the group consisting of fish oil and fish meal.

22. A method for increasing freezability of an oocyte of an animal, comprising the step of feeding said animal a ╧ë-3 fatty acid-containing component, in an amount effective to increase freezability of the oocyte, by substantially maintaining at least one fertility factor.

23. The method of claim 22, wherein said fertility factor is decreased embryo mortality.

24. The method of claim 22, wherein said fertility factor is oocyte fertility.

25. The method of claim 22, wherein said fertility factor is number of eggs.

26. The method of claim 22, wherein said fertility factor is quality of eggs.

27. The method of claim 22, wherein said fertility factor is hatchability.

28. The method of claim 22, wherein said animal includes an agriculturally useful animal.

29. The method of claim 22, wherein said animal includes poultry.

30. The method of claim 22, wherein said animal includes a turkey.

31. The method of claim 22, wherein said animal includes a lower mammal.

32. The method of claim 31 , wherein said lower mammal is selected from the group consisting of animals from the ovine and bovine species.

33. The method of claim 22, wherein said animal includes a non-mammalian aquatic animal.

34. The method of claim 22, wherein said animal includes a human.

35. The method of claim 22, wherein said ╧ë-3 fatty acid-containing component is selected from the group consisting of a source of omega 3, 18:3 omega 3 (LNA), 20:5 omega 3 (EPA), 22: 5 omega 3 (DP A) and 22: 6 omega 3 (DHA) fatty acids and mixtures thereof.

36. The method of claim 22, wherein said ╧ë-3 fatty acid-containing component is of plant origin.

37. The method of claim 22, wherein said ╧ë-3 fatty acid-containing component is of animal origin.

38. The method of claim 22, wherein said ╧ë-3 fatty acid-containing component is of single cell oil origin.

39. The method of claim 36, wherein said plant source is selected from the group consisting of linseeds and purslane.

40. The method of claim 37, wherein said animal source is selected from the group consisting of fish oil and fish meal.

41. A dietary supplement to increase freezability of animal sperm, comprising an element from the group consisting of a source of omega 3, 18:3 omega 3 (LNA), 20:5 omega 3 (EPA), 22: 5 omega 3 (DP A) and 22: 6 omega 3 (DHA) fatty acids and mixtures thereof.

42. The dietary supplement of claim 41, wherein said animal includes an agriculturally useful animal.

43. The dietary supplement of claim 41 , wherein said animal includes poultry.

44. The dietary supplement of claim 41 , wherein said animal includes a turkey.

45. The dietary supplement of claim 41 , wherein said animal includes a lower mammal.

46. The dietary supplement of claim 41 , wherein said animal includes a non- mammalian aquatic animal.

47. The dietary supplement of claim 41, wherein said animal includes a human.

48. The dietary supplement of claim 41, wherein said source of omega 3 is a plant source.

49. The dietary supplement of claim 41, wherein said source of omega 3 is an animal source.

50. The dietary supplement of claim 41, wherein said source of omega 3 is a single cell oil source.

51. The dietary supplement of claim 48, wherein said plant source is selected from the group consisting of linseeds and purslane.

52. The dietary supplement of claim 49, wherein said animal source is selected from the group consisting of fish oil and fish meal.

53. A dietary supplement to increase freezability of animal oocytes, comprising an element from the group consisting of a source of omega 3, 18:3 omega 3 (LNA), 20:5 omega 3 (EPA), 22: 5 omega 3 (DP A) and 22: 6 omega 3 (DHA) fatty acids and mixtures thereof.

54. A method for increasing hypothermic storage of a gamete of an animal, comprising the step of feeding said animal a ╧ë-3 fatty acid-containing component, in an amount effective to increase hypothermic storage of sperm, by substantially maintaining at least one fertility factor.

55. The method of claim 54, wherein said fertility factor is sperm viability.

56. The method of claim 54, wherein said fertility factor is sperm motility.

57. The method of claim 54, wherein said fertility factor is ability to fertilise a gamete from the opposite gender.

58. The method of claim 54, wherein said animal includes an agriculturally useful animal.

59. The method of claim 54, wherein said animal includes poultry.

60. The method of claim 54, wherein said animal includes a turkey.

61. The method of claim 54, wherein said animal includes a lower mammal.

62. The method of claim 54, wherein said animal includes a non-mammalian aquatic animal.

63. The method of claim 54, wherein said animal includes a human.

64. The method of claim 54, wherein said gamete is a sperm.

65. The method of claim 54, wherein said gamete is an oocyte.

66. The method of claim 54, wherein said hypothermic storage includes chilling injury resistance.

67. The method of claim 54, wherein said ╧ë-3 fatty acid-containing component is selected from the group consisting of a source of omega 3, 18:3 omega 3 (LNA), 20:5 omega 3 (EPA), 22: 5 omega 3 (DP A) and 22: 6 omega 3 (DHA) fatty acids and mixtures thereof.

68. The method of claim 54, wherein said ╧ë-3 fatty acid-containing component is a plant source.

69. The method of claim 54, wherein said ╧ë-3 fatty acid-containing component is an animal source.

70. The method of claim 54, wherein said ╧ë-3 fatty acid-containing component is a single cell oil source.

71. The method of claim 68, wherein said plant source is selected from the group consisting of linseeds and purslane.

72. The method of claim 69, wherein said animal source is selected from the group consisting of fish oil and fish meal.

73. A method for increasing hypothermic storage of an oocyte of an animal, comprising the step of feeding said animal a ╧ë-3 fatty acid-containing component, in an amount effective to increase hypothermic storage of the oocyte, by substantially maintaining at least one fertility factor.

74. A dietary supplement to increase hypothermic storage of animal sperm, comprising an element from the group consisting of a source of omega 3, 18:3 omega 3 (LNA), 20:5 omega 3 (EPA), 22: 5 omega 3 (DP A) and 22: 6 omega 3 (DHA) fatty acids and mixtures thereof.

75. The dietary supplement of claim 74, wherein said animal includes an agriculturally useful animal.

76. The dietary supplement of claim 74, wherein said animal includes poultry.

77. The dietary supplement of claim 74, wherein said animal includes a turkey.

78. The dietary supplement of claim 74, wherein said animal includes a lower mammal.

79. The dietary supplement of claim 74, wherein said animal includes a non- mammalian aquatic animal.

80. The dietary supplement of claim 74, wherein said animal includes a human.

81. The dietary supplement of claim 74, wherein said hypothermic storage includes chilling injury resistance.