Graham J. Wishart

Storage of poultry semen.

Methods of semen collection and artificial insemination (AI) in poultry, requirement for diluents, methods of liquid and frozen storage of avian semen and evaluation of spermatozoa after storage for fertilizing ability are reviewed. Frozen storage of semen from non-domestic birds is also briefly discussed.

Fatty acid composition, glutathione peroxidase and superoxide dismutase activity and total antioxidant activity of avian semen.

This work demonstrates that spermatozoa from five avian species (chicken, turkey, guinea fowl, duck and goose) are all characterised by high proportions of polyunsaturated fatty acids, from 46 (turkey) to 55% (duck) of total. For each of the species, the most abundant fatty acids were arachidonic (20:4n-6) and docosatetraenoic (22:4n-6) acids, representing between 22 (turkey) and 40% (chicken) of total. Significant activities of the major isozymes of superoxide dismutase and glutathione peroxidase, which protect against the peroxidation associated with high degree of fatty acid unsaturation, were found in spermatozoa from all species. The seminal plasma also had these activities and showed additional mechanisms for protecting spermatozoa from peroxidation. In general terms, these lipid and enzyme proteins were similar between the five avian species and different from those reported for mammalian sperm.

Effect of Vitamin E and Selenium Supplementation of Cockerel Diets on Glutathione Peroxidase Activity and Lipid Peroxidation Susceptibility in Sperm, Testes, and Liver

The phospholipids of avian spermatozoa are characterized by high proportions of arachidonic (20:4n-6) and docosatetraenoic (22:4n-6) fatty acids and are therefore sensitive to lipid peroxidation. α-Tocopherol and glutathione peroxidase [GSH-Px] are believed to be the primary components of the antioxidant system of the spermatozoa. The present study evaluates the effect of vitamin E and vitamin E plus Se supplementation of the cockerel diet on GSH-Px activity, vitamin E accumulation, and lipid peroxidation in the spermatozoa, testes, and liver. At the beginning of the experiment 75 Rhode Island Red cockerels were divided into five groups, kept in individual cages, and fed a wheat-barley-based ration balanced in all nutrients. Supplements fed to the different groups were as follows: vitamin E, 0, 20, 200, 20, and 200 mg/kg to groups 1–5, respectively, with groups 4 and 5 also receiving 0. 3 mg Se/kg. The vitamin E supplementation produced increased levels of α-tocopherol in semen, testes, and liver. The inclusion of the Se into the cock diet had a significant (P < 0.01) stimulating effect on GSH-Px activity in seminal plasma, spermatozoa, testes, and liver. The increased vitamin E concentration in the spermatozoa was associated with a reduction in their susceptibility to lipid peroxidation. Similarly, the increased GSH-Px activity provided enhanced protection against lipid peroxidation.

Quantitative aspects of sperm:egg interaction in chickens and turkeys

Spermatozoa embedded in the outer perivitelline layer and points of hydrolysis (holes) produced by spermatozoa in the inner perivitelline layer of chicken and turkey eggs were found to be evenly distributed and linearly correlated (r = 0.80 for both species) throughout the layers from most regions of the egg, except from those directly over the germinal disc, in which there were more holes. In turkey eggs there appeared to be relatively fewer perivitelline spermatozoa, since many had degenerated beyond recognition. In eggs from both species, there were approximately 25 times more holes mm-2 in the inner perivitelline layer from over the germinal disc region than that from other regions of the egg. The relationship between these two frequencies could also be described as linear (r = 0.81 for chicken and 0.78 for turkey eggs), although there was some evidence for a saturation effect for holes over the germinal disc. The fertile status of eggs was shown to be a function of all of the above parameters. Eggs from both species had a 50% probability of being fertile when around 3 spermatozoa penetrated the inner perivitelline layer over the germinal disc and showed maximum fertility when more than 6 spermatozoa penetrated this region. Spermatozoa in the outer perivitelline layer and holes in the inner perivitelline layer from regions other than over the germinal disc could also be used to predict fertility, although with less certainty. Since the number of spermatozoa interacting with the egg reflects the numbers of those stored in the uterovaginal sperm storage tubules, the relationships derived in this work should be useful for understanding how fertility in chickens and turkeys is a function of oviducal sperm storage and transport.

Evidence for a species-specific barrier to sperm transport within the vagina of the chicken hen

Following their insemination into the vagina of chicken hens, turkey spermatozoa did not appear to reach the ovum within the upper magnum or infundibulum and were only occasionally found within the sperm storage tubules at the uterovaginal junction. Turkey spermatozoa were able to populate chicken uterovaginal sperm storage tubules as (or more) efficiently as fowl spermatozoa in uterovaginal junction tissue in vitro. They also populated uterovaginal junction sperm storage tubules in vivo after insemination directly into the uterovaginal region. Thus, a barrier to foreign spermatozoa appears to exist within the vagina of the chicken and not at the level of the uterovaginal junction sperm storage tubules. The nature of this barrier is not known; however it can be shown that while chicken and turkey spermatozoa have similar morphological features and motility characteristics, they have distinct surface antigenicity. Recognition of surface antigenicity by a localised immunological mechanism may be the basis of sperm selection within the hens vagina.

Poultry artificial insemination technology in the countries of the former USSR

A considerable volume of research on fertility in poultry, especially that associated with artificial insemination and related technologies, has been undertaken and published by scientists working ...

PRESENCE OF PROTEIN PHOSPHATASE TYPE 1 AND ITS INVOLVEMENT IN TEMPERATURE-DEPENDENT FLAGELLAR MOVEMENT OF FOWL SPERMATOZOA

Even in the presence of ATP, the motility of demembranated fowl spermatozoa was negligible at the avian body temperature of 40°C. Motility could be restored by the addition of calyculin A, okadaic acid, specific inhibitors of phosphatase type 1 (PP1) and PP‐2A, and inhibitor 1 or inhibitor 2, which are specific inhibitors of protein phosphatase type 1 (PP1). Demembranated spermatozoa, stimulated by calyculin A or okadaic acid, lost their motility following the addition of 1 mM CaCl2, but this was restored gradually by the stepwise addition of EGTA. Immunoblotting of sperm extract using an antibody to PP1 revealed a major cross‐reacting protein of 36–37 kDa, which corresponded to the molecular weight of the known catalytic subunit of PP1. These results suggest that PP1 present in the fowl sperm axoneme may be involved in the inhibition of fowl sperm motility at 40°C via Ca2+‐dependent regulatory systems.

Identification of perivitelline N-linked glycans as mediators of sperm-egg interaction in chickens

This study demonstrates that carbohydrates play an essential role in sperm-egg interactions in birds. Sperm-egg interaction was measured in vitro as the ability of spermatozoa to hydrolyse a small hole in the inner perivitelline layer, the equivalent of the mammalian zona pellucida. Preincubation with Triticum vulgaris lectin (WGA) and succinyl-WGA (S-WGA) at 10 microgram ml(-1) resulted in complete inhibition of sperm-egg interaction, whereas at the same concentration a range of other lectins (Canavalia ensiformis (Con A), Arachis hypogea (PNA), Ulex europaeus II (UEA II), Solanum tuberosum (STA), Tetragonolobus purpureas (LTA) and Pisum sativum (PSA)) were unable to inhibit sperm egg interaction significantly, although fluorescein-labelled derivatives of these lectins were found to stain the inner perivitelline layer. Significant inhibition of sperm-egg interaction was achieved by the addition of N-acetyl-D-glucosamine and fucoidin to the assay mixture; however, D-glucose, D-galactose, D-fucose and L-fucose had no significant effect on sperm-egg interaction. Pretreatment of the inner perivitelline layer with N-glycanase significantly reduced sperm-egg interaction, whereas treatment with O-glycanase had no effect. These results demonstrate that N-linked glycans play an essential role in sperm-egg interaction in chickens.

The effect of pH on the motility of spermatozoa from chicken, turkey and quail

At 40 degrees C in a NaCl-based buffer the motility of spermatoza from chicken, turkey and quail was inhibited at pH values below 7.8, 7.2 and 7.2, respectively. At these pH values the percentage motile and velocity of spermatoza were relatively low, but the motility became vigorous when the pH was raised by 0.2 units and increased even more following further alkalinization. Spermatozoa from all three species stored motionless at pH 6.0 for 3 h could be reactivated by dilution in an alkaline solution (pH 9.0). These findings support the hypothesis that a change in the environmental pH could be implicated in the suppression and stimulation of sperm motility during oviducal sperm storage and transport.

Demonstration that the removal of sialic acid from the surface of chicken spermatozoa impedes their transvaginal migration

Staining of ejaculated chicken spermatozoa with lectin from FITC-conjugated Limulus polyphemus indicated a uniform distribution of terminal sialic acid residues on surface-associated glycoproteins throughout all regions of the spermatozoa. Treatment of spermatozoa with neuraminidase resulted in complete disappearance of this lectin affinity without any visible change in the viability of spermatozoa, as indicated by assessment in vitro of their motility, ATP content and ability to exclude eosin. Neuraminidase-treated spermatozoa were also severely limited in their ability to populate the uterovaginal sperm storage tubules after intravaginal insemination, although they were able to perform this function as well as untreated control spermatozoa after insemination directly into the uterovaginal region. These results demonstrate the presence of a mechanism that recognises sperm surface characteristics which inhibit transport of spermatozoa through the vagina of the chicken hen, but not the entry of spermatozoa into the sperm storage tubules. We hypothesize that cleavage of sperm surface sialic acid residues may increase antigenicity of spermatozoa in the vagina, resulting in their destruction by an immunologically-based sperm-selection mechanism.