Regulation of acrosome reaction of fowl spermatozoa: evidence for the involvement of protein kinase C and protein phosphatase-type 1 and/or -type 2A.

The signal transduction pathways involved in the regulation of the acrosome reaction and motility of fowl spermatozoa were investigated. The motility and acrosomal integrity of fowl spermatozoa in TES/NaCl buffer, with or without homogenised inner perivitelline layers (IPVL), prepared from laid fowl eggs, was almost negligible at 40 degrees C. In the presence of 2 mmol CaCl(2)/l at 40 degrees C, motility became vigorous and the acrosome reaction was stimulated when IPVL was added. In the absence of Ca(2+), motility was stimulated by the addition of calyculin A and okadaic acid, both specific inhibitors of protein phosphatase-type 1 (PP1) and -type 2A (PP2A), but Okadaic acid, which is a weaker inhibitor of PP1, did not completely restore motility at 40 degrees C. However, the acrosome reaction was significantly and equally stimulated in a dose-dependent manner by both inhibitors in the range of 10-1000 nmol/l, when spermatozoa were incubated with IPVL but without Ca(2+). These inhibitors did not stimulate the acrosome reaction in the absence of IPVL. The vigorous motility of spermatozoa, stimulated by the addition of Ca(2+), was reduced gradually as the concentrations of SC-9, a selective activator of protein kinase C (PKC), were increased and a similar SC-9-induced inhibition was observed in the acrosome reaction in the presence of Ca(2+) and IPVL. These results confirm that IPVL is necessary for the activation of the acrosome reaction in fowl spermatozoa and that Ca(2+) plays an important role in the stimulation of motility and acrosomal exocytosis. Furthermore, it appears that the intracellular molecular mechanisms for the regulation of acrosome reaction of fowl spermatozoa are different from those for the restoration of motility, i.e., protein dephosporylation involving PP1 and/or PP2A in the former, and PP1 alone in the latter case. In addition, the activation of PKC may contribute to a decrease in the flagellar movement and acrosome reaction of fowl spermatozoa.

Effects of calpain and Rho-kinase inhibitors on the acrosome reaction and motility of fowl spermatozoa in vitro.

At the avian body temperature of 40 degrees C, intact fowl spermatozoa require Ca(2+) for the initiation of motility and a combination of both Ca(2+) and homogenized inner perivitelline layer (IPVL) together to induce the acrosome reaction. Within the range of 1-100 micromol/l, neither PD 150606 (a Ca(2+)-dependent calpain inhibitor) nor Y-27632 (an inhibitor of Ca(2+)-dependent Rho-kinase) were able to inhibit the acrosome reaction induced by the presence of Ca(2+) and IPVL. However, PD 150606, although not Y-27632, was able to inhibit sperm motility initiated by Ca(2+), as well as motility initiated by calyculin A -- a specific inhibitor of protein phosphatases, which also initiates sperm motility at 40 degrees C. The addition of PD 150606 did not reduce the ATP concentrations of intact spermatozoa, nor the motility of demembranated spermatozoa. Immunoblot analysis of sperm extract using a polyclonal antibody against calpain 12 revealed a cross-reacting protein of approximately 80 kDa. These results suggest that Rho-kinase is not involved in the regulation of the acrosome reaction or of motility in fowl spermatozoa. In contrast, calpain appears to be involved in the regulation of flagellar movement, but not izn that of the acrosome reaction. Furthermore, it seems that endogenous calpain is present in the cytoplasmic matrix and/or the plasma membrane, but not retained in the axoneme and/or accessory cytoskeletal components.

Protein phosphatase-type 2B is involved in the regulation of the acrosome reaction but not in the temperature-dependent flagellar movement of fowl spermatozoa.

The motility and acrosomal integrity of fowl spermatozoa in TES/NaCl buffer, with or without homogenized inner perivitelline layers (IPVL) prepared from laid fowl eggs, was almost negligible at 40 degrees C. However, motility became vigorous even at 40 degrees C when 2 mmol CaCl2/l was added, and the acrosome reaction was also stimulated in the presence, but not in the absence, of IPVL. The presence of deltamethrin or fenvalerate, specific inhibitors of protein phosphatase-type 2B (PP2B), did not permit the restoration of motility at 40 degrees C but, in the presence of IPVL, these compounds stimulated the acrosome reaction in a dose-dependent manner in the range of 1-1000 nmol/l. These results suggest that IPVL is necessary for the activation of the acrosome reaction in fowl spermatozoa and that Ca2+ plays an important role in the stimulation of motility and acrosomal exocytosis. Furthermore, it appears that the intracellular molecular mechanisms for the regulation of the acrosome reaction of fowl spermatozoa are different from those for the restoration of motility, i.e. protein dephosphorylation by PP2B in the former but not in the latter case.

Weekly monitoring of broiler breeder flock mating efficiency by sperm transfer into eggs.

1. Sample fertility and the median number of points of hydrolysis produced by spermatozoa in the perivitelline layer from the germinal disc area were determined in samples of 60 eggs taken weekly from each of two commercial broiler breeder flocks. 2. Flock fertility remained above 90% from weeks 30 to 45, after which it fell in both flocks, reaching 85% in Flock A by week 51 and 76% in Flock B by week 55. 3. Sample fertility, as assessed by the Kosin test, followed a similar trend, but showed more variation; the same was true for the proportion of eggs with at least one perivitelline hole. 4. In Flock A, the median number of perivitelline holes in samples increased from 145 in week 30 to reach a maximum of 323 on week 39, thereafter falling to 109 in week 51; for Flock B, the equivalent figures for weeks 30, 36 and 55 were 160, 266 and 29, respectively. A quadratic model confirmed that the weekly sample median perivitelline holes peaked at weeks 40 and 37 in Flocks A and B, respectively. 5. The results show that transfer of spermatozoa by males into females and subsequently into eggs begins to decline 8 (Flock A) to 9 (Flock B) weeks before it is noticeable as a significant reduction in flock fertility and that mating efficiency, unlike fertility, is never in apparent equilibrium, but rises to a peak before 40 weeks and then falls. 6. The pattern of sperm transfer suggests that the reduction in fertility of broiler flocks could well be for social or for physiological reasons other than those associated with 'ageing'.

Semen quality in captive Houbara bustard, Chlamydotis undulata undulata.

Semen quality in captive-bred Houbara bustards, Chlamydotis undulata undulata, was assessed during three consecutive breeding seasons. In any one season, sperm quality, in terms of the proportion of eosin-permeable spermatozoa and of spermatozoa with abnormally large nuclei, varied among individual males, but not among their ejaculates. Neither the proportion of spermatozoa with large nuclei, nor those permeable to eosin were related to the total sperm output of males. The fertilizing ability of males was related to their mean seasonal proportion of eosin-permeable spermatozoa, but not the proportion of spermatozoa with large nuclei. The ranking of males on the basis of the proportion of spermatozoa with large nuclei in their ejaculates was significantly positively correlated between seasons, although ranking on the basis of sperm eosin-permeability was not. The cause or consequence of producing spermatozoa with large nuclei (and excess DNA) remains to be elucidated, but appears to be a trait that is characteristic of houbara bustard males that is maintained between breeding seasons.

Assay of sperm quality in the domestic fowl by MTT reduction.

1. An assay which measures the capacity of spermatozoa from the domestic fowl to reduce the colourless tetrazolium dye MTT to its highly coloured purple formazan pigment has been developed and characterised. 2. The assay has several advantages over a previous tetrazolium-based sperm quality assay: it functions without the unstable phenazine methosulphate and toxic cyanide reagents and, following the reductive reaction, the sperm suspension is solubilised to produce optically clear solutions without centrifugation. 3. For samples of semen from individual males, MTT-reduction is strongly correlated with INT-reduction, which has been previously shown to be a useful predictor of sperm fertilising ability.

Application of an MTT reduction assay for assessing sperm quality and predicting fertilising ability of domestic fowl semen.

1. The ability of domestic fowl spermatozoa to reduce MTT tetrazolium to its coloured formazan was compared with other tests of sperm quality and fertilising ability. 2. MTT reduction was highly correlated with sperm ATP content (r2 = 0.85); sperm mobility (r2 = 0.62.); sperm:perivitelline layer interaction (r2 = 0.80) and fertilizing ability (r2 = 0.83). 3. The simple, robust, MTT-reduction assay may therefore be used to select male chickens on the basis of their sperm quality and thus potential fertilising ability.

Assessing the efficiency of mating in broiler breeder flocks by enumerating the spermatozoa which penetrate the inner perivitelline layer over the germinal disc.

1. The frequency distribution of points of hydrolysis produced by spermatozoa in the perivitelline layer from directly over the germinal disc was examined in 60 samples of 60 eggs from commercial broiler breeder flocks. 2. Typically, these distributions were positively skewed, although log transformation of the data revealed 2 populations: one representing eggs which contained no evidence of spermatozoa and another in which the data were, generally, normally distributed. 3. Problem flocks with low fertility had more eggs without evidence of spermatozoa and, compared to control flocks with acceptable fertility, a lower median and mean before and after log transformation, respectively. 4. In 4 flocks studied between 30 to 55 weeks of age, the median number of points-of-hydrolysis in samples of eggs fell from around 200 at peak to less that 20 at 55 weeks, whilst the mean proportion of fertile eggs laid by the whole flocks fell from 94% at peak to around 79% at 55 weeks. 5. A log-linear relationship was demonstrated between flock fertility and the median points-of-hydrolysis from the inner perivitelline layer over the germinal disc in samples of 60 eggs (R2=0.79). 6. The main advantages of this system for measuring sperm-in-eggs are that it is technically simple and presents a more expanded scale than fertility, so that an estimation of whole flock fertility can be derived from a sample of 60 eggs.

Induction of acrosomal exocytosis in chicken spermatozoa by inner perivitelline-derived N-linked glycans.

In birds, the ovum is surrounded by a glycoprotein coat known as the inner perivitelline layer (IPVL), which is analogous to the mammalian zona pellucida and, as such, is the site of initial sperm binding and induction of acrosomal exocytosis (the acrosome reaction). In this study, we demonstrate that oligosaccharides isolated from chicken-IPVL glycoproteins are capable of inducing the acrosome reaction in chicken spermatozoa. Preparations containing only O-linked glycans were unable to induce the acrosome reaction whereas N-linked oligosaccharides released from the IPVL by PNGaseF treatment could induce the acrosome reaction. Addition of galactose to terminal N-acetyglucosamine residues suppressed the acrosome reaction-inducing capacity of the oligosaccharide preparation; however, this capacity could be restored by co-incubation with beta-galactosidase. This evidence suggests that the acrosome reaction-inducing factor is probably an N-linked oligosaccharide with terminal N-acetyl-glucosamine residues.

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.

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.

Localization of calcium and zinc in the sperm storage tubules of chicken, quail and turkey using X-ray microanalysis.

Sperm storage tubules from the utero-vaginal junction of chickens, quails and turkeys were analysed for calcium and zinc using X-ray microanalysis of ultra-rapidly frozen tissue in a scanning electron microscope. This technique enabled the tubular fluid surrounding the stored spermatozoa and the intracellular content of the cells of the sperm storage tubules to be analysed separately and, by using standards with known concentrations, their elemental concentrations were estimated. The mean (+/- SEM) concentration of calcium in the tubular fluid from chickens, quails and turkeys was 17 +/- 3, 19 +/- 3 and 17 +/- 4 mmol kg(-1) wet weight, respectively. The intracellular calcium concentration of the cells of the tubules did not differ significantly from these values and was also similar in the mucosal epithelial cells of the utero-vaginal junction. Zinc was localized in the cells of turkey sperm storage tubules and tubular fluid, but at low concentrations. No zinc could be detected in corresponding structures from chickens and quails. The concentration of calcium in the tubular fluid is within the range known to inhibit the motility of spermatozoa, supporting this function for calcium during storage. Zinc is known to depress turkey sperm metabolism and it may also be involved in inducing quiescence of spermatozoa during storage in this species.

Temperature-dependent inhibition of motility in spermatozoa from different avian species.

This study demonstrates that the pattern of temperature-dependent inhibition of chicken sperm motility at 40 degrees C in vitro, and its release by calcium, is also found in drake spermatozoa and, partially, in turkey spermatozoa. However, no such temperature-dependent inhibition was found in spermatozoa from Japanese quail and Houbara bustard, for which physiological levels of calcium at 40 degrees C had an inhibitory and no effect on sperm motility, respectively. Thus, on the basis of this evidence on the regulation of avian sperm motility in vitro, the hypothesis that oviducal sperm storage tubules might immobilise spermatozoa by providing a calcium-free environment in vivo does not appear to be universally applicable to all species of birds.

Measuring sperm:egg interaction to assess breeding efficiency in chickens and turkeys.

Systems used to measure fertility in poultry have themselves presented a major impediment to progress in maintaining or improving fertility. Generally, these systems have been time-consuming, quantitatively inadequate, or both. A simplistic illustration of the basis of the problem is that if six fertile eggs were laid by a turkey hen during 1 wk after insemination, then all we know is what happened to six sperm: they fertilized the eggs. If 100 million sperm were inseminated, then information on the other 999,999,994 is missing. A better approach for quantitating breeding efficiency is to estimate the numbers of sperm that interact with the egg in the infundibulum. These can be identified in laid eggs, as sperm in the outer perivitelline layer (OPVL sperm), or holes produced by sperm in the inner perivitelline layer (IPVL holes). Eggs can contain up to 250,000 OPVL sperm, so the scale improves on binary estimation of fertilization status. The number of spermatozoa interacting with the perivitelline layer is related to the artificial insemination (AI) dose, the number of oviducal sperm, and the probability of fertilization, not just for one egg, but for subsequent eggs laid by the same hen. Practical applications of sperm:egg interaction measurements include: replacement of fertility trials for evaluation of semen; general fertility evaluation; and monitoring breeding efficiency of commercial turkey and broiler breeders. Furthermore, studies of sperm transfer into eggs raise interesting questions about the efficiency of turkey hens' response to AI or mating frequency of broiler hens in commercial flocks.

Variability in the size of the nucleus in spermatozoa from Houbara bustards, Chlamydotis undulata undulata.

Semen collected from 3-year-old male Houbara bustards contained large proportions (6-40%) of spermatozoa with large nuclei. In these spermatozoa, the length of the nucleus was up to twice the mean length of the nucleus in normal spermatozoa. The lengths of the acrosome, midpiece and flagella were all normally distributed, but the length of the nucleus formed a bimodal distribution. The proportion of spermatozoa with large nuclei varied among males, but not among different semen samples collected from the same male throughout the breeding season. The proportion of motile spermatozoa with large nuclei was half that of normal spermatozoa, but their velocity was significantly greater. After insemination into females, spermatozoa with large nuclei were observed in the outer perivitelline layer of eggs laid, indicating that they were stored and transported within the oviduct and reached the egg at about the time of fertilization. Furthermore, there was no difference in the ability to produce viable progeny in females that were mated with males producing greater proportions of spermatozoa with large nuclei compared with those producing 'normal' spermatozoa. Thus, the abnormal spermatozoa did not appear to impede fertility. There were no signs of triploidy in the males that produced spermatozoa with large nuclei, or in their progeny, as demonstrated by the size of erythrocytes. Therefore, it appears that the spermatozoa with large nuclei were the result of aberrant spermatogenesis.

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.

Quantification of a sperm-egg interaction for estimating the mating efficiency of broiler breeder flocks.

1. The frequency distribution of points of sperm hydrolysis (or holes) produced per unit area of the inner perivitelline layer was examined in samples of approximately 60 laid eggs, taken on the same day from each of 19 flocks of broiler breeder hens. 2. The holes counted in samples of perivitelline layer from eggs varied from 0 to greater than 100; lower numbers being found in eggs from flocks with lower fertility. 3. The median frequency of holes in the inner perivitelline layer was strongly correlated (r = 0.92) with the median frequency of spermatozoa found trapped in the corresponding outer perivitelline layer. 4. The median frequency of holes in the inner perivitelline layer and of spermatozoa in the outer perivitelline layer were both strongly correlated (r = 0.80 and 0.77, respectively) with flock fertility. 5. It is suggested that counting 'holes' in the inner perivitelline layer of laid eggs is a more convenient method for assessing breeding efficiency and predicting flock fertility than counting spermatozoa trapped in the outer perivitelline layer.

Evaluation of semen from individual male domestic fowl by assessment of sperm: perivitelline interaction in vitro and in vivo.

1. Spermatozoa in semen samples from 8 individual male domestic fowls were shown to have a differential and characteristic ability to hydrolyse holes in the inner perivitelline layer from laid eggs in an in vitro assay. 2. The number of holes produced by samples of spermatozoa per unit area of inner perivitelline layer in vitro was linearly correlated with sperm ATP content (r = 0.85) and motility (r = 0.76). 3. The number of holes formed in the inner perivitelline layer in vitro was also linearly correlated with the numbers of holes formed in the inner perivitelline layer of eggs fertilised in vivo, in inseminated hens (r = 0.90); and was correlated logarithmically with the proportion of fertile eggs laid by these hens.

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.