Preferential attachment of cock spermatozoa to the perivitelline layer directly over the germinal disc of the hen's ovum.

In vitro incubation of cock spermatozoa with perivitelline layer (PL) from recently ovulated ova of the hen resulted in binding of spermatozoa to the PL and activation of the acrosome reaction. A simple quantitative technique was developed for assessing these events. Following incubation of the PL (0.5 cm2 sections) with spermatozoa, the PL section was rinsed and stained with Schiff's reagent. Microscopic examination revealed holes in the PL that were assumed to be sites of spermatozoa penetration. Utilizing this technique, a correlation was demonstrated between sperm concentration and the number of spermatozoa attaching to the PL and undergoing an acrosome reaction. Pre-treatment of spermatozoa with solubilized PL inhibited spermatozoa binding to pieces of intact PL. The PL overlying the germinal disc and a similarly sized section of PL from another area of the ovum were removed and incubated separately with spermatozoa (1 x 10(5) sperm/100 microliters). Spermatozoa showed preferential attachment and digestion of the PL from the germinal disc area (809 sperm/mm2) as compared to PL from other areas of the ovum (608 sperm/mm2). Spermatozoa attached to the PL in a circular, doughnut-shaped fashion in the area directly over the germinal disc.     

In vitro penetration assay of boar sperm fertility: effect of various factors on the penetrability of immature pig oocytes

The present study was designed 1) to examine the influence of cumulus cells, ovary storage time and oocyte size on the penetrability of immature pig oocytes, and 2) to investigate the effect of 2 methods of treating the semen from different boars on the inter-assay variability of homologous in vitro penetration tests of boar sperm fertility. In Experiment 1, cumulus oocyte complexes, oocytes with spontaneous loss of the cumulus cells during collection, and oocytes mechanically stripped of cumulus cells were used. No differences were observed in oocyte penetrability among the 3 types of oocyte, although mechanical removal of the cumulus caused an increase (P < 0.005) in the degeneration rate compared with the other oocyte types. In Experiment 2, the oocytes were recovered from ovaries kept in PBS (30 degrees C) for 2, 4 or 6 h after slaughter of prepuberal gilts. Ovary storage did not modify the penetrability of oocytes but increased (P < 0.02) their degeneration rates. In Experiment 3, the diameters of fresh oocytes were determined after co-incubation with spermatozoa. They were classified into 4 groups according to diameter: A) < 105 microm, B) 105-115 microm, C) 116-120 microm and D) > 120 microm. Oocytes from Groups C and D exhibited higher (P < 0.05) penetrability than oocytes from the other groups. In Experiment 4, stored, diluted spermatozoa from 4 boars were pretreated by centrifugation at 50 x g for 3 min and subsequent concentration of the supernatants at 1,200 x g for 3 min. The pellets were treated (washed twice and preincubated for 40 minutes) before co-incubation with immature oocytes or used directly as untreated samples (unwashed and non-preincubated). A boar effect (P < 0.001) was evident for the parameters of in vitro penetration, independently of sperm treatment. When the oocytes were inseminated with untreated spermatozoa, the effects of the replicate and the boar-by-replicate interaction on the variability in oocyte penetrability were not significant. The results of this study indicate that the use of standardized immature pig oocytes and stored untreated, diluted spermatozoa can provide a useful method for optimizing the homologous in vitro penetration (hIVP) assay of boar fertility.     

Relative efficacy of conventional sperm parameters and sperm penetration bioassay to assess bull fertility in vitro

Frozen semen samples from 10 bulls were thawed and actively motile sperm recovered using a swim-up technique. Calcium ionophore A23187 at 0.5 microM concentration was used for 1 min to induce the acrosome reaction in the sperm. Mature female golden hamsters were superovulated with 50 IU of equine chorionic gonadotrophin followed 56 h later with 75 IU of human chorionic gonadotrophin. The cumulus mass was recovered 17 h after hCG treatment by puncturing the oviducts in the infundibulum region. Subsequently, cumulus cell mass and zona pellucida were digested by 0.1% hyaluronidase and 0.1% trypsin, respectively, to yield zona-free hamster eggs (ZFE). A sperm penetration bioassay was performed by coincubating capacitated sperm at 5 X 10(6) concentration and ZFE for 3 h at 38 degrees C in an air incubator. The conception rate of the bulls was based of an average of 82.6 cows per bull with pregnancy status confirmed by rectal palpation. It was found to be strongly correlated (p < 0.01, r = 0.723) with fertilization percentage, whereas percent motile sperm, percent viable sperm and percent sperm with intact acrosomes were not significantly correlated with the conception rate (r = 0.210, -0.021 and -0.468, respectively). Results of the present study suggest that the sperm penetration bioassay can be reliably used to test the fertilizing potential of bull sperm in vitro.     

Effects of sperm preparation and bull fertility on in vitro penetration of zona-free bovine oocytes

The objectives of this study were to develop and validate a zona-free bovine oocyte penetration assay for detecting relative differences in bovine sperm fertility and to determine the effect of different sperm preparation methods on oocyte penetration. Oocytes were incubated with heparin-capacitated spermatozoa which either were or were not induced to acrosome-react with lysophosphatidylcholine. Heparin-capacitated spermatozoa treated with lysophosphatidyl-choline penetrated more oocytes and had more penetrations per oocyte than spermatozoa capacitated in heparin but not induced to acrosome-react with lysophosphatidylcholine. Spermatozoa stained with Hoechst 33342, fluorescein isothiocyanate or tetramethyl rhodamine isothiocyanate, alone or in combination, penetrated similar numbers and percentages of zona-free bovine oocytes as the similar to non-stained spermatozoa. When spermatozoa from the same ejaculate were stained with either fluorescein isothiocyanate or tetramethyl rhodamine isothiocyanate and competed in penetrating the same oocytes, the number of penetrations generated by the 2 differently stained spermatozoa was similar. Spermatozoa from bulls of differing in vivo fertilities were labeled with different fluorescent dyes, and their relative abilities to penetrate the same oocytes were assessed. Comparisons between spermatozoa from high and low fertility bulls demonstrated that high fertility spermatozoa had a significant oocyte penetrating advantage over low fertility spermatozoa in 13 of 16 paired competitions. We concluded that the results of the competitive penetration of zona-free bovine oocytes by fluorochrome-labeled spermatozoa from bulls of different fertilities were indicative of their relative in vivo fertility.     

Human sperm injection into the perivitelline space (SI-PVS) of hamster oocytes: Effect of sperm pretreatment by calcium-ionophore A23187 and freezing-thawing on the penetration rate and polyspermy

Calcium-ionophore A23187 and freezing-thawing were used as sperm treatments before human sperm injection into the perivitelline space (SI-PVS) of hamster oocytes. The penetration rate (PR) was higher when SI-PVS was performed with calcium-ionophore-treated (28%) or frozen-thawed (51%) sperm than with untreated sperm (8%). Optimal PR occurred when five calcium-ionophore-treated (57%) or frozen-thawed (71%) sperm were injected under the zona pellucida. When the sperm:egg ratio was 1:1, PR was higher for calcium-ionophore-treated (18.5%) or frozen-thawed (27.8%) sperm than for untreated sperm (0.0%). Calcium-ionophore sperm treatment had no effect on the polyspermic oocyte rate (POR) or the mean number of swollen sperm nuclei per penetrated oocyte (Pd) or per injected sperm (SR). This may result from premature oocyte activation induced by Ca-ionophore. However, POR was higher with frozen-thawed (74%) than with untreated (50%) or Ca-ionophore-treated (50%) sperm. Whatever the sperm treatment, there was a trend toward a lower SR as the number of injected sperm increased. Cytoplasmic regulation of polyspermy in the hamster oocyte is discussed.     

Age and social position moderate the effect of stress on fertility

There is now compelling evidence that psychosocial stress is a cause of reproductive suppression in humans. However, women continue to conceive in the harshest conditions of war, poverty, or famine, suggesting that suppression can be bypassed. The reproductive suppression model (RSM) proposes that natural selection should favor factors that reliably predict conditions for reproduction. In this study, we examine two such factors, age and social position, in women undergoing fertility treatment. We hypothesized that stress-related reproductive suppression would be more likely in younger compared to older women and in women in lower compared to higher social positions. The final sample consisted of 818 women undergoing fertility treatment. Psychosocial stress and sociodemographic data were collected prior to the start of treatment (Time 1), whereas fertility, as indexed by pregnancy or live birth, was assessed 12 months later (Time 2). The results showed that younger women were four times more likely to suppress than older women, and that unskilled and manual workers were more likely to suppress than those in middle social positions (e.g., white collar workers). However, significant associations between stress and fertility were also observed for women in higher social positions (e.g., professionals and executives). The findings provide support for the RSM.     

The effect of glutathione on the motility and fertility of frozen bull sperm

Adding glutathione (GSH, 5 mM) to frozen bull sperm increased the motility of the thawed sperm and decreased the release of aspartate aminotransferase from the sperm. After 3 h of incubation at 37°C, the addition of GSH increased the percentage of normal acrosomes and the motility of the spermatozoa. GSH also increased the fertility of the thawed bull sperm.     

Quantification of spermatozoa in the sperm-storage tubules of turkey hens and the relation to sperm numbers in the perivitelline layer of eggs

This study was conducted to determine the number of spermatozoa residing in the oviduct sperm-storage tubules (SST) and the relationship between these numbers and the number of spermatozoa embedded in the perivitelline layer of oviductal eggs after a single insemination of 200 x 10(6) spermatozoa. The SST of hens inseminated within one week before the expected onset of egg production were filled faster (4 h vs. 2 days) and possessed more spermatozoa (4.1 vs. 2.0 x 10(6)) than the SST of hens inseminated after the onset of egg production. Furthermore, for hens in egg production, significantly fewer spermatozoa were recovered from the SST if the hen was inseminated within 2 h before or after oviposition than if inseminated more than 2 h before or after the oviposition. There was a strong positive correlation between the number of spermatozoa in the SST and the number of spermatozoa embedded in the perivitelline layer of the oviductal eggs (r = 0.85, p less than 0.01). These data show that the population of spermatozoa actually accepted by the SST is quite small relative to the number of spermatozoa inseminated and that maximum sperm-storage is achieved when the hen is inseminated just prior to the onset of egg production. It is suggested that the sperm-storage capacity of the oviduct and the quality of the semen sample can be estimated on the basis of numbers of spermatozoa embedded in the egg perivitelline layer.     

Dietary chromium deficiency effect on sperm count and fertility in rats

Male rats raised on a low chromium diet containing less than 100 ppb chromium had decreased sperm counts and decreased fertility at age 8 months compared to the Cr-supplemented controls. Decreased sperm cell production and fertility were not apparent at age 4 months. At age 7–8 months the frequency of conception was 25 percent or less and the sperm count of the low chromium males was approximately 50 percent of that of the Cr-supplemented rats.     

Neuropeptide Y effect on food intake in broiler and layer chicks

Broiler chicks eat more food than layer chicks. In this study, we examined the involvement of orexigenic peptide neuropeptide Y (NPY) in the difference in food intake between broiler and layer chicks (Gallus gallus). First, we compared the hypothalamic mRNA levels of NPY and its receptors (Y1 and Y5 receptors) between these strains at 1, 2, 4, and 8 days of age. Daily food intake was significantly higher in broiler chicks than layer chicks after 2 days of age. However, the hypothalamic NPY mRNA level was significantly lower in broiler chicks than layer chicks except at 8 days of age. In addition, the mRNA levels of NPY receptors were also significantly lower in broiler chicks than layer chicks at 2 and 4 days of age (Y1 receptor) or 2 days of age (Y5 receptor). These results suggest that the differences in the expressions of hypothalamic NPY and its receptors do not cause the increase in food intake in broiler chicks. To compare the orexigenic effect of NPY between broiler and layer chicks, we next examined the effects of central administration of NPY on food intake in these strains. In both strains, central administration of NPY significantly increased food intake at 2, 4 and 8 days of age. All our findings demonstrated that the increase in food intake in broiler chicks is not accompanied with the over-expression of NPY or its receptor.     

Combined effect of DHA and alpha-tocopherol enrichment on sperm quality and fertility in the turkey

The aim of the present study was to characterize the dietary effects of n-3 LC-PUFA and alpha-tocopheryl acetate (vE) on the quality, phospholipid fatty acid composition, alpha-tocopherol content (alpha-T) and in vitro susceptibility to lipid peroxidation in turkey semen. Fertility of fresh semen was also evaluated. Male turkeys were randomly divided and fed either a control diet or a fish oil and vE rich diet (FO diet) from 40 to 60 weeks of age. The FO diet increased the proportion of n-3 fatty acids in spermatozoa and as a consequence the (n-3)/(n-6) ratio also increased. These changes did not affect the proportion of n-9 PUFAs, particularly of C22:3n-9, in semen. The sperm content of alpha-T was dependent by the dietary supplementation of the vitamin and the sperm content was more than doubled supplying 120 mg kg(-1) of feed to the males compared to the 60 mg kg(-1) of feed in the control diet. In agreement with the major content of alpha-T in spermatozoa collected from the FO group were significantly less susceptible to in vitro induced oxidation. The reproductive capacity of the male breeders was not affected by the diet; however the result is considered of some relevance for field conditions where even very small changes have economic interest being applied to large bird population.     

Selenium content of livers from sex-linked dwarf and normal broiler breeders

Plasma concentrations of cGH, T₃, and T₄ were not different between dwarf and normal broiler breeders. Normal hens had a liver selenium content of 710±35 ng/g, and dwarf hens 656 ±nine ng/g (n=8). Following injections into a wing vein of different doses (1.5, 3, 6, 12, and 24 μg/kg) of the hypothalamic hormone TRH, GH was increased after 15 min. This effect seemed to last longer in dwarf chickens. Plasma concentrations of T₃ increased significantly 1 h after TRH in normal hens, but TRH was ineffective in raising T₃ levels in dwarf animals. The selenium content of livers obtained following decapitation after 2 h was also increased in normal hens up to 902±42 ng/g using the highest dose of TRH (24 μg/kg). This seemed not to be the case for dwarf animals. A much smaller. number of hepatic cGH receptors was also found in dwarf hens, whereas the affinity of the hepatic GH receptor was not influenced by the genotype. It is concluded that the sex-linked dwarf hens are unable to increase their hepatic T₄ into T₃ conversion following a TRH challenge probably because of a deficiency in hepatic GH receptors. The lower content of selenium in dwarfs and their inability to increase its uptake after TRH seem therefore to support the hypothesis that selenium has a direct role in the activity of the 5′-deiodinase complex.     

Relationships between the results of a modified sperm penetration test and a swim-up technique and the fertility of ram semen

Two relatively simple techniques for the objective measurement of semen quality, and which are easy to apply in the field or laboratory, have been developed: a modified sperm penetration test read by the naked eye and a colorimeter-based swim-up technique. Both the modified sperm penetration and swim-up methods produced promising results in prediction of ram fertility; the modified sperm penetration test was correlated (P<0.05) with a 48-day nonreturn rate and a 60-day conception rate; the correlation between a swim-up velocity and the 60-day conception rate approached significance (r=0.483; 0.1>P>0.05).     

Timing of sperm transport, sperm penetration and cleavage in the rat

Times of sperm entry into the oviduct from the uterus, into the ampulla from the isthmus; of sperm penetration into oocytes, and of cleavage, were determined using three mating regions. Time intervals and their errors of estimation were calculated. Spermatozoa were first found in the isthmus of the oviduct no earlier than 15 minutes after coitus, but required four hours to ascend the oviduct to the ampulla. The rate of sperm arrival was equal to the rate of sperm penetration, i.e., about 3 sperms/hour. Time of cleavage in vivo was 20.6 hours after sperm penetration in ad libitum mated animals. In culture, oocytes cleaved at exactly the same time as in vivo. Delaying sperm arrival to the site of fertilization (by delaying mating) shortened the time interval between median time of sperm penetration and median time of cleavage. It was concluded that the time of cleavage of the oocyte reflects primarily the time of sperm penetration, but is also influenced by the postovulatory age of the oocyte.     

The effect of oleanolic acid on sperm motion characteristics and fertility of male Wistar rats

The study was designed to examine the effect of oleanolic acid on cauda epididymal sperm motion using a computer-aided sperm analysis system and to elucidate the relationship between sperm motion and fertility, as a tool for contraceptive studies. Oleanolic acid-polyvinylpyrrollidone suspension was orally administered to adult male Wistar rats for 30 days, followed by a 14-day drug withdrawal from half of the rats in the group. Control rats received only polyvinylpyrrollidone. All males were mated with untreated females. Treated males failed to impregnate females, whereas control and oleanolic acid withdrawn males achieved 100% pregnancies. Sperm motion analysed on the Sperm Motility Quantifier (SMQ) showed significant differences in linearity (P < 0.001) and wobble (P < 0.01) between control and treated groups. However, the curvilinear velocities were not significantly different (P > 0.05) among all the groups. Sperm motility patterns verified differences among kinematic parameters.     

Effects of ethanol ingestion on sperm monosaccharides and fertility

Chronic alcohol abuse is often associated with reproductive disorders. Sperm monosaccharides play an indispensable role in sperm-egg interactions and fertilization. Ethanol (3 g/kg body weight as 25%, v/v) was given by gastric intubation twice daily for 30 days while in another group, rats which had been treated with ethanol were withdrawn from treatment for a further period of 30 days, in order to assess the reversibility of the ethanol-induced effects. Epididymal ethanol content, sperm monosaccharides and the fertility of ethanol treated and ethanol withdrawn rats were assessed. Ethanol ingestion caused a significant decrease in sperm monosaccharides suggesting defective glycosylation of sperm surface proteins. Sperm monosaccharides and fertility were returned to normal following the withdrawal of ethanol. Ethanol-induced changes in sperm monosaccharides may be one of the reasons for the reduced fertility of ethanol treated rats.