The inner perivitelline layer sperm hole assay: Use of filter paper rings for the isolation of the perivitelline layer overlying the germinal disc and new observations on its morphology

The inner perivitelline layer (IPL) sperm hole assay provides an objective assessment of the number of sperm penetrating the hen's ovum at the germinal disc. That number will provide insight into numerous parameters concerning fertility, including, but not limited to, the probability of the ovum being fertilized (true fertility), the duration of fertility, an assessment of mating activity, the effect of inseminating in vitro-stored semen, and the influence of spiking a breeder flock. In the current paper, we discuss the use of a filter paper ring to isolate and process the IPL overlying the germinal disc (PL disc) in 5 species of domestic birds. The morphology of the PL disc is also described to assist those using the sperm hole assay during sperm hole counting. The filter ring approach was successfully used with chicken, turkey, duck, goose, and Japanese quail eggs; the PL discs remained intact and stretched across the filter ring and sperm holes, when present, were clearly discernible. Morphologically, the PL disc appeared to be a silhouette of the germinal disc, including the germinal vesicle observed in follicular oocytes. The fibers forming the IPL had a honeycomb-like arrangement that could be mistaken for actual sperm holes in faintly stained slides. Given its diversity of applications and relative simplicity using the filter ring, it is recommended that the sperm hole assay be part of a quality control program at the hatchery. Already used in the scientific community, this filter ring method has the additional advantage that the blastoderm may be collected before the staining process.     

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.     

Sperm-egg penetration in laying breeder flocks: a technique for the prediction of fertility.

1. An experiment was conducted to evaluate indices of fertility including the sperm penetration (SP) assay as a technique for the prediction of fertility. Forty-eight males consisting of White Leghorn (WL), New Hampshire (NH), Iraqi Brown (IBr) and Iraqi Barred (IBa) (12 males each) and 64 WL hens were divided at random into 4 groups of 4 replicates of 3 males and 4 females each. 2. At the beginning of each week semen was collected from males and pooled by breed of male. Hens in each breeding group were inseminated once weekly, by breeding group, for 4 consecutive weeks with pooled semen from WL, NH, IBr and IBa males (WLxWL, NHxWL, IBrxWL and IBaxWL). 3. The differences in percentage of dead sperm, acrosomal abnormalities, mass motility, individual motility and spermatocrit between the experimental breeds demonstrated the superiority of WL and NH males in all these quantitative characters of the semen. On the other hand, WL hens inseminated with spermatozoa from NH males had significantly more sperm-egg penetration (SP) holes than WL hens inseminated with spermatozoa from other breeds of males. The breed of males used for insemination affected fertility, hatchability and embryonic mortality. 4. The highest fertility and hatchability and lowest embryonic mortality were observed in eggs laid by hens inseminated with spermatozoa from WL and NH males in comparison with hens inseminated with spermatozoa from Iraqi males. 5. There was a strong positive correlation between SP values and fertility for WLxWL, NHxWL, IBrxWL and IBaxWL. The correlation for all breeds combined was also significant. In addition, SP was also positively correlated with hatchability and negatively correlated with embryonic mortality.     

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.     

Interrelationships Between Apoptosis and Fertility in Bull Sperm

Male fertility, the ability of sperm to fertilize and activate the egg and support early embryogenesis, is vital for mammalian reproduction. Despite producing adequate numbers of sperm with normal motility and morphology, some males suffer from low fertility whose molecular mechanisms are not known. The objective was to determine apoptosis in sperm from high and low fertility bulls and its relationship with male fertility. DNA damage, phosphatidylserine (PS) translocation, and expression of pro- and anti-apoptotic proteins (BAX and BCL-2) in the sperm were determined using TUNEL, Annexin V, and immunoblotting approaches, respectively. Amounts of apoptotic spermatozoa were 2.86 (± 1.31) and 3.00 (± 0.96) in high and low fertility bulls, respectively (P=0.548), and were not correlated with fertility. There was a negative correlation between early necrotic spermatozoa and viable spermatozoa (r = –0.99, P<0.0001). Fertility scores were correlated with live spermatozoa detected by eosin-nigrosin test and necrotic spermatozoa determined via flow cytometry (r = –0.49, P<0.006 and r = –0.266, P<0.0113, respectively). BAX level was higher in low fertile group than high fertile group; however, this difference was not statistically significant due to the variations of bull samples (Bull 1–3 vs. Bull 4–5) in low fertile group (P<0.283). BCL-2 was not detectable in any of the sperm samples. The results shed light onto molecular and cellular underpinnings of male fertility.     

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'.     

Effects of acrosomal conditions of frozen-thawed spermatozoa on the results of artificial insemination in Japanese Black cattle

The purposes of this study were to examine the relationship between male artificial insemination (AI) fertility and sperm acrosomal conditions assessed by new and conventional staining techniques and to identify possible reproductive dysfunctions causing low conception rates in AI using frozen-thawed spermatozoa with poor acrosomal conditions in Japanese Black bulls. We investigated individual differences among bulls in the results concerning (1) acrosomal conditions of frozen-thawed spermatozoa as assessed by not merely peanut agglutinin-lectin staining (a conventional staining technique) but also immunostaining of acrosomal tyrosine-phosphorylated proteins (a new staining technique), (2) routine AI using frozen-thawed spermatozoa as assessed by pregnancy diagnosis, (3) in vivo fertilization of frozen-thawed spermatozoa and early development of fertilized eggs as assessed by superovulation/AI-embryo collection tests and (4) in vitro fertilization of frozen-thawed spermatozoa with oocytes. The percentages of frozen-thawed spermatozoa with normal acrosomal conditions assessed by the abovementioned staining techniques were significantly correlated with the conception rates of routine AI, rates of transferable embryos in superovulation/AI-embryo collection tests and in vitro fertilization rates. These results are consistent with new suggestions that the distribution of acrosomal tyrosine-phosphorylated proteins as well as the acrosomal morphology of frozen-thawed spermatozoa are AI fertility-associated markers that are valid for the prediction of AI results and that low conception rates in AI using frozen-thawed spermatozoa with poor acrosomal conditions result from reproductive dysfunctions in the processes between sperm insemination into females and early embryo development, probably failed fertilization of frozen-thawed spermatozoa with oocytes.     

Review on Effects of Fescue Grass Ergot Alkaloids in the Horse and Preliminary Study on Effect of Fescue Grass Ergot Alkaloid in the Stallion

Feed with ergot alkaloids ingested by horses has a deleterious clinical and economic impact on the industry. The clinical manifestation of the effects in mares is early embryonic mortality, abortions, prolonged gestation, dystocia, thickened (edematous) or retained placental membranes, agalactia, and increased rates of newborn mortality. For the stallion, very little is known, although ergot alkaloids decrease the ejaculate volume. However, a large number of breeding stallions graze endophyte infected (E+) pastures. This is especially true in the southeastern United States, and clinically we do not perceive that any stallions have any defined problems that could be attributed to ergot alkaloids. However, the number of spermatozoa produced by any stallion might mask the effects. The effects on fertility may be more subtle and only evident with sperm cell manipulation such as chilling or freezing. A preliminary study was performed on six breeding stallions fed feed containing infected fescue seed. We were not able to determine any significant (P < .05) detrimental effects on sperm motility, number morphology, and sperm morphology when compared with controls.     

Relationship Between Motility and Mitochondrial Functional Status in Canine Spermatozoa

Inner mitochondrial membrane potential (IMM) is considered a sensitive indicator for the energetic status and motility of spermatozoa. The relationship between sperm motility parameters evaluated by Computer Assisted Sperm motility Analyzer and plasma membrane integrity and IMM assessed by triple staining (PI/SYBR-14 and JC-1) was evaluated in 10 dogs of unknown fertility. Sperm motility showed large variations ranging from 10% to 98%. Proportion of viable sperm cells and of spermatozoa with high IMM ranged from 74% to 99% and from 53% to 87%, respectively. The presence of a high IMM assessed by JC-1 was more strongly correlated to sperm viability ( r  = 1) than to sperm motility ( r  = 0.778). Our results indicate that JC-1 is suitable for detection of IMM changes in canine spermatozoa, but it should always be associated with an objective motility analysis to avoid incorrect evaluation of potential sperm fertility. Ejaculates with a low motility rate showed an unexpectedly high proportion of sperm with high IMM, suggesting that mitochondrial respiration could not be sufficient to support sperm motility, although it may be important for sperm survival in the female genital tract.     

Observations on the patterns of embryonic mortality over the laying season of pheasants.

1. Embryonic mortality of domestic pheasant (Phasianus colchinus) eggs was recorded for a 10-week laying season for two groups of laying hens maintained under mating ratios of 8:1 and 12:1 (females:males). 2. Mating ratio had no significant effect on embryonic mortality between 1 and 4 or 5 and 20 d but values for these categories doubled as the laying season progressed, both rising to 8% to 9% of fertile eggs. 3. Higher embryonic mortality was observed between 21 and 25 d for the eggs from the 8:1 mating ratio (14.5% of fertile eggs compared with 12.1%) but this was unaffected by week of setting. 4. Reasons for these patterns of embryonic mortality were not clear and further research is needed to determine the factors affecting embryonic mortality in pheasants.     

Selection responses in duration of fertility and its consequences on hatchability in the intergeneric crossbreeding of ducks

1. From 1992 to 2003, selected (S) and control lines (C) of the laying Brown Tsaiya duck (Anas platyrhynchos) were simultaneously maintained under the same standardised conditions of feeding and management. 2. The selection objective was to increase the number of fertile eggs after a single artificial insemination (AI) with pooled Muscovy semen. From generations G1 to G11, 2452 and 2022 female ducks, in S and C lines, respectively, were measured and recorded. In the S line, the percentage selected varied between 20.2 and 34.3% in females and between 7.2 and 20.8% in males. 3. Selection for number of fertile eggs had a correlated effect of increasing the parameter tau of the logistic curves which fitted the daily variations (d 2 to 15) in fertility or hatchability on the basis of eggs set. The differences S-C for the estimates of the times of half maximal fertility and hatchability increased by 0.41 and 0.37 d per generation between G1 and G11, respectively. 4. The highest increases of fertility per day rates after a single AI were observed between d 5 and 11. Moreover, in the selected line, fertility rate was higher than, or equal to, 90% in d 2 from G8. The same tendencies were observed for the changes in the evolution of hatchability on the basis of eggs set. 5. Selection increased fertility and hatchability according to the egg set rates, especially for d 2 to 8 after AI. Hatchability of fertile eggs was not impaired, confirming that selection for one AI per week was possible in this strain of laying ducks.     

Evidence of sperm storage in the female ostrich

OBJECTIVE: To determine the length of time following mating that fertile eggs can be laid by an ostrich (Struthio camelus). DESIGN: A clinical reproductive problem in a pair of breeding ostriches provided the opportunity to mate the birds at intervals of 5 to 8 days and assess the fertility of the eggs laid. PROCEDURE: Following prolapse of the phallus of the male ostrich during the breeding season, the pair were immediately separated. The hen was reintroduced to the cock at intervals of 5 to 8 days over a 6 week period for supervised mating. Records were kept of dates of mating and laying, number of eggs laid, egg weights, and fertility determined by candling after 2 weeks incubation. RESULTS: Over the 6 week period, 10 eggs were laid, of which 8 were fertile and 2 infertile. Fertile eggs weighed 1020 to 1285 g (mean 1143). The two infertile eggs weighed 1160 and 925 g. Six fertile eggs were laid 2 to 7 days after mating. The remaining two fertile eggs were laid the same days that mating occurred, suggesting that fertilization resulted from the last matings 5 and 8 days previously, or from earlier matings. CONCLUSION: Sperm storage occurs in ostrich hens and fertile eggs can be laid for at least 5 to 8 days after copulation. Further studies are required to demonstrate the maximum period during which stored sperm are capable of successful fertilization.     

Chromosome analysis of early embryonic mortality in layer and broiler chickens

1. Twenty-three lines of chickens, obtained from grandparent stocks of 4 Australian breeding companies, were analysed to determine the incidence of early embryonic mortality attributable to chromosome abnormalities. The lines included 10 layer strains, consisting of 6 White Leghorn, 2 New Hampshire and 2 Australorp lines, and 13 broiler lines. 2. A total of 10,730 eggs was examined after 3 d incubation; of these 9746 (90.8%) were fertile. Abortive embryonic development was observed in 1379 (14.1%) of the fertile eggs. This consisted of 952 (69.0%) dead and dying embryos, including 646 malformed and 427 (31.0%) membranes without embryos. 3. Early embryonic mortality was found to vary from 9.8 to 26.8% (average 16.4%) in broiler lines and from 8.0 to 27.9% (average 11.9%) in layer lines. 4. Among 898 abortive embryos analysed, 112 had abnormal chromosomes consisting of 27 haploids, 38 haploid-euploids, 24 triploids, 16 diploid-polyploids, 4 aneuploids, 2 tetraploids and 1 translocation. 5. In broilers and layers respectively, chromosome abnormalities were responsible for 4.4 to 28.1% (average 11.8%) and 7.4 to 25.0% (average 13.4%) of the early embryonic mortality. 6. The overall frequency of chromosome abnormalities in all fertile eggs varied from 0.7 to 3.7% for the broiler lines and 0.7 to 3.4% for the layer lines.     

Quantitation of the fertilising ability of fresh compared with frozen and thawed fowl spermatozoa

The fertilising abilities of freshly-diluted and frozen and thawed fowl semen were compared by inseminating a wide range (0.2 to 1600 X 10(6] of doses of spermatozoa. These data demonstrated that there was a non-linear relationship between numbers of spermatozoa and the probability of fertilisation. It was concluded that a typical fertility trial, which assesses the percentage of fertile eggs laid by groups of hens inseminated with a fixed dose of semen, was inadequate for comparing the quality of semen samples. An alternative fertility trial is described, which involves assessing the numbers of spermatozoa required to fertilize a particular percentage of eggs laid. This modified method showed that the quality of frozen and thawed semen, in terms of its fertilising ability, was reduced to 1.6% of that of fresh semen.     

Physiological changes in fowl and turkey spermatozoa during in vitro storage

1. Although early work on semen storage has been rather empirical in approach, only basic research can provide a framework of biological mechanisms from which improvements in the techniques of cryopreservation and liquid semen storage can progress logically. 2. A major drawback in this work has been the lack of adequate tests for quantitating and differentiating aspects of 'fertility'. 3. Basic research has now provided techniques for assessing: sperm fertilising ability in terms of numbers of fertile eggs; the efficiency of hens' oviducts at accepting and retaining spermatozoa, and sperm 'quality' as motility, metabolism and plasma membrane patency. 4. These techniques may be used for a more critical assessment of the effects of both cryopreservation and liquid semen storage on sperm function, although the integrity of sperm surface proteins may be a more sensitive variable which has yet to be measured. 5. Further improvements in sperm cryopreservation technology are best approached through an understanding of the fundamental cellular and molecular changes which take place during freezing; thus far little is known of such changes in avian spermatozoa. 6. The ideal milieu for maintaining spermatozoa in liquid semen storage should mimic the environment of the oviducal sperm storage tubules; elucidation of the factors involved in progressing steadily.     

Sperm Supply and Egg fertilization in the Ostrich (Struthio camelus)

We used egg break‐out and spermatozoa trapped in the perivitelline layer of eggs to test the hypothesis that sperm supply and egg fertilization rate are high in the ostrich. Egg fertilization status was determined at break‐out by the appearance of the germinal disc (GD) and then the perivitelline layer overlying the GD region was collected to count sperm (Sperm_OPVL) under fluorescence following staining with 4′,6′‐diamidino‐2‐phenyindole (DAPI). The study was carried out on commercial ostrich farms over two laying seasons. In the first year, 229 eggs from nine randomly chosen ostrich pens comprising pairs, trios (two females, one male) and larger groups were collected for 1 week of every month of laying. Eggs contained 253 ± 18 Sperm_OPVL/mm² of the GD (mean ± SEM; range 0–1330). Egg fertilization rate averaged 89.4 ± 3.4% and varied from 78.6 to 98.2% between pens. Month had no effect on sperm supply or egg fertilization status. Eggs from paired birds (sex ratio 1 : 1) had less sperm in the GD than the eggs from pens with a higher sex ratio. In the second year, 150 eggs from seven pens, each containing only one male and either one, two or three females, were studied for 2 weeks at the beginning (winter), middle (spring) and end (summer) of laying. Eggs contained 364 ± 45 Sperm_OPVL/mm² of the GD (range 0–2880). Season had no effect on sperm supply or egg fertilization. The number of Sperm_OPVL varied between pens, assumed to be due to variation between individual males. The number of Sperm_OPVL increased as the sex ratio increased only when very high‐ranking males were excluded from the analysis. Egg fertilization rate was 94.4 ± 3.1% but varied from 64.0 to 100% between pens. Egg fertilization was not affected by season or sex ratio. Low fertilization rates were observed in two pens and appeared related to the lack of synchrony between timing of laying and sperm production in the first, and lack of mating in the second pen. We conclude that ostrich flocks generally have high rates of egg fertilization and any infertility is associated with lack of sperm supply.     

Assessment of Sperm Apoptosis in Cryopreserved Bull Semen After Swim-up Treatment: A Flow Cytometric Study

The techniques used to prepare bovine spermatozoa for in vitro fertilization, to enhance the percentage of motile sperm cells include the swim-up (SU) method, among others. The objective of the present study was to evaluate the phosphatidylserine (PS) translocation and plasma membrane integrity as the indicator of apoptosis and necrosis in post-thaw bull sperm after SU treatment using annexin V-fluorescein isothiocyanate (FITC)/propidium iodide (PI) assay. A flow cytometric method was employed to measure apoptosis levels on frozen-thawed bull spermatozoa. The assay detects PS translocation across the plasma membrane using a fluorescein-labelled annexin-V and PI. By using the annexin V/PI assay four different subpopulations of sperm were observed: (i) a population of apoptotic sperm, labelled with annexin V-FITC but not with PI; (ii) a population of early necrotic spermatozoa, sperm labelled with annexin-FITC and PI; (iii) a population of necrotic sperm, labelled with PI but not with annexin-FITC; and (iv) a population of fully viable sperm cells, sperm not labelled with annexin V-FITC and without PI. Results clearly indicated that SU technique itself could have an adverse effect on the spermatozoa membrane stability. It has also been found, significant differences between bulls in the levels of apoptotic sperm, after SU treatment.     

Effect of vaginal douching and different semen extenders on bacterial load and fertility in turkeys

1. A study on artificial insemination of Beltsville Small White turkeys investigated the effect on bacterial load and fertility of vaginal douching with diluents containing Gentamicin 400 microg/ml and different semen extenders. 2. Irrespective of the extenders used, vaginal douching with Gentamicin reduced the microbial load of the vagina with resultant improvement in fertility and hatchability and corresponding reduction in embryonic mortality. 3. Eggs from hens inseminated with semen extended with Beltsville Poultry Semen Extender (BPSE) diluent along with vaginal douching showed a trend towards higher per cent fertility and per cent hatchability of total and fertile eggs set compared to other extenders, though this was non-significant.     

The effect of collection method and housing system on semen production and fertility of Alabio drakes

Three methods of semen collection from Alabio drakes were compared: the use of an artificial vagina (AV), electro ejaculation (EE) and manual massage (MM). For the latter two methods, drakes were housed in individual cages or in groups in floor pens, while for the AV method all drakes were kept in individual cages. Training success rates of drakes for the three collection methods were similar. Drakes housed in cages produced significantly more semen with a higher sperm concentration, which maintained fertility longer than semen from floor-penned drakes. The average semen volume per ejaculate, sperm concentration and total number of sperm per ejaculate were highest for the AV method while the other two methods did not differ. The average duration of fertility was significantly longer for the AV method (8.8 d) than the EE (6.5 d) or MM methods (5.1 d). The proportion of fertile eggs for 7 d following the insemination of 2 X 10(8) spermatozoa was also highest for the AV method. Although greater skill was required from the operator, the AV method is recommended for general use because of its significantly higher semen yield and longer duration of fertility.