Assessment of sperm characteristics post-thaw and response to calcium ionophore in relation to fertility in Swedish dairy AI bulls

The present study examined the relationship between bull sperm characteristics post-thawing, after swim-up, and after challenge to calcium ionophore in relation to fertility (56-d nonreturn rates) after artificial insemination (AI). Spermatozoa from 25 semen batches derived from 15 Swedish Red and White AI bulls were evaluated with regard to post-thaw motility, membrane integrity, and migration through a swim-up procedure. The swim-up separated spermatozoa were assessed in terms of sperm concentration, viability and capacitation status as well as their response to exogenous calcium ionophore (A23187). Acrosome reactions were evaluated by fluorescence microscopy and flow cytometry. Sperm motility and viability post-thawing were significantly correlated with fertility. For the swim-up separated semen, significant correlations to nonreturn rates were found for concentration, viability, number of viable spermatozoa and sperm capacitation status (Pattern F and Pattern B). The only parameter significantly correlated to fertility after the ionophore challenge was the percentage of acrosome-reacted spermatozoa with remaining equatorial fluorescence, as assessed by fluorescence microscopy, but not by flow cytometry. The regression analysis showed that combining the results of sperm membrane integrity assessment post-thawing with those of capacitation status after swim-up provided the best prediction of fertility. The accuracy of prediction did not improve when these parameters were combined with the percentage of spermatozoa in which acrosome reaction was induced by ionophore challenge.     

A novel automated fluorometric assay to evaluate sperm viability and fertility in dairy bulls

The artificial insemination (AI) industry is in need of an objective and rapid, but inexpensive method to evaluate frozen thawed bull semen ejaculates. This study presents a new fluorescence method that uses an automatized fluorometer and fluorophore stain propidium iodide that stains only those cells with damaged membranes. The fluorescence of the semen sample and the totally killed subsample were measured simultaneously, and viability was calculated. Every semen batch was analyzed before use in AI. For fertility evaluation, the nonreturn rates (NR%) obtained from 92,120 inseminations with the analyzed batches were recorded from 166 bulls (436 batches). This study confirms a 3.9% better NR% for the Finnish Holstein-Friesian breed than for Finnish Ayrshire. There was a clear seasonality in NR%: it differed (5.3%) significantly, being best in summer to autumn (June to October) and lowest in winter (January to March). The fluorometer method was fast and easy. The correlation between the total number of viable spermatozoa in an insemination dose and field fertility was low but significant (r = 0.051, P = 0.016), suggesting that the plasma membrane integrity evaluation can serve as a cost-beneficial quality control method of frozen-thawed semen at bull stations.     

Assessment of sperm quality through fluorometry and sperm chromatin structure assay in relation to field fertility of frozen-thawed semen from Swedish AI bulls

We investigated fluorometry to study sperm viability and flow cytometry to study sperm chromatin structure. We also assessed sperm quality after thawing relative to field fertility after AI as shown by 56-day non-return rates (56-d NRR) Frozen-thawed semen samples were obtained from 20 Swedish Red and White bulls (1 to 3 semen batches/bull) and the fertility data were based on 6,369 AIs. Fluorometry enabled simultaneous detection of sperm viability and concentration in Hoechst 33258-stained semen samples. Sperm chromatin structure assay (SCSA) evaluated denaturability of sperm nuclear DNA in situ after acid treatment. The intensity of fluorescence in non-permeabilized samples was negatively (r = -0.60, P < 0.001) correlated with microscopically-assessed sperm viability, and the fluorescence of permeabilized semen samples significantly (r = 0.67, P < 0.001) correlated with sperm concentration as assessed by hemocytometry. From the fluorescence output, the calculated percentage of damaged cells was negatively (r = -0.71, P < 0.001) correlated with the number of live cells derived from the microscopic assessment of sperm viability and concentration. This variable was significantly correlated with fertility results both at batch (r = -0.39, P < 0.05), and bull (r = -0.57, P < 0.01) levels. The SCSA variables SDalphat and COMPalphat were significantly (r = -0.59-0.64, P < 0.001) correlated with sperm viability variables after thawing but only the COMPalphat correlated significantly (r = -0.53, P < 0.05) with fertility results and solely at the bull level. The results indicate that fluorometric assessment is in good agreement with other practiced procedures and can be performed with sufficient accuracy. The SCSA may be a valuable complement for routinely practiced microscopic evaluation of sperm morphology of AI bull semen     

Assessment of sperm quality traits in relation to fertility in boar semen

Background  

Several studies have been published where sperm plasma membrane integrity correlated to fertility. In this study we describe a simple fluorometer-based assay where we monitored the fluorescence intensity of artificially membrane-ruptured spermatozoa with a fixed time staining with fluorescent DNA dyes.     

In vitro assessment of sperm from bulls of high and low field fertility

The aim of this study was to investigate the reasons for differences in field fertility of bulls following insemination with frozen-thawed semen. The study was carried out in two separate parts over two years and comparisons were made between 5 high and 4 low fertility Holstein Friesian bulls as determined by their either 90 day non-return rate (Year 1) or calving rate (Year 2). Two high fertility Limousin bulls were included in Year 1 for comparative purposes. The ability of sperm from each bull to penetrate artificial mucus was assessed (Year 1 = 7 replicates; Year 2 = 5 replicates). Glass capillary tubes (2 per bull per replicate) were filled with artificial mucus and incubated with sperm stained in 1% Hoechst 33342 for 30 min at 37 °C. The number of sperm were subsequently counted at 10 mm intervals along the tube between 40 and 80 mm markers. Sperm mitochondrial activity of each bull was assessed by the MTT (3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyl-tetrazolium bromide) assay (4 replicates in each year). Sperm were incubated with MTT for 1 h at 37 °C following which the absorbance of formazan was read using a spectrophotometer. Sperm viability after thawing was assessed for each bull using a live/dead sperm viability kit (Year 1 = 3 replicates; Year 2 = 4 replicates). A minimum of 250 cells were assessed per bull in each replicate and classified as either live or dead. Finally, the ability of sperm to fertilize oocytes in vitro and their ability to develop to blastocyst stage embryos were assessed (5 replicates in each year involving 220 to 306 oocytes per bull). Data transformation to normalize residuals was required for mucus sperm penetration (square root) and IVF (cleavage and blastocyst rate) results (arcsin). The mean number of sperm counted at each 10 mm mark between 40 and 80 mm was higher in the high fertility (56.0; 95% CI 39.5 to 75.3) compared to the low fertility (42.9; 95% CI 29.3 to 59.1) Holstein Friesian bulls but the difference did not reach formal significance (P = 0.09). Fertility status had no effect on the ability of sperm to reduce MTT to formazan (mean absorbance 0.34 ± 0.051 and 0.30 ± 0.044) or on the percentage of live sperm per straw (mean 47.3 ± 5.47 and 32.4 ± 4.66) for high and low fertility Holstein Friesian bulls respectively. Oocyte cleavage rate following insemination with sperm from high fertility Holstein Friesian bulls was significantly higher than with sperm from low fertility Holstein Friesian bulls [76.7% (95% CI 60.9 to 89.4) and 55.3 (95% CI 40.4 to 69.7) respectively, P = 0.04]. There was no significant effect of bull fertility on blastocyst rate [34.7% (95% CI 21.1 to 49.6) and 24.2 % (95% CI 14.1 to 36.0) for the high and low fertility Holstein Friesian bulls, respectively; P = 0.2]. In conclusion, sperm from high fertility bulls tended to be more effective in penetrating artificial mucus and to have an increased ability to fertilize oocytes in vitro; however, once fertilization occurred subsequent embryo development was not significantly affected by fertility status.     

Relationship between heparin binding to spermatozoa and the fertility of dairy bulls

The presence of heparin in in vitro media has been implicated in improved fertility parameters of bull spermatozoa. In a previous study, Zhang et al. (25) obtained an estimate of bull nonreturn rates based on spermatozoal concentration, motility and zona pellucida binding (24). The objective of this study was to test for a relationship between fertility parameters previously estimated for the same batch of cryopreserved semen (25) and amount of heparin bound to spermatozoa. 3H-heparin binding to spermatozoa was assessed by radioimmunoassay, and statistical correlations were drawn to previously measured sperm characteristics. Preliminary experiments established optimal binding conditions of 25 degrees C, and 60 min incubation with 3H-heparin at a concentration of 50,000 cpm. 3H-heparin bound to an average of 2.2 x 10(6) receptors/cell with a Kd of 2.0 x 10(-7) M. The total 3H-heparin bound to spermatozoa from different bulls was significantly different (P<0.003). However, the total 3H-heparin bound to spermatozoa was not correlated with any measured sperm parameter, including zona pellucida binding, embryo cleavage and blastocyst formation, and 56-day nonreturn rates (P>0.19). Thus, the total amount of heparin bound to the surface of spermatozoa may not be relevant to fertilizing ability.     

Sperm morphology and fertility of progeny-tested AI dairy bulls in Sweden

Use of bull semen with high levels of sperm abnormalities, reflecting genital dysfunction, is not recommended for artificial insemination (AI) since it would most likely lead to subfertility. Sperm quality, including sperm morphology, may deteriorate with increasing age of the bull thus becoming a source of concern when using older, progeny-tested AI bull sires. Although a relationship between sperm morphology and fertility after AI in progeny-tested bull sires has been reported, it is yet unclear which sperm abnormalities are most critical. This constituted the core aim of a 22-month long retrospective study in proven (aged 60-84 months at the start of the study) AI sires of the Swedish Red (SR, n=8) and Swedish Holstein (SLB, n=4) breeds where their semen (107 freezing batches in total, built by a single ejaculate (n=3) or pooling two consecutive ejaculates (n=104) collected at 1-3 months interval), were subjected to detailed morphological examinations on wet- and dry, stained smears. Attention was paid to between- and within-bull variations with regard to presence and level of sperm abnormalities. Sperm morphology differed significantly between sires and ejaculates, with 6/12 sires having ejaculates containing >10% of morphologically deviating sperm head shapes, a commonly used threshold for young AI bulls in Sweden. However, with the exception of pear-shaped or narrow-at-the-base anomalies, the mean values for individual defects were always within the limits expected for a normal bull sire, and were therefore considered acceptable. The percentage of morphologically normal spermatozoa was positively related to fertility, whose output differed significantly among bulls. Among sperm abnormalities, the proportion of morphologically deviating sperm head shapes were negatively correlated with fertility, pear-shaped sperm heads in particular. In conclusion, the relationship between sperm morphology and fertility after AI calls for frequent (2-3 months interval) detailed assessments of sperm morphology in AI stud bull sires.     

Flow cytometric evaluation of sperm parameters in relation to fertility potential

Most laboratory methods used to evaluate semen quality have not correlated highly with fertilizing capacity. The discovery of a variety of fluorochromes and compounds conjugated to fluorescent probes has enabled a more widespread analysis of sperm attributes, and in conjunction with the flow cytometer, permit the evaluation of a large number of spermatozoa. A number of characteristics of sperm integrity, viability and function can be assessed by flow cytometry. The DNA status of spermatozoa has been determined using the metachromatic properties of acridine orange (AO). AO staining, when used in the sperm chromatin structure assay (SCSA), correlates with fertility in a number of species. DNA fragmentation can also be assessed using the terminal deoxynucleotidyl transferase-mediated dUTP nick-end labeling (TUNEL) assay, which identifies DNA strand breaks by labeling free 3'-OH termini with modified nucleotides. The status of the sperm acrosome can be determined using fluorescently labeled lectins and LysoTracker Green DND-26, a fluorescent acidotropic probe. Capacitation status has been observed through calcium-mediated changes using chlortetracycline (CTC) or by changes in membrane fluidity monitored by the binding of the fluorescent amphiphilic probe, Merocyanine 540. Fluorescently labeled annexin-V, C6NBD and Ro-09-0198 can also be used to detect changes in membrane phospholipid distribution. Cell viability can be determined using the propensity of propidium iodide (PI), ethidium homodimer-1 (EthD-1) or Yo-Pro-1 to permeate damaged membranes. These are generally more adaptable to clinical flow cytometry than the bisbenzimide membrane impermeable stain, Hoechst 33258, which excites in the ultraviolet range and requires UV laser equipment. Mitochondrial function can be determined using rhodamine 123 (R123) and MitoTracker Green FM (MITO) and 5,5',6,6'-tetrachloro-1,1',3,3'-tetraethylbenzimidazolyl-carbocyanine iodide (JC-1). Flow cytometry is a tool that may be used in the future to monitor many new potential markers of sperm function.     

The usefulness of combining traditional sperm assessments with in vitro heterospermic insemination to identify bulls of low fertility as estimated in vivo

To date, no single in vitro assessment can estimate bull fertility. This research was aimed at evaluating the ability of a series of laboratory assessments to assign 50 Holstein Friesian bulls grouped as low (ER-NRR<-1.5), medium (-0.5相似文献   

Relationships among frozen-thawed sperm characteristics assessed via the routine semen analysis, sperm functional tests and fertility of bulls in an artificial insemination program

Frozen semen specimens from 22 Holstein bulls representing a wide range of field fertility levels or nonreturn rates (NRR) were used in this study. Semen specimens were thawed at 37 degrees C for a minimum of 30 sec, followed by assessment via a routine semen analysis (RSA) and other sperm functional tests. The RSA was performed by assessing sperm count, motility and morphological characteristics. Other sperm functional tests were performed by assessing the acrosomal membrane integrity, sperm penetration into the cervical mucus and the sperm membrane functional integrity. Following assessment of sperm characteristics, the fertility data of the various bulls were compared to the RSA and the functional tests results. Bulls of high and low fertility were similar in terms of sperm count and progressive motility (P > 0.05). Other characteristics measured by the RSA and functional tests were significantly higher in high fertility bulls (P < 0.05). Correlation coefficients among the various sperm characteristics and fertility of bulls were highly significant (P < 0.01). The highest correlation coefficients between sperm characteristics and fertility were obtained for motility (r = 0.53; P < 0.01), normal morphology (r = 0.59; P < 0.01) and swollen spermatozoa (r = 0.57; P < 0.01). Analysis of specific sperm swelling patterns showed that those patterns considered to reflect maximal sperm swelling were indicative of high fertility.     

Use of a new computerized system for evaluation of spermatozoal motility and velocity characteristics in relation to fertility levels in dromedary bulls

Three ejaculates from each of 14 dromedary bulls were collected at 7-day intervals and diluted to 50 x 10 (6) spermatozoa per ml with sodium citrate (2.9%) seminal extender. Spermatozoal concentrations, motility percentages and velocity measures were evaluated by a new computerized cell motion analyzer (CMA, medical Technologies Montreux SA, Switzerland) for assessing fertility rates in such animals. Greatest variability in concentrations and motility percentages was generally attributed to animal effect within the first ejaculate. Spermatozoal concentrations and kinematic variables in particular percentages of progressive motility, amplitude of lateral head displacement (ALH) and linearity (LIN) percentage were strongly correlated (P<0.01) with fertility rates of dromedary bulls. The CMA-derived measurements of velocity straight line (VSL), velocity curve line (VCL), and velocity averaged line (VAP) were significantly affected by the type of spermatozoal tracks. The present results evidenced that the CMA is a reliable system for determining spermatozoal concentrations, motility percentages and velocity measures and is considered as an accurate and rapid method for evaluating and predicting fertility in the one-humped camel bulls.     

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.     

The contribution of bulls and cows to the seasonal differences in the fertility of dairy cattle in Israel

Semen collected from dairy Israeli-Friesian bulls in the winter and summer was pelleted-frozen and used for inseminations in the collection season, in the other season, and after a 12-month storage period.The semen quality, as assessed by the percentage of ejaculates with initial poor motility and low sperm density and the percentage of spermatozoa surviving the freezing-thawing process, and those revealing acrosomal and tail abnormalities after thawing, was slightly and non-significantly lower in summer than in winter.The fertility of semen collected in both seasons was not affected by a 12-month storage period. The use of winter semen in summer did not improve the low fertility of cows in that season compared with those inseminated with summer semen.It appears from the results of this study that the summer decrease in the fertility of the inseminations is due to the adverse effect of this season on the fertility of the cows.     

Rapid adjustments of sperm characteristics in relation to social status

Sperm competition models predict that males typically mating in disfavoured roles should be selected to compensate for their disadvantage by investing more into sperm. We studied the effect of rapid changes in social status on ejaculate investments during experimental trials with an externally fertilizing teleost--the Arctic charr (Salvelinus alpinus). We document that males becoming dominant produce less sperm with lower velocity, but have higher sex steroid concentrations than subordinate males. These differences in sperm characteristics seem mainly to result from a decreased investment in sperm among fish that become dominant compared to pre-trial levels. Moreover, these adjustments of sperm production and sperm velocity seem not to be traded against sperm longevity. Our results support theoretical models of sperm competition, as males forced to mate in disfavoured roles seem to invest more into ejaculate quality than males in favoured roles. Additionally, we are the first to report that males, in a species with status-dependent shifts in reproductive tactics, have evolved rapid tactic specific adjustments of sperm production and sperm velocity corresponding to what could be predicted from their reproductive roles.     

Association between mRNA abundance of functional sperm function proteins and fertility of Holstein bulls

Although the existence of a complex population of mRNA in sperm is well documented, its role has not been completely elucidated. The objective of this study was to determine the relationship of mRNA abundance of sperm specific proteins and sire conception rates (SCR; a fertility index) in Holstein bulls. Samples of sperm from a single collection from commercial Holstein bulls (N = 34) were used to evaluate relative mRNA expression of adenylate kinase (AK) 1, integrin beta (IB) 5, Doppel, nerve growth factor, tissue inhibitors of metalloproteinases (TIMP) 2, lactate dehydrogenase C 1, small nuclear ribonucleoprotein polypeptide N, outer dense fiber 2, and phospholipase C zeta (PLCz) 1 in sperm. With the exception of lactate dehydrogenase C 1 and outer dense fiber 2, the mRNA abundances of these proteins were greater (P < 0.05) for high fertility (> +2 to ≤ 4 SCR) bulls compared with average (≥ 2 to ≤ +2) and low fertility (> −2 to ≤ −4) bulls. Of all the multivariate regression models tested, a combination of AK1, IB5, TIMP2, small nuclear ribonucleoprotein polypeptide N, and PLCz1 accounted for 97.4% of the variance in SCR scores. In the absence of PLCz1, the combination of AK1, IB5, Doppel, nerve growth factor, TIMP, and small nuclear ribonucleoprotein polypeptide N accounted for 96.6% of the variance in SCR scores. In addition, immunocytochemistry confirmed that the sperm-specific protein markers evaluated in this study were present in sperm. In conclusion, frozen-thawed semen from bulls with higher AK1, IB5, TIMP, small nuclear ribonucleoprotein polypeptide N 2 and PLCz1 mRNA abundances in the sperm had greater correlations with sire fertility index and may possess greater probabilities of siring calves.     

Zona-penetration in vitro test for evaluating boar sperm fertility

We investigated the capacity of porcine sperm-zona binding and penetration by using bioassay to differentiate between spermatozoa from fertile and subfertile boars. Semen was collected from Large White boars grouped into categories of fertile and subfertile (n=5 per each group) according to the results of artificial insemination. Boars in both groups showed similarly hyperactivated sperm motility at insemination (44.72 and 43.03% respectively) regardless of the lower percentage of progressive motility observed in the ejaculates of subfertile boars. At in vitro insemination, a high proportion of the sperm population (43.76%) in the subfertile boars was without acrosomes, while in the fertile boars this proportion was only 24.35%. The sperm penetration rate of fertile boars reached 66.03% while that of subfertile boars was only 25.08%. In conclusion, the results of our study showed that the penetration rate by boar spermatozoa of the zona pellucida can be used to predict fertility and/or as an in vitro standard for describing porcine semen characteristics.     

Moribund sperm in frozen-thawed semen, and sperm motion end points post-thaw and post-swim-up, are related to fertility in Holstein AI bulls

The objectives were to compare testicular physical characteristics and post-thaw sperm characteristics and their associations with fertility in Holstein bulls used for AI. Ten Holstein bulls (4-5 y old) were classified as either high-fertility (HF) or low-fertility (LF; n = 5 each), based on adjusted 56-d non-return rates [non-return rate (NRR); range (mean ± SD): 55.6 ± 4.6 to 71.8 ± 1.3%). Testicular physical characteristics were not significantly different between the two groups. Four ejaculates were collected from each bull and cryopreserved. Several indexes of sperm motion (based on computer-assisted sperm analysis) at post-thaw and post-swim-up were correlated with NRR. Sperm from HF bulls were in transition to a hyperactivated motility pattern, whereas those from LF bulls had only a forward progressive motility pattern. In HF vs LF bulls, there was a greater percentage of viable sperm after thawing (60.6 ± 9.7 vs 49.5 ± 8.0%, P < 0.05) and after swim-up (70.9 ± 11.0 vs 63.0 ± 8.8%, P < 0.01); these two end points were positively correlated with fertility (r = 0.45, P < 0.01 and r = 0.78; P < 0.01, respectively). Furthermore, in HF vs LF bulls, the ratio of sperm recovered after swim-up to viable sperm in post-thaw semen was higher (P < 0.001), and the proportion of moribund sperm expressed as a percentage of live sperm differed (12.6 ± 3.4 vs. 16.4 ± 3.1%, P < 0.001) and was negatively correlated (r = −0.33, P < 0.05) with fertility. In conclusion, fertility of Holstein bulls maintained in a commercial AI center was not predicted by testicular physical characteristics, but it was associated with differences in moribund sperm in the inseminate, as well as characteristics of sperm post-thaw and after swim-up.     

Assessment of in vitro sperm characteristics after flow cytometric sorting of frozen-thawed bull spermatozoa

The effect of processing prior to sex-sorting, re-freezing and thawing of frozen-thawed bull spermatozoa on in vitro sperm characteristics was investigated. Frozen-thawed bull spermatozoa (three bulls; three ejaculates per bull) were prepared for sorting by washing (FT-WASH) or gradient centrifugation (FT-GRADIENT) and evaluated for motility and forward progressive motility (FPM) after processing, staining, sorting and incubation (3 h; 37 degrees C). After frozen-thawed samples were processed and analyzed using a high-speed cell sorter, aliquots were removed and re-frozen and thawed (FTF-WASH; FTF-GRADIENT). Non-sorted frozen-thawed spermatozoa (FT-CONTROL) were also re-frozen and thawed (FTF-CONTROL). Spermatozoa from all treatments were assessed for penetration of an artificial cervical mucus at 0 h after sorting or thawing, and for motility, FPM and acrosomal status after 3-h incubation (37 degrees C). Frozen-thawed spermatozoa prepared by gradient centrifugation before sorting were sorted more efficiently than washed samples (P < 0.05). However, after sorting (FT) or thawing (FTF) and incubation, the percentage of motile spermatozoa and FPM rating was lower for GRADIENT than WASH (21.5 +/- 3.39%; 1.4 +/- 0.16 FPM versus 48.6 +/- 4.02%, 2.6 +/- 0.16 FPM; P < 0.01). Frozen-thawed sorted spermatozoa (FT) penetrated in greater numbers (151.0 +/- 19.50 spermatozoa) and distance (56.3 +/- 5.11 mm) in the artificial cervical mucus and had a higher proportion of motile spermatozoa (65.5 +/- 2.77%) and FPM rating (2.8 +/- 0.12) after incubation than spermatozoa that had been re-frozen and thawed after sorting (FTF: 14.0 +/- 3.67 spermatozoa, 21.6 +/- 3.05 mm, 12.2 +/- 1.31% and 1.2 +/- 0.10 FPM, respectively; P < 0.001). Regardless of processing prior to sorting, frozen-thawed sorted and non-sorted spermatozoa migrated similar distances in the artificial cervical mucus (FT-WASH: 60.0 +/- 1.2 mm; FT-GRADIENT: 57.2 +/- 0.76 mm; FT-CONTROL: 51.7 +/- 0.69 mm). The results of this preliminary study suggested that frozen-thawed bull spermatozoa can be efficiently sorted into high purity X- and Y-chromosome enriched samples with retained functional capacity.     

Evaluation of fertility status of Murrah buffalo bulls in vitro using zona-free hamster eggs

Cryopreserved semen samples from 10 Murrah buffalo bulls were used for sperm penetration bioassay using zona-free hamster oocytes. The samples were evaluated for sperm motility, viability and acrosome integrity. Actively motile spermatozoa recovered by the swim-up technique were capacitated using calcium ionophore A(2 3 1 8 7). Mature female golden hamsters were superovulated with 50 IU PMSG followed 56 h later by 75 IU hCG. Cumulus mass, recovered by puncture of oviducts at the infundibulum region, was treated with 0.1% hyaluronidase and 0.1% trypsin to obtain zona-free oocytes. After coincubation of zona-free oocytes with capacitated buffalo spermatozoa, scoring was done as fertilization percentage and fertilization index. The correlation coefficients with conception rate were statistically significant with fertilization percentage (r = 0.588, P < 0.05) and fertilization index (r = 0.660, P < 0.01). However, conventional parameters like viability, motility and acrosome integrity showed poor correlation with conception rate.