水牛精液品质的研究

[目的]本研究测定了摩拉水牛、尼里水牛和槟榔江水牛的射精量、精子密度、精子活力和畸形率。[方法]精液品质按照《牛冷冻精液》标准和《牛冷冻精液生产技术规程》进行测定。[结果]结果得出,摩拉水牛公牛的射精量为5.1±2.37ml（n=2688）、精子密度为7.8±3.68亿/ml、原精活力57.4±16.42%;冷冻精液解冻活力平均为36.0±2.13%（n=1680）、畸形率为20.62±11.04%（n=873）;尼里水牛公牛的射精量为5.3±1.99ml（n=1370）、精子密度为8.4±3.54亿/ml、原精活力62.7±12.51%;冷冻精液解冻活力平均36.5±2.43%（n=1102）、畸形率为15.2±5.71%（n=481）;槟榔江水牛公牛的射精量为5.6±2.26ml（n=271）、精子密度为8.6±5.13亿/ml、原精活力65.2±9.19%;冷冻精液解冻活力平均为37.2±2.48%（n=239）、畸形率为19.6±6.08%（n=110）。[结论]本研究获得了水牛精液品质的基础数据。     

Arachidic Acid in Extender Improves Post‐thaw Parameters of Cryopreserved Nili‐Ravi Buffalo Bull Semen

Cryopreservation process reduces lipids and phospholipids from buffalo bull spermatozoa. It was therefore hypothesized that supplementation of fatty acid to extender may improve the post‐thaw quality of buffalo semen. The objective was to evaluate the effect of arachidic acid supplementation in extender on post‐thaw quality of buffalo bull (Bubalus bubalis) spermatozoa. Semen was collected from three adult Nili‐Ravi buffalo bulls of similar age group with artificial vagina (42°C) for 3 weeks (replicate). Qualified semen ejaculates (n = 18) were split into four aliquots and diluted in tris–citric acid extender containing 0.0 (control), 5.0, 10.0 and 20.0 ng/ml at 37°C having approximately 50 × 10⁶ spermatozoa/ml. Diluted semen was cooled to 4°C in 2 h and equilibrated for 4 h at 4°C. Cooled semen was filled in 0.5‐ml straws at 4°C, kept on liquid nitrogen vapours for 10 min and plunged in liquid nitrogen for storage. Thawing of frozen semen was performed after 24 h at 37°C for 30 s. Sperm progressive motility (%) was improved in a dose‐dependent manner by supplementing arachidic acid at 5.0, 10.0 and 20.0 ng/ml compared with control. Structural and functional integrity of sperm plasma membrane (%), number of acrosome‐intact live sperm (%) and sperm chromatin integrity (%) were better (p < 0.05) in extender having 5.0 ng/ml of arachidic acid compared with control. At 10.0 ng/ml, these values did not vary (p > 0.05) from those at 5.0 ng/ml. Further improvement in structural and functional integrity of sperm plasma membrane, number of acrosome‐intact live sperm and chromatin integrity was observed at 20.0 ng/ml of arachidic acid in extender. In conclusion, arachidic acid supplementation in extender improved the post‐thaw quality parameters of cryopreserved Nili‐Ravi buffalo bull spermatozoa. Among the arachidic acid concentrations studied, maximum improvement in post‐thaw semen quality parameters was observed at 20.0 ng/ml.     

Effect of Oviductal Proteins on Structural and Functional Characteristics of Cryopreserved Sperm in Murrah Buffaloes*

The present study was undertaken to elucidate the effect of non‐luteal oviductal proteins on sperm characteristics in Murrah buffaloes. Oviducts from healthy buffaloes were collected immediately after slaughter and the oestrous cycle phase was determined as either luteal or non‐luteal based on ovarian morphology. Non‐luteal oviducts (n = 80) were flushed from the isthmic end of the oviduct with PBS, fluid was centrifuged at 10 000 g at 4°C for 20 min and then dialysed and clarified. The supernatant obtained was lyophilized to concentrate the protein and stored at ?20°C till use. Sixteen good quality ejaculates from four Murrah buffalo bulls were collected using an artificial vagina. After fresh semen analysis, each ejaculate was split into two parts and extended in Tris–citrate–egg yolk glycerol dilutor. Part I of the split ejaculate was treated with non‐luteal oviductal proteins at the dose rate of 1 mg/ml of diluted semen, while part II remained as control. The extended semen was equilibrated for 4 h at 5°C, filled in 0.5 ml French straws, exposed to LN₂ vapour, plunged into LN₂ and then stored at ?196°C. The equilibrated and frozen–thawed semen was evaluated for sperm motility, viability, acrosomal integrity, cervical mucus penetration test and hypo‐osmotic sperm swelling test (HOST). In frozen–thawed semen, the percentage of sperm motility, viability and acrosomal integrity was significantly (p < 0.05) higher in the treatment group compared to the control group. The incorporation of non‐luteal oviductal proteins in the extender increased the ability of sperm to penetrate cervical mucus both after equilibration and the freeze‐thaw process. Similarly, the proportion of sperm with intact plasma membrane, as revealed by HOST values, was also significantly (p < 0.05) higher in the treatment group (32.6%) than the control group (27%) in frozen–thawed semen. It was inferred that incorporation of non‐luteal whole oviductal fluid proteins improved the sperm quality in frozen–thawed semen in Murrah buffaloes.     

Seasonal variation in semen quality of bulls and correlations with metabolic and endocrine parameters

Sperm output and semen quality of 17 bulls sampled over 12 months showed minimal output in mid-winter and late summer and minimum quality in late summer. Monthly measurements of luteinising hormone and testosterone concentration in plasma and testosterone concentration in semen were made over 12 months. Serum non-esterified fatty acids (NEFA), albumin and total protein were also measured for the final seven months of this period. Plasma testosterone showed a strong negative correlation with sperm numbers two months hence but not in the current month. Plasma testosterone by bull and semen testosterone by month was also correlated with sperm output. Plasma luteinising hormone three months and serum total protein two months prior was positively correlated with sperm numbers ejaculated and the normality of sperm morphology, possibly by affecting the luteinising hormone dependent A0 to A1 spermatogonial division. NEFA was correlated with initial and post freezing/thawing motility in the current month, possibly by affecting membrane stability. The value of examining the bull in diagnosing infertility of cows where nutritional stress may have occurred is suggested, as is the use of albumin/total protein and NEFA measurements as a prognostic aid for time to return to normality of function of such bulls.     

Effect of Semen Collection Methods on the Quality of Pre‐ and Post‐thawed Bali Cattle (Bos javanicus) Spermatozoa

This study was conducted to evaluate the response of Bali bulls (Bos javanicus) to different semen collection methods and their effects on fresh and post‐thawed semen quality. The collection methods employed were electro‐ejaculation (EE), transrectal massage (RM) and RM followed by EE (RM + EE). A total of 25 untrained Bali bulls (age between 2 and 4 years old) were subjected to the different semen collection methods. Fresh semen samples from all the 25 bulls were evaluated for volume, pH, general motility, live/dead ratio and abnormality using the conventional method. For fresh and frozen samples collected by EE and RM from 10 bulls, computer‐assisted semen analysis system was used for precise quantitative measurement of motility, velocity and forward progression. Accucell photometer was used to measure sperm concentration in all samples, regardless fresh and frozen. Semen samples were obtained 100% of the attempts using EE, 84% using RM and 96% using RM + EE. There were no differences among the collection methods for fresh semen quality characteristics, including motility, morphology and viability, but pH and volume were higher for EE than RM and RM + EE. Higher sperm concentration was observed in semen collected by RM than the other two methods. Different age groups (2–3 and >3–4 years old) of the bulls did not show significant differences in volume, pH, sperm concentration, percentages in motility, live/dead ratio and normal sperm morphology. The quality of semen for general and progressive motility, VAP, VSL and VCL and acrosomal integrity after thawing was higher for RM than EE. In conclusion, Bali bulls appeared to respond best to EE and the combination of RM + EE than RM, as a method of semen collection, with a shorter time of stimulation required. Differences in age of the Bali bulls did not affect the semen quality.     

Fertility evaluation in Egyptian buffalo bulls using zona pellucida binding and in vitro fertilization assays

This study aimed to develop a system of in vitro assays based on zona pellucida binding and in vitro fertilization for predicting male fertility in buffalo bulls. Frozen–thawed semen from nine bulls was tested for motility, viability index, acrosomal integrity, zona pellucida binding and in vitro fertilizing ability. Differences in post-thaw sperm motility between bulls were not significant. Differences in viability indices and percentage of spermatozoa with detached acrosome between bulls was highly significant (P < 0.001). Sperm attached per ovum, fertilization rates and polyspermy percentages varied significantly (P < 0.01) among buffalo bulls. A significant (P < 0.01) positive correlation coefficient of 0.69 was evident between normal acrosome and sperm attached per ovum, while between normal acrosome and fertilization efficiency it was 0.72. Sperm from different buffalo bulls differs in their ability to bind and fertilize oocytes. This study provides a basis to predict and maximize the in vitro fertilization performance of individual bulls.     

Comparison of two methods of seminal plasma removal on buffalo (Bubalus bubalis) sperm cryopreservation

Cryopreservation causes damage to spermatozoa, and methods minimizing this damage are therefore needed. Although much discussed, seminal plasma removal has become an alternative to improve sperm quality and viability after freezing and has been applied to different species in attempt to obtain good results. The objective of this study was to evaluate semen quality in buffaloes submitted to two methods for seminal plasma removal (filtration and centrifugation). Semen samples were collected from seven Murrah buffalo bulls (Bubalus bubalis) once a week for 8 weeks. Each ejaculate was divided into three groups: control (presence of seminal plasma), centrifugation and filtration. Sperm kinetics was evaluated with the computer‐assisted sperm analysis (CASA) system. Plasmalemma and acrosomal membrane integrity, mitochondrial membrane potential and reactive oxygen species (ROS) were measured by flow cytometry, and lipid peroxidation was evaluated by the thiobarbituric acid reactive substances (TBARS) assay. Seminal plasma removal did not improve sperm kinetics compared to the control group. Centrifugation increased the number of cells with damaged acrosomal membranes (0.77 ± 0.05) and filtration caused greater plasmalemma and acrosomal membrane damage (22.18 ± 1.07). No difference in the mitochondrial membrane potential was observed between groups. In contrast, ROS production was higher in the centrifugation group compared to the control and filtration groups, although no differences in TBARS formation were detected. In conclusion, seminal plasma removal did not improve the quality of thawed buffalo semen compared to control in terms of sperm kinetics, membrane integrity, mitochondrial membrane potential or lipid peroxidation.     

Biochemical studies into variation and repeatability of glutathione concentrations in bovine and bubaline semens

Repeatability (r) value of glutathione (GSH) content was estimated in semen of Tharparkar, Red Dane, their crosses, and Murrah buffalo bulls. Mean GSH values were higher in bovine bull semen as compared to mean GSH values in bubaline bull semen. The r of GSH concentration for the pooled data was 0.1278. This trait is 12.78% repeatable. GSH value in semen of bovine and bubaline bull differed insignificantly. R estimates are expressed for selection of bulls of higher fertility and semen quality.     

Effect of cattle-specific estrus molecules on libido and semen production of zebu bulls under tropical climate

Zebu bulls are a shy breeder and they exhibit optimum libido in the presence of females with estrus phase. Continuous semen collection with the use of male dummy leads to lack of adequate sexual stimulation. Therefore, the present study was designed to test the effect of estrus-specific molecule(s) for effective sexual preparation of donor bulls. The bulls were divided into normal and poor libido group, five bulls in each group by taking 1-month control study data after collecting the information of individual bull’s sexual behaviour during semen collection by regular semen collector. The bulls were never being exposed to female animals and semen was collected by an artificial vagina. The ten animals were exposed to a glycerol-water solution (50/50 v:v) as control and then exposed to estrus-specific molecules one by one. The estrus-specific molecules like squalene, 1-iodoundecane, acetic acid, coumarin, propionic acid, oleic acid, and 2-butanone were purchased from Sigma-Aldrich Company, USA, and the molecules were solubilised individually in a non-pressurised aerosol dispenser as 1.0% concentration in glycerol-water solution (50/50, v:v). Identical bulls were used as the control and exposed to each molecule one by one by giving a refractory period of 14 days. A nasal spray of acetic acid or 2-butanone significantly (p < 0.05) reduced reaction time (RT) and total time taken to ejaculate (TTTE) in normal libido bull group. Semen volume, sperm concentration, and the total number of sperm per ejaculation obtained did not show significant improvement in the normal libido group of bulls after the application of estrus-specific molecules as compared to the control. In poor libido group, acetic acid, oleic acid, and 2-butanone application showed significant (p < 0.01) improvement in RT and TTTE as compared to the control group, whereas semen production variables like sperm concentration and total sperm output per ejaculation increased significantly (p < 0.05) except semen volume. There was significant (p < 0.01) reduction in RT (%) and TTTE (%) after the application of acetic acid followed by 2-butanone and oleic acid. The sperm concentration and total sperm output per ejaculation were more after the application of each molecule but significant increase (p < 0.05) in sperm concentration was observed with 2-butanone (11.42%), acetic acid (11.42%), and oleic acid (10.13%), whereas total sperm output per ejaculation increased significantly (p < 0.05) only after the application of acetic acid and 2-butanone (24.75% and 26.84%). Hence, it can be concluded that acetic acid, 2-butanone, and oleic acid are effective for better sexual preparation of Sahiwal bulls and total sperm output per ejaculation.

     

Kisspeptin injection improved the semen characteristics and sperm rheotaxis in Ossimi ram

The aim of the present study was to investigate the effect of kisspeptin-10 (Kp10) injection on semen characteristics, testosterone (T) production and sperm rheotaxis using microfluidic devices in immature ram. Computer-assisted sperm analysis (CASA) with controlled flow velocity was used to explore the kinetic parameters of sperm and positive rheotaxis (PR %). PR % was defined as the number of PR sperms over the number of motile sperms. Healthy Ossimi rams were randomly divided into two groups; a saline-treated control group and Kp10-treated one (5 µg/kg body weight). Treatments were given by intramuscular injection once a week for 1 month. After 1 month, the semen was collected and evaluated weekly for 6 weeks, while the blood samples were collected every 2 weeks for the next 8 weeks. Semen properties were significantly affected by Kp10 injection (p < .01). The Kp10 increased the volume, sperm concentration and percentages of live sperm compared with those of control. Additionally, sperm trajectories and rheotaxis get improved by the injection of Kp10 with time. Furthermore, kisspeptin improved the secretion of testosterone levels throughout the period of study. In conclusion, injections of the Kp10 had a positive impact on semen characteristics as well as improved sperm rheotaxis of Ossimi rams in subtropics.     

Effects of Hydrogen Peroxide (H2O2) on Fresh and Cryopreserved Buffalo Sperm Functions During Incubation at 37°C In Vitro

The magnitude of damage to buffalo spermatozoa during incubation with different levels of H₂O₂ was assessed. A total number of 24 ejaculates from four Murrah buffalo bulls were analysed in the study. Each ejaculate was split into two parts (part I and II). Part I was extended in Tris–egg yolk–citrate extender (20% egg yolk:7% glycerol), equilibrated (4 h at 5°C) and cryopreserved in 0.5‐ml French straws and stored in liquid nitrogen. The other part was utilized for fresh semen studies. The sperm in fresh, equilibrated and frozen–thawed semen was separated by centrifugation (1500 g ; 15 min) and were washed with sperm TALP. The sperm cells were re‐suspended in incubation TALP at the rate of 10⁸ sperm cells per millilitre and incubated with 0, 10, 25, and 50 μm H₂O₂ per ml at 37°C. Sperm motility, viability and intact acrosome percentages were assessed at 15‐min intervals up to 60 min of incubation. Lipid peroxidation levels of sperm were assessed at 0 and 60 min of incubation. The results of the experiment revealed that sperm motility decreased drastically during incubation with H₂O₂. Among the different levels of H₂O₂, the 50‐μm H₂O₂‐incorporated group had significantly (p < 0.05) higher malonaldehyde (MDA) level than the other groups. In the 50‐μm H₂O₂‐incorporated group, the MDA levels in fresh, equilibrated and frozen–thawed semen after incubation for 60 min were 961.6 ± 12.7, 991.8 ± 10.3 and 1234.9 ± 9.6 nm per 10⁹ spermatozoa respectively. An inverse relationship was observed between sperm motility, viability, intact acrosome percentages and concentration of H₂O₂ and duration of incubation. The decrease in sperm functions with duration of incubation and concentration of H₂O₂ was significantly (p < 0.05) higher in frozen–thawed than fresh and equilibrated spermatozoa.     

Effect of organic and inorganic selenium supplementation on semen quality and blood enzymes in buffalo bulls

The present study aimed to evaluate the effect of organic and inorganic selenium (Se) supplementation on semen quality and blood serum profiles of buffalo bulls. Nine mature buffalo bulls were divided into three groups: control (non‐supplemented); organic Se (10 mg Sel‐Plex®/head twice weekly) and inorganic Se (10 mg sodium selenite/head twice weekly). Semen was collected twice a week for 3 months during Se supplementation. Semen properties were evaluated from fresh ejaculate. Moreover, fructose concentration, aspartate and alanine transaminase (AST and ALT) activities, total protein and total cholesterol were assayed in seminal plasma. Additionally AST, ALT, testosterone and Se levels were determined in the blood serum. Results showed that Se supplementation significantly (P < 0.05) influences the semen parameters during 3 months of treatment. Organic Se significantly (P < 0.05) increased the percentage of viable sperms compared to inorganic Se and the control group. Fructose concentration was significantly higher (P < 0.05) in the seminal plasma of organic Se‐treated bulls. Serum testosterone and Se concentrations were significantly (P < 0.05) increased in the Se supplemented groups than the control group. In conclusion, Se supplementation improved the parameters of buffalo bull semen and more precisely, organic Se was more effective for the improvement of semen quality and some blood components than inorganic Se.     

Development of an in vitro oviduct epithelial explants model for studying sperm–oviduct binding in the buffalo

In this study, we developed an in vitro model for studying sperm–oviduct binding in the buffalo. Oviduct explants were prepared by overnight culture of epithelial cells in TCM‐199 medium under 5% CO₂ at 38.5 °C. Cryopreserved spermatozoa from buffalo bulls (n = 4) were incubated with the oviduct explants, and the sperm–oviduct explants complex was stained with JC‐1. The effect of sperm concentration (2, 3 and 4 million), size of the oviduct explants (<0.2, 0.2–0.3, 0.3–0.4 and >0.4 mm²) and time of incubation (1 hr and 4 hr) on binding index (BI—number of sperm bound to unit area of explants) was studied. No significant difference was observed in the BI among <0.2, 0.2–0.3 and 0.3–0.4 mm² size of explants; however, the BI decreased significantly (p < .05) when the size of explants exceeded 0.4 mm². The BI decreased significantly (p < .05) when the sperm concentration was increased to 4 million, while the duration of incubation did not have any significant effect on the BI. The interaction of bulls with explants size, sperm concentration and incubation time was not significant. The developed assay has the potential to be used as an in vitro model for studying sperm–oviduct binding in the buffalo.     

Variation in sperm cryosurvival is not modified by replacing the cryoprotectant glycerol with ethylene glycol in bulls

Breed and sire differences in sperm cryosurvival have been noted, with negative implications for sperm cryobanking and assisted reproduction programmes. This study hypothesized that these differences could be modified by using lower molecular weight cryoprotectants. Therefore, the effect of replacing glycerol (GLY) with ethylene glycol (EG) on differential cryosurvival of semen from two Sanga cattle breeds (Mashona vs. Tuli) was determined. Three to five ejaculates were collected from each of ten bulls (3-8 years) by electro-ejaculation, diluted in three Tris-egg yolk extenders (Triladyl^®, 7% GLY-based and 7% EG-based) and evaluated for sperm motility, viability and morphology at three time periods (fresh – 0 hr, pre-freeze – 4 hr and post-thaw). Tuli bulls produced larger (11.8 ± 0.31 ml vs. 8.5 ± 0.38 ml) and more concentrated ejaculates of lower fresh semen quality. Breeds differed across time for motility and morphology, but not viability. Mashona bull semen had significantly higher motility and normal morphology values at each sampling time. Bulls classified as poor freezers had lower concentration (0.70 ± 0.09 × 10⁹ sperm/ml vs. 1.37 ± 0.10 × 10⁹ sperm/ml), sperm motility index (SMI, 35.0 ± 3.4 % vs. 67.8 ± 2.1 %) and viability (69.7 ± 1.1 % vs. 75.7 ± 1.0 %) compared to good freezers. Maintenance of semen quality by GLY and EG did not differ between breeds, poor and good freezers, or age groups. The interaction breed by extender across time did not reach statistical significance for all variables. The study revealed that bull and breed variation in sperm quality and cryosurvival is not modified by replacing GLY with EG, suggesting that cryostress tolerance of sperm may be under control of mechanisms other than differential response to GLY cytotoxicity.     

Melatonin supplementation improved cryopreserved Thai swamp buffalo semen

The production of reactive oxygen species (ROS) during cryopreservation process impairs the sperm characteristics and fertilizing ability. However, melatonin, an antioxidant, could protect spermatozoa against this cell damage during cryopreservation. Therefore, we attempted to evaluate whether the melatonin supplementing in the semen extender could improve the sperm quality of swamp buffalo during cryopreservation. The semen collected from six swamp buffalo bulls were diluted with tris-citrate egg yolk extender supplementing with 0, 0.1, 0.5, 1.0, 2.0 and 3.0 mM of melatonin. The parameters of sperm viability and motility were evaluated using computer-assisted semen analyser (CASA) after cryopreservation on days 1, 7, 15 and 30. The group supplemented with 1.0 mM melatonin exhibited the higher viability after cryopreservation on days 1, 7, 15 and 30 with 58.346 ± 2.1^a, 57.586 ± 2.0^a, 55.082 ± 1.8^a and 55.714 ± 1.8^a, respectively, and showed the best results of motility parameters. However, higher concentration of melatonin at 3.0 mM impaired all the parameters. In conclusion, the addition of melatonin at 1 mM to semen extender could exert the best protection against sperm damage in swamp buffalo bull during cryopreservation.     

Divergent development of testosterone secretion in male zebu (Bos indicus) and crossbred cattle (Bos indicus x Bos taurus) and buffaloes (Bubalus bubalis) during growth

Delayed pubertal development and low fertility of Bos indicus x Bos taurus crossbred male cattle and domestic buffaloes is hardly understood hence, a sensitive enzymeimmunoassay (EIA) was developed using the second antibody-coating technique and testosterone-3-O-carboxymethyloxime-horseradish peroxidase conjugate as a label for determination of testosterone in blood plasma. The EIA was validated by standard criteria. Blood samples were collected by venipuncture from growing male cattle (Karan Fries and Sahiwal) and buffalo (Murrah) and testosterone was estimated using the EIA procedure. Plasma testosterone concentrations increased significantly (P < 0.05) with advancing age. Testosterone concentrations were significantly (P < 0.01) higher in Sahiwal males in comparison to Karan Fries males. The low testosterone levels in crossbred than Sahiwal could imply that crossbred males have either not stabilized genetically or not adapted well in Indian climatic conditions resulting in poor libido and poor semen quality. The low testosterone levels in Murrah buffalo males may be the possible reason for delayed maturity in this species. The direct, sensitive EIA validated for estimating the plasma testosterone concentration was reliable for studying the testosterone profile in blood plasma of males. The results suggest that there could be a requirement for higher testosterone secretion by males during early stages of growth for attaining early sexual maturity.     

Assessment of Different Functional Parameters of Frozen–Thawed Buffalo Spermatozoa by Using Cytofluorimetric Determinations

Flow cytometry is a useful tool that provides an accurate, objective and rapid evaluation of semen quality. The use of this technique could significantly improve the quality of buffalo semen samples used in artificial insemination. This study was carried out to evaluate, by flow cytometry, frozen–thawed buffalo spermatozoa quality parameters such as sperm viability by SYBR‐14/propidium iodide staining; mitochondrial function by JC‐1 potentiometric probe; sperm chromatin stability (SCSA) by acridine orange; and acrosome reaction (AR) by FITC‐PNA staining. Semen samples from five Italian Mediterranean buffalo bulls were used. Sperm viability was not different between bulls and ranged from 33.4% to 43.6%. A consistent rate (55.1 ± 10.8%) of sperm cells showed high mitochondrial membrane potential (Δψ^high), with no significant differences between subjects. Sperm chromatin structure assay differed significantly between the five buffalo bulls; moreover, data showed high stability within each buffalo. DNA fragmentation indexes (DFI), such as %‐DFI, ‐DFI, SD‐DFI, were 11.2 ± 8.6, 153.3 ± 24.6 and 81.6 ± 21.2, respectively. Regarding AR, the percentage of acrosome‐reacted live (ARL) and acrosome‐reacted dead (ARD) spermatozoa was 0.3 ± 0.2 and 15.3 ± 5.5, respectively. This functional parameter differed significantly between buffalo bulls and showed high stability. Following to Ca²⁺ ionophore A23187 for 3 h, AR significantly differed between subjects and was characterized by an increase in both ARL (10.8%) and ARD population (22.0%). This study indicates that flow cytometry could be a useful tool for a quick multiparametric evaluation of sperm quality in buffalo. In particular, SCSA and AR resulted in sperm functional parameters sensitive enough for the diagnosis of frozen‐thawed semen fertilizing potential.     

Effect of Supplementation of Taurine or Trehalose in Extender on Immunolocalization of Tyrosine Phosphoproteins in Buffalo and Cattle (Karan Fries) Cryopreserved Spermatozoa

The present study assessed the effects of incorporation of Taurine or Trehalose in extender on immunolocalization of tyrosine phosphoproteins, Cryocapacitation and other sperm quality parameters (motility, viability and membrane integrity) in post‐thawed sperm from Buffalo (Murrah) and Cattle (Karan Fries). Six ejaculates from six individual bulls from both species were chosen at random and split into four aliquots: one aliquot without dilution (fresh sample), another diluted in egg yolk tris‐citrate (EYTC) extender and the rest of aliquots with EYTC dilution supplemented with taurine (50 mm ) or trehalose (100 mm ), respectively, and cryopreserved. Following cryopreservation, semen were thawed and assessed for standard semen quality parameters. Extent of capacitation in cryopreserved spermatozoa was measured by inducing in vitro acrosome reaction followed by dual staining. Immunolocalization of tyrosine phosphoproteins was carried out by immunocytochemistry using primary antibody clone pT‐154 (anti‐phosphotyrosine antibody) and FITC‐conjugated secondary antibody. Immunofluorescent signals were analysed for level of protein tyrosine phosphorylation in spermatozoa. Post‐thaw semen evaluation showed supplementation of taurine or trehalose to EYTC extender significantly (p < 0.05) increased motility, viability and membrane integrity of spermatozoa in both species. Percentage of cryocapacitated spermatozoa was significantly (p < 0.05) higher in cattle as compared to buffalo and degree of cryocapacitaion of spermatozoa decreased significantly (p < 0.05) upon supplementation of additives in both the species. It was also found that tyrosine phosphoproteins were localized differentially in fresh and cryopreserved spermatozoa. Supplementation of taurine or trehalose to freezing extender changed the localization of tyrosine phosphoproteins in cryopreserved spermatozoa similar to fresh in both the species. The results obtained clearly indicated that supplementation of taurine or trehalose to EYTC prior to cryopreservation improves Buffalo and Cattle sperm quality in terms of cryocapacitation and immunolocalization of tyrosine phosphoproteins during freezing–thawing process.     

Is neutral genetic diversity related to quantitative variation in semen traits in bulls?

Conservation decisions based on neutral genetic diversity have been observed to promote retention of useful quantitative variation in biological populations. An experiment was undertaken to determine the association between microsatellite marker polymorphisms and phenotypic variation in semen production and cryosurvival traits in bulls. Thirty-five ejaculates were collected from ten bulls of two breeds and evaluated before and after cryopreservation for several semen traits. The bulls were also genotyped using a set of sixteen bovine-specific microsatellite marker loci. Fixation indices (F_ST), heterozygosity and Nei's genetic distance measures were computed from allele frequency data for each of the bulls. Molecular and phenotypic data were used to compute tri-distance matrices for the ten bulls and correlated using Mantel's test in GenAIEx 6.5. The study revealed extensive heterogeneity in semen traits, heterozygosity and F_ST values among the bulls. Large pairwise phenotypic and genetic distances were also observed. Correlation between pairwise genetic distances and phenotypic distances was significant and highly positive for sperm viability (r = .61, p < .001) and moderately positive for sperm motility (r = .40–42, p < .05) variables. For sperm morphology, ejaculate volume and sperm concentration, correlation with genetic distances was positive, low and not significantly different from zero (p > .05). A tendency for a triangular-shaped relationship between genetic and phenotypic distances for post-thaw motility and viability traits was also observed. Accordingly, association with neutral genetic diversity was absent for semen production traits and moderate to highly positive for sperm cryosurvival traits. Given these findings, conservation decisions based on neutral genetic diversity may capture variation in some adaptive traits, but not others.     

Assessment of buffalo (Bubalus bubalis) sperm DNA fragmentation using a sperm chromatin dispersion test

Chromosomal fragmentations or damage in sperm DNA has considerable value in determination of semen quality. However, rapid and/or simple method to assess sperm DNA integrity in buffalo has apparently not been reported. In the present study, SCD was used for the first time in buffalo bulls for assessment of sperm DNA fragmentation. A modified SCD protocol, under bright field microscope was developed and validated by comparison with other routine tests which can be used for processing of samples. The DNA fragmentation index (DFI) from SCD was correlated with semen quality parameters viz. viability (r=-0.68, p<0.05), membrane integrity (r=-0.74, p<0.05) and capacitation status (r=-0.69, p<0.05). The amount of DNA fragmentation assessed by SCD was highly correlated (R=0.874, p<0.05) with results of acridine orange test (AOT), a traditional method of assessing DNA damage. There were no significant differences between two observers with regards to scoring dispersion patterns. Therefore, the SCD test can be routinely used for detection of DNA fragmentation in buffalo sperm, with potential for replacing conventional time consuming tests.