Cryoprotectant effect of trehalose in extender on post‐thaw quality and in vivo fertility of water buffalo (Bubalus bubalis) bull spermatozoa

This study was designed to ascertain the cryoprotectant effects of different concentrations of trehalose [0 (T0), 25 (T25), 35 (T35), 45 (T45) mm ], egg yolk [20% (E20), 15% (E15) v/v] and glycerol [7% (G7), 5% (G5) v/v] in Tris‐citric acid‐based extender on post‐thaw quality and in vivo fertility of buffalo bull spermatozoa. Twenty‐five ejaculates were collected from five bulls and split into four parts. After that, the split ejaculates from each of the bull were diluted either in T0E20G7 (control) or T25E20G5 or T35E15G5 or T45E15G5 extender. Finally, the sperm suspension was frozen in 0.54‐ml French straws. Post‐thaw sperm total motility (%), progressive motility (%), rapid velocity (%), average path velocity (μm/s), straightline velocity (μm/s), curvilinear velocity (μm/s), linearity (%), plasma membrane and acrosome integrities (%) were higher (p < .05) in T45E15G5 extender as compared to other treatment groups and control. The fertility rate (56.8% versus 41.3%) was higher (p < .05) in buffaloes inseminated with semen doses cryopreserved in extender containing T45E15G5 combination of cryoprotectants than the control. In conclusion, addition of 45 mm trehalose along with 15% egg yolk and 5% glycerol in extender improves the post‐thaw quality and in vivo fertility of buffalo bull spermatozoa.     

l‐Cysteine improves antioxidant enzyme activity,post‐thaw quality and fertility of Nili‐Ravi buffalo (Bubalus bubalis) bull spermatozoa

The effects of l ‐cysteine in extender on antioxidant enzymes profile during cryopreservation, post‐thaw quality parameters and in vivo fertility of Nili‐Ravi buffalo bull spermatozoa were studied. Semen samples from 4 buffalo bulls were diluted in Tris–citric acid‐based extender having different concentrations of l ‐cysteine (0.0, 0.5, 1.0, 2.0 and 3.0 mm ) and frozen in 0.5‐ml French straws. The antioxidative enzymes [catalase, super oxide dismutase and total glutathione (peroxidase and reductase)] were significantly higher (P < 0.05) at pre‐freezing and post‐thawing in extender containing 2.0 mm l ‐cysteine as compared to other groups. Post‐thaw total motility (%), progressive motility (%), rapid velocity (%), average path velocity (μm s^?1), straight line velocity (μm s^?1), curvilinear velocity (μm s^?1), beat cross frequency (Hz), viable spermatozoa with intact plasmalemma (%), acrosome and DNA integrity (%) were higher with the addition of 2.0 mm l ‐cysteine as compared to other groups (P < 0.05). The fertility rates (59 versus 43%) were higher (P < 0.05) in buffaloes inseminated with doses containing 2.0 mm of l ‐cysteine than in the control. In conclusion, the addition of 2.0 mm l ‐cysteine in extender improved the antioxidant enzymes profile, post‐thaw quality and in vivo fertility of Nili‐Ravi buffalo bull spermatozoa.     

Addition of pomegranate juice (Punica granatum) in tris‐based extender improves post‐thaw quality,motion dynamics and in vivo fertility of Nili Ravi buffalo (Bubalus bubalis) bull spermatozoa

The aim of the present study was to determine the protective effects of pomegranate juice in tris‐based extender on semen parameters, computer‐assisted sperm analysis (CASA) motion characteristics and field fertility of post‐thawed Nili Ravi buffalo (Bubalus bubalis) bull spermatozoa. Two consecutive ejaculates/collection from each of the five adult Nili Ravi buffalo bulls were collected with artificial vagina at 42°C for a period of 7 weeks, diluted in extender containing different concentrations of pomegranate juice (0.0%, 2.5%, 5%, 7.5% and 10%). Diluted samples were packed and frozen in 0.54 ml French straws. The addition of 10% pomegranate juice in extender significantly improved post‐thaw sperm morphology (%), motilities (CASA total motility, progressive motility (%) as well as VAP, VSL, VCL, STR, DAP, DSL) compared to the control group (p < 0.05). Plasma membrane, acrosome membrane and DNA integrity were significantly higher in extender with 10% pomegranate juice than the control group (p < 0.05). Field fertility rate (60.39% vs. 46.53%) was higher (p < 0.05) in extender with 10% pomegranate juice as compared to the control. It is therefore concluded that the addition of 10% pomegranate juice in tris‐based extender improves post‐thaw semen parameters, CASA motion dynamics and field fertility in Nili Ravi buffaloes.     

Freezability of water buffalo bull (Bubalus bubalis) spermatozoa is improved with the addition of curcumin (diferuoyl methane) in semen extender

Effects of curcumin as antioxidant in extender were evaluated on freezability of buffalo spermatozoa. Semen from each of the five bulls (n = 3 replicates, six ejaculates/bull, a total of 30 ejaculates) was diluted in Tris‐citric acid extender containing curcumin (0.5, 1.0, 1.5 or 2.0 mM) or control. At pre‐freezing and post‐thawing, total antioxidant contents (μM/L) and lipid peroxidation levels (μM/ml) were higher (p < .05) and lower (p < .05) respectively, with 1.5 and 2.0 mM compared to 0.5 and 1.0 mM curcumin and control. At post‐thawing, progressive motility (PM, %) and rapid velocity (RV, %) were higher (p < .05) with 1.5 mM compared to other doses of curcumin and control (except in case of RV, 1.5 was similar with 1.0 mM). Kinematics (average path velocity, μm/s; straight‐line velocity, μm/s; curved‐line velocity, μm/s; straightness, %; linearity, %), in vitro longevity (%, PM and RV) and DNA integrity (%) at post‐thawing were higher (p < .05) with 1.5 mM compared to control. At post‐thawing, supravital plasma membrane integrity (%) and viable spermatozoa with intact acrosome (%) were higher with 1.5 compared to 2.0 mM curcumin and control. We concluded that freezability of water buffalo spermatozoa is improved with the addition of 1.5 mM curcumin in extender.     

Use of post‐thaw semen quality parameters to predict fertility of water buffalo (Bubalus bubalis) bull during peak breeding season

This study was designed to predict the fertility of water buffalo bull using post‐thaw semen quality parameters during peak breeding season. Thirty ejaculates were collected from five bulls with artificial vagina and cryopreserved. At post‐thaw, semen was analysed for motility parameters, velocity distribution, kinematics, DNA integrity/fragmentation, viability, mitochondrial transmembrane potential, morphology, plasma membrane and acrosome integrity. Data of 514 inseminations were collected for estimation of in vivo fertility. Pearson's correlation coefficients showed that progressive motility (PM), rapid velocity, average path velocity, straight line velocity, straightness, supravital plasma membrane integrity, viable spermatozoon with intact acrosome or with high mitochondrial activity were correlated with in vivo fertility (r = .81, p < .01; r = .85, p < .01; r = .64, p < .05; r = .73, p < .05; r = .57, p < .05; r = .88, p < .01; r = .84, p < .01 and r = .81, p < .01 respectively). Step forward multiple regression analysis showed that the best single predictor of fertility was PM. However, combinations of semen quality parameters to predict fertility were better as compared to single parameter. In conclusion, fertility of buffalo bull can be predicted through some of the post‐thaw in vitro semen quality tests during peak breeding season.     

Seasonal variation in semen quality of swamp buffalo bulls （Bubalus bubalis） in Thailand

Aim： To test the hypothesis that season affects the semen quality of swamp buffalo （Bubalus bubalis） bulls used for artificial insemination （AI） under tropical conditions in Thailand, as it does in Bos taurus and Bos indicus. Methods： Clinical and andrological examinations, and monitoring of semen production and quality were carried out on five mature, healthy swamp buffalo AI bulls in Thailand from July 2004 to the end of June 2005. Sperm output, motility, morphology and plasma membrane integrity （PMI） were compared between three seasons of the year （rainy, i.e. July-October; winter, i.e. November-February; and summer, i.e. March-June） with distinct ambient temperature and humidity. Results： All bulls were diagnosed as clinically healthy and with good libido throughout the study. Ejaculate volume, pH, sperm concentration, total sperm number and initial sperm motility did not differ between seasons, whereas PMI and the relative proportion of morphologically normal spermatozoa were highest in summer and lowest in winter （P 〈 0.05）. Buffalo age, week of collection and season influenced sperm morphology （P 〈 0.05-0.001）. Among morphological abnormalities, only proportions of tail defects were affected by season, being highest in the rainy season and lowest in summer （P 〈 0.001）. In conclusion, climatic changes did not seem to largely affect semen sperm output or viability. Although the proportions of PMI and tail abnormalities were affected by season, they were always below what is considered unacceptable for AI bull sires. Conclusion： Seasonal changes did not appear to cause deleterious changes in sperm quality in swamp buffalo AI-sires in tropical Thailand.     

Study on correlation of sperm quality parameters with antioxidant and oxidant status of buffalo bull semen during various stages of cryopreservation

This investigation was carried out to study the correlation of sperm quality parameters with antioxidant and oxidant status of buffalo bull semen during various stages of cryopreservation. Semen samples were evaluated for sperm parameters (mass motility [MM], concentration [CON], progressive motility [PM], viability [VIB], acrosomal integrity [AI] and hypo‐osmotic swelling [HOS] response), antioxidants (superoxide dismutase [SOD], catalase [CAT], glutathione peroxidase [GPx] and total antioxidant capacity [TAC]) and oxidants (Lipid peroxidation [LPO] and reactive oxygen species [ROS]) at fresh, pre‐freeze and post‐thaw stages. Sperm parameters (PM, VIB, AI and HOS response) and antioxidants (SOD, CAT and TAC) were significantly (p < .05) reduced at fresh stage, and oxidants (LPO and ROS) were significantly (p < .05) increased at pre‐freeze and post‐thaw stages. At fresh stage, MM was negatively correlated with LPO (p < .05), and CON was positively correlated with SOD, TAC and CAT, negatively correlated with LPO and CAT was positively (p < .01) correlated with VIB and HOS response. At pre‐freeze stage, CAT was positively correlated with PM and AI (p < .05), and AI was negatively (p < .05) correlated with ROS. At post‐thaw stage, CAT was positively correlated with PM, VIB, HOS response and AI,, and LPO was negatively correlated with HOS, AI and VIB. The study of correlations of these parameters at different preservation stages with bull fertility may play an important role in developing models for predicting future fertility of bulls in the absence of conception rate data.     

Antioxidant activity of Nigella sativa Seeds Aqueous Extract and its use for cryopreservation of buffalo spermatozoa

The free radical scavenging activity (RSA) of Nigella sativa extract and its efficiency for cryopreservation of buffalo spermatozoa was investigated. In experiment 1, Nigella sativa extract was prepared and evaluated for RSA using 2,2‐diphenyl‐1‐picrylhydrazyl assay. The results showed increased pattern of RSA at 1%–5% of Nigella sativa extract. In experiment 2, buffalo semen from three bulls (24 ejaculates) was incubated at 0%, 0.1%, 0.3%, 0.5%, 1%, 1.5%, 2%, 3%, 4%, 5% and 6% extract to assess in vitro tolerability to Nigella sativa in terms of progressive motility (PM). Buffalo spermatozoa showed tolerance to all levels; rather, sperm PM was increased at 1%–4% extract. In experiment 3, semen from three bulls (24 ejaculates) was cryopreserved with 0%, 1%, 2%, 3%, 4% and 5% of Nigella sativa extract. Sperm PM and plasma membrane integrity (PMI) were evaluated after dilution and cooling, while PM, PMI, viability and DNA integrity were evaluated after thawing. Nigella sativa extract at 4% in extender improved (p < .05) post‐dilution, post‐cooling and post‐thaw sperm quality. In conclusion, Nigella sativa extract at all concentrations (1%–6%) showed antioxidant activity and its supplementation at 4% in extender improved buffalo sperm quality at all stages of cryopreservation.     

Isolation and enrichment of type A spermatogonia from pre‐pubertal buffalo ( Bubalus bubalis) testis

The aim of this study was to isolate and subsequently enrich type A spermatogonia from pre‐pubertal buffalo testis. Two‐step enzymatic digestion was used to isolate spermatogonia from 10 to 14 months pre‐pubertal buffalo calves, resulting in maximal release of spermatogonia from the seminiferous tubules. After enzymatic digestion, the type A spermatogonia were subsequently enriched by differential plating and Percoll gradient centrifugation. The identity of type A spermatogonia was determined by light microscopy and further characterised by Dolichos biflorus agglutinin, a specific marker for bovine type A spermatogonia by fluorescence‐activated cell sorting analysis. After enzymatic isolation, the cell suspension contained about 27% of type A spermatogonia, which was enriched up to 71% with >70% cell viability. Further flow cytometric analysis showed the presence of THY1+ cells (cells expressing thymocyte differentiation antigen 1), suggesting that THY1 is a conserved marker of the undifferentiated spermatogonial cells in buffalo. The isolation of the enriched type A spermatogonia from buffalo testis opens ways to study the further biochemical characteristics of this important class of germ cells in this species.     

Relationship between the proportion of capacitated spermatozoa present in frozen-thawed bull semen and fertility with artificial insemination

The objective of the present study was to confirm the presence of prematurely capacitated spermatozoa in frozen-thawed bull semen and to investigate the relationship of premature capacitation to the fertility of the respective semen. Twenty batches of frozen semen from young AI bulls of the Swedish Red and White breed with known fertility (expressed as 56-day non-return rates; 56 d-NRR) were tested using a Chlortetracycline (CTC) assay to assess capacitation status in frozen-thawed spermatozoa. The status of capacitation, as evidenced in this experiment, was further tested based on the hypothesis that capacitated spermatozoa present in frozen-thawed semen should undergo the acrosome reaction (AR) on co-incubation with homologous zona pellucida (ZP) glycoproteins. The percentage (mean +/- SEM) of uncapacitated, capacitated and acrosome-reacted spermatozoa in the frozen-thawed semen (n = 20) were 49.3 +/- 11.9, 36.3 +/- 8.3 and 14.2 +/- 11.9, respectively. On co-incubation with ZP, there was a significant increase (p = 0.001) in the proportion of spermatozoa undergoing the AR compared to the control with a concurrent decrease in the proportion of capacitated spermatozoa, suggesting that a proportion of capacitated spermatozoa were undergoing the AR. The proportion of viable, uncapacitated spermatozoa present in the frozen-thawed semen was correlated to the 56 d-NRR (n = 20, r = 0.5, p = 0.03). In conclusion, a proportion of spermatozoa in frozen-thawed semen was capacitated and the proportion of viable, uncapacitated spermatozoa present in semen was positively correlated to fertility.     

Programmable fast‐freezing method improves the post‐thaw motion dynamics,integrities of plasmalemma,mitochondrial transmembrane,DNA and,acrosome, and in vivo fertility of water buffalo (Bubalus bubalis) spermatozoa

The effects of freezing methods (FR1, nonprogrammable/static, 5 cm above liquid nitrogen [LN₂] for 10 min, plunging in LN₂; FR2, programmable medium, +4°C to ?15°C at 3°C min^?1, from ?15 to ?80°C at 10°C min^?1 and final holding for 1 min at ?80°C, plunging in LN₂; FR3, programmable fast, from initial holding at +4°C for 2 min, from +4°C to ?20°C at 10°C min^?1, from ?20°C to ?100°C at 30°C min^?1, final holding for 1 min at ?100°C and plunging in LN₂) were assessed on post‐thaw in vitro quality and in vivo fertility of water buffalo spermatozoa. Mean sperm progressive motility (%), rapid velocity (%), average path velocity (μm s^?1), straight line velocity (μm s^?1), curved line velocity (μm s^?1), integrities (%) of plasmalemma, mitochondrial transmembrane, DNA and acrosome were higher (p < .05) in samples cryopreserved with FR3 compared to FR1 and FR2. Similarly, in vivo fertility (%) of buffalo spermatozoa was higher (p < .05) with FR3 than FR1 (%; 68.0 versus 50.0). We concluded that programmable fast‐freezing method (FR3) improves the post‐thaw in vitro quality and in vivo fertility of water buffalo spermatozoa.     

Identification of the uterine factor(s) which induce the acrosome reaction in buffalo (Bubalus bubalis) spermatozoa

This study characterizes factors present in uterine fluid from oestrous buffaloes which induce the acrosome reaction in buffalo spermatozoa. Characterization was performed by dialysis, heat treatment (90 degrees C, 30 min) and deproteinization of uterine fluid. The % motility and viability of sperm was maintained better in unfractionated uterine fluid than in dialysed fluid. Heating and deproteinization of the dialysed uterine fluid significantly reduced the % motility and viability of sperm. The percentage of sperm showing different stages of the acrosome reaction (swelling, and vesiculation) was significantly higher for sperm incubated in dialysed than in neat uterine fluid. There was no significant difference in the % of sperm showing acrosome shedding in both dialysed and neat uterine fluid. Heat treatment destroyed the ability of uterine fluid to induce the acrosome reaction. Sperm incubated in the protein precipitate from uterine fluid showed all stages of the acrosome reaction. Thus, the uterine factor(s) responsible for inducing the acrosome reaction in buffalo sperm was associated with the non-dialysable, albumin-like protein fraction and was heat-labile.     

Effect of calmodulin-like protein from buffalo (Bubalus bubalis) seminal plasma on Ca2+, Mg2+-ATPase of purified plasma membrane of buffalo spermatozoa

Summary. Buffalo sperm heads and tails were cleaved by sonication and isolated in relatively pure proportions i.e. 95% and 98% respectively, by discontinuous sucrose density-gradient centri-fugation. Purified plasma membranes from the isolated sperm heads and tails were obtained by hypotonic treatment and brief sonication followed by discontinuous sucrose density-gradient centrifugation. Ca²⁺, Mg²⁺-ATPase activity was evident in plasma membrane from sperm heads and tails, although activity was greater in the latter. A calmodulin-like protein isolated from buffalo seminal plasma increased the Ca²⁺, Mg²⁺-ATPase of plasma membrane from the sperm heads and tails by 128 and 136% respectively. Based upon the data obtained here and elsewhere (Sidhu & Guraya, 1989a) a model is proposed which explains regulation of Ca²⁺ in buffalo spermatozoa and implicates calmodulin-like protein and Ca²⁺, Mg²⁺-ATPase in sperm acrosome reaction.     

Comparison of three different extenders on Murrah buffaloes (Bubalus bubalis) semen freezability

The use of frozen semen for artificial insemination is the main approach utilised for the genetic improvement of most domesticated species. The advantages include lower transportation costs, continuous availability of semen, fewer occurrences of sexually transmitted diseases and the incorporation of desirable genes in a relatively short amount of time. Nevertheless, the use of frozen semen in buffalo herds remains limited due to the loss of sperm quality when buffalo semen is frozen. So, the goal of this study was to evaluate the pre‐ and post‐cryopreservation quality of buffalo semen diluted in three distinct freezing media: Tris‐egg yolk, Botu‐bov® (BB) and ACP‐111®. Thirty‐two ejaculates from four bulls were analysed in terms of kinetics, morphology and sperm viability by epifluorescence microscope. Thawed samples were also evaluated for capacitation‐like damage, DNA fragmentation and plasma and acrosomal membrane integrity using flow cytometry. The Tris‐egg yolk and BB® extenders yielded better results than the ACP‐111® extender for kinetics parameter (total motility, progressive motility and percentage of rapid cells). However, semen samples were similar for parameters evaluated by flow cytometry. Taken together, the data indicate that in comparison with Tris‐egg yolk and BB extender, ACP‐111® can also be used as an extender for buffalo semen cryopreservation.     

Immunohistochemical distribution of S-100 protein and subunits (S100-alpha and S100-beta) in the swamp-type water buffalo (Bubalus bubalis) testis

The distribution and localization of S-100 protein (S-100) and its subunits (S100-alpha and S100-beta) in the testis of swamp-type water buffalo were investigated using immunohistochemistry. S-100 was detected in the Sertoli cells in the convoluted seminiferous tubules, modified Sertoli cells lining the terminal segment of the seminiferous tubules and in the intratesticular excurrent ducts (straight tubules and rete testis). S100-beta showed the same distribution and localization with that of S-100. However, the cytoplasmic extension of the Sertoli cells in S100-beta staining showed less staining intensity compared with that of S-100. S100-alpha showed a positive staining only in the modified Sertoli cells of the terminal segment of the seminiferous tubule. Endothelial cells of blood vessels were also positive with the proteins while the Leydig and spermatogenic cells showed a negative reaction. The localization of S-100 in the testis of the water buffalo was in parallel with that of other artiodactyls which supports the hypothesis that this protein is a multifunctional protein. S100-beta in the Sertoli cells suggests that this protein is involved in establishing blood-testis barrier. Its presence in the modified Sertoli cells and in the epithelium of the excurrent ducts suggest secretory and absorptive function, respectively. Meanwhile, S100-alpha, which was detected only in the modified Sertoli cells, is involved in the secretory activity of these cells that are related to exocrine function.     

Testicular lesions and epididymal sperm parameters in the Iranian river buffalo (Bubalus bubalis)

The aim of this study was to evaluate the testicular lesions and their effects on the epididymal sperm parameters in the Iranian river buffalo (Bubalus bubalis). Total numbers of 117 scrota from the pubertal buffalo were provided from the local slaughterhouse. The samples were evaluated for morphological parameters and any macro‐ or microscopic lesions. The sterile swabs from the testis parenchyma were subjected to microbiology culture. The epididymal spermatozoon was analysed for concentration, progressive motility and abnormalities. The results showed 34.2% fibrotic adhesions between parietal and visceral layers of tunica vaginalis that was significantly different among seasons (P < 0.05). The cases of unilateral cryptorchidism and bilateral Sertoli cell tumour were detected, with no spermatozoa in the respected epididymides. Microscopic examination showed 13.25% (31/234) lesions including general (51.61%; 16/31) and multifocal (29.03%; 9/31) degenerations as well as interstitial orchitis (9.68%; 3/31) and the Sertoli cell tumour (6.45%; 2/31). No relationship between the lesions and the bacterial isolation (n = 6) was detected. The sperm parameters and morphological parameters of the testis were under influence of microscopic lesions (P < 0.05). In conclusion, the testicular macro‐ and microscopic lesions may have a noticeable contribution in the Iranian buffalo fertility.     

Tyrosine phosphorylation of a 38-kDa capacitation-associated buffalo (Bubalus bubalis) sperm protein is induced by L-arginine and regulated through a cAMP/PKA-independent pathway

The aim of the present study was to determine the effect of L-arginine on nitric oxide (NO*) synthesis, capacitation and protein tyrosine phosphorylation in buffalo spermatozoa. Ejaculated buffalo spermatozoa were capacitated in the absence or presence of heparin, or L-arginine or N(omega)-nitro-L-arginine methyl ester (L-NAME), an inhibitor of nitric oxide synthase (NOS) for 6 h. Capacitating spermatozoa generated NO* both spontaneously and following stimulation with L-arginine and L-NAME quenched such L-arginine-induced NO* production. Immunolocalization of NOS suggested for existence of constitutive NOS in buffalo spermatozoa. L-Arginine (10 mm) was found to be a potent capacitating agent and addition of L-NAME to the incubation media attenuated both L-arginine and heparin-induced capacitation and suggested that NO* is involved in the capacitation of buffalo spermatozoa. Two sperm proteins of M(r) 38 000 (p38) and 20 000 (p20) were tyrosine phosphorylated extensively by both heparin and L-arginine. Of these, the tyrosine phosphorylation of p38 was insensitive to both induction by cAMP agonists as well as inhibition by a protein kinase A (PKA) inhibitor. Further, most of these L-arginine-induced tyrosine phosphorylated proteins were localized to the midpiece and principal piece regions of flagellum of capacitated spermatozoa and suggested that sperm flagellum takes active part during capacitation. These results indicated that L-arginine induces capacitation of buffalo spermatozoa through NO* synthesis and tyrosine phosphorylation of specific sperm proteins involving a pathway independent of cAMP/PKA.     

Survival of buffalo bull spermatozoa: effect on structure and function due to alpha‐lipoic acid and cholesterol‐loaded cyclodextrin

Sperm survival depending upon integral membranes and function is imperative for fertilization. This study was designed to augment survival of buffalo spermatozoa using alpha‐lipoic acid (ALA) and cholesterol‐loaded cyclodextrin (CLC) during cryopreservation. Semen was frozen using 0, 0.5, 1, 1.5, 2 and 2.5 mmol L^?1 ALA (experiment 1) and ALA or CLC separately or together (experiment 2). Semen was assessed for post‐thaw motility, plasma membrane integrity (PMI), intact acrosome and plasma membrane (IACR‐IPM) and DNA integrity at 0, 1.5, 3 and 4.5 hr of incubation. In experiment 1, use of 0.5 mmol L^?1 ALA enhanced the sperm cryosurvival and post‐thaw longevity than other groups up to 4.5 hr of incubation, and this concentration of ALA was used in second experiment with CLC. The results revealed higher (p < .05) sperm survival function and time of sperm attributes due to use of ALA than CLC and control. However, the sperm quality did not improve (p > .05) when ALA was combined with CLC. In conclusion, survival of buffalo bull spermatozoa during freeze‐thawing and post‐thaw incubation can be enhanced more with ALA than CLC or control, followed by CLC than control. However, there is no synergistic effect on survival of buffalo bull spermatozoa due to ALA and CLC.     

Comparison of Tris egg yolk‐based,Triladyl® and Optixell® extender on post‐thaw quality,Kinematics and in vivo fertility of Nili Ravi Buffalo (Bubalus bubalis) bull spermatozoa

Our objectives were to ascertain the comparison of Tris egg yolk‐based, Triladyl ^® and Optixell ^® extender on postthaw quality, CASA parameters and in vivo fertility of Nili Ravi buffalo (Bubalus bubalis) bull spermatozoa. Semen samples (n = 35) from five bulls were diluted in Tris egg yolk‐based, Triladyl ^® , Optixell ^® extender and frozen in 0.50 ml French straws. Postthaw sperm CASA motility (%) was higher (p < 0.05) in Optixell extender as compared to Triladyl and Tris egg yolk‐based extender. Although sperm progressive motility (%), morphology (%), average path velocity (μm/s), straight line velocity (μm/s), curvilinear velocity (μm/s), amplitude of lateral head displacement (μm), beat cross‐frequency (Hz), straightness (%), length of curvilinear path (μm), length of average path (μm), intact plasma and acrosome membrane (%), and DNA integrity (%) were higher (p < 0.05) in spermatozoa cryopreserved in Optixell ^® extender as compared to Tris egg yolk‐based and Triladyl ^® extender. The fertility rates (68.18%, 45.45%, 55.4%) were higher (p < 0.05) in buffaloes inseminated with semen doses frozen in Optixell extender than the Tris egg yolk‐based and Triladyl ^® extender respectively. It is concluded that Optixell ^® extender improves postthaw semen quality and fertility in Nili Ravi buffaloes.     

G‐Protein‐Coupled Chemokine Receptor Gene in Lumpy Skin Disease Virus Isolates from Cattle and Water Buffalo (Bubalus bubalis) in Egypt

Lumpy skin disease virus (LSDV), sheep poxvirus (SPV) and goat poxvirus (GPV) are the most serious poxviruses of ruminants. In this study, we analysed the G‐protein‐coupled chemokine receptor (GPCR) genes of LSDV isolates from cattle and water buffalo (Bubalus bubalis) in Egypt during the summer of 2011. Multiple alignments of the nucleotide sequences revealed that the water buffalo LSDV isolate differed from the cattle isolate at four nucleotide positions, and both isolates had nine nucleotide mutations from the reference strain, Egyptian tissue culture‐adapted cattle LSDV/Ismailyia88 strain. Compared with the GPCR sequences of SPV and GPV strains, a 21 nucleotide insertion and a 12 nucleotide deletion were identified in the GPCR genes of our used isolates and other LSDVs. The amino acid sequences of GPCR genes of our isolates contained the unique signature of LSDV (A₁₁, T₁₂, T₃₄, S₉₉ and P₁₉₉). Phylogenetic analyses showed that the GPCR genes of cattle and water buffalo LSDVs were closest genetically, indicating a potential transmission of cattle LSDV to water buffalo.