M. Honparkhe

Minimization of apoptosis-like changes in cryopreserved buffalo bull sperm by supplementing extender with Bcl-2 protein

Aim: This study was aimed at evaluating the anti-apoptotic effects of Bcl-2 protein in cryopreserved buffalo bull sperm. Materials and Methods: A total 10 ejaculates from two buffalo bulls (5 each) were collected using artificial vagina method, and semen was evaluated using a standard protocol. Semen was extended by Tris egg yolk extender supplemented with Bcl-2 protein at 5, 10, and 15 µM. Semen was cryopreserved at ultra-low temperature using traditional vapor freezing method. Pre-freeze and post-thaw semen samples were evaluated for percent motility, viability, hypo-osmotic swelling test (HOST) reactive sperms; status of mitochondrial membrane activity and status of sperm phospholipase A1 and phospholipase A2 activity. Results: There were no significant effects of Bcl-2 protein supplementation on pre-freeze sperm quality. Percent motility and active mitochondria in post-thaw Bcl-2 supplemented and control groups were also similar. However, viable sperms were significantly (p<0.05) higher (74.29±4.23%) in Bcl-2 supplemented group (5 µM) as compared to control (51.6±5.77%). The proportion of HOST reactive sperms was also higher (63.1±6.73%) in Bcl-2 supplemented (5 µM) group as compared to control (50.7±6.98%). The sperm with low PLA activity (non-apoptotic) was significantly (p<0.05) higher in all the supplemented doses of Bcl-2 protein, i.e., at 5 µM (73.42±5.79%), 10 µM (75.51±6.22%), and 15 µM (74.78±5.89%) as compared to control (60.23±4.45%). We found that Bcl-2 protein supplementation at 5 µM dose improved the post-thaw semen quality indicated by higher viability, HOST reactive sperms, and sperm with low PLA activity (non-apoptotic sperms). Conclusion: Bcl-2 protein supplementation exerts its protective effect on spermatozoa against apoptosis-like changes developed during cryopreservation.

Size of dominant follicle, plasma progesterone and estradiol levels on the day of ovulation and subsequent conception rate in buffalo (Bubalus bubalis) following modified ovsynch + CIDR protocol

The study was designed to compare the size of dominant follicle (DF) achieved, plasma progesterone decline and plasma estradiol level on the day of estrus and subsequent conception in buffaloes subjected to modified ovsynch-based regimen in buffaloes. In estradiol-based protocol (n = 15) on day 0 (beginning of experiment), buffaloes were administered controlled internal drug release (CIDR) device (1.38 g P4) and concurrently received 1.5 mg estradiol-17β in 1.5 ml canola oil (i.m.). On day 9, CIDR was removed and a PGF2α analogue (500 µg, i.m.) administered. On day 11, buffaloes were administered gonadotropin releasing hormone (GnRH) analogue (20 µg, i.m.) and inseminated on day 11 and day 12. Ovsynch-based group (control, n = 15) received GnRH on day 0 in place of estradiol-17β, CIDR insert for 7 instead of 9 days and the remaining protocol and insemination procedures were same as treatment group. The diameter of DF, plasma progesterone and estradiol levels were not different between the groups. The first service conception rate (FSCR) was higher (p < 0.05) in estradiol-based group than in control (53.33 vs. 33.33%, respectively). In conclusion, replacement of first GnRH with estradiol-17β in ovsynch plus CIDR protocol and increasing CIDR exposure by two days leads to higher FSCR.

Improvement in cryosurvival of buffalo bull (Bubalus bubalis) sperm by altering freezing rate within critical temperature range

Objective: To optimize the cryopreservation of buffalo bull semen by altering freezing rates within critical temperature range (4 °C to -60 °C). Methods: A total of 20 ejaculates each from 5 Murrah buffalo bulls were cryopreserved using programmable biofreezer in 2 phases. In the 1st phase, 9 freezing rates were applied at -2, -5, -10, -20, -30, -40, -50, -60 or -4 °C/min (control) from 4 °C to -15 °C ; at -40 °C/min from -15 °C to -60 °C. In the 2nd phase, a fixed freezing rate was applied at -30 °C /min from 4 °C to -15 °C. Six freezing rates were applied at -10, -20, -30, -40 (control), -50 or -60 °C/min from -15 °C to -60 °C. The freezing from -60 °C to -140 °C were fixed at -50 °C/min in both the phases. Post thaw semen quality was assessed in terms of motility, viability, membrane integrity (hypo-osmotic swelling test), sperm abnormalities, and active mitochondria. Data were arc sine transformed and analyzed through one-way analysis of variance using SPSS software. Results: In the 1st phase, percent individual motility, progressive motility and viability were similar among various protocols. Percent hypo-osmotic swelling reactive sperm was higher with freezing at -30 °C/min. In the 2nd phase, percent individual motility, viability and hypo-osmotic swelling reactive sperm was higher with freezing at -50 °C /min. Sperm head abnormalities were lower at -30 °C /min in the 1st phase, but were similar among the protocols of the 2nd phase. Percent active mitochondria were higher at -30 °C /min in the 1st phase and at -50 °C/min in the 2nd phase. Conclusions: The optimum post thaw semen quality of buffalo bull could be obtained by applying freezing rate at -30 °C/min (4 °C to -15 °C) and at -50 °C/min (-15 °C to -140 °C ), followed by plunging of straws in into liquid nitrogen for storage.

Comparison of follicular dynamics, superovulatory response, and embryo recovery between estradiol based and conventional superstimulation protocol in buffaloes (Bubalus bubalis).

Aim: To evaluate the follicular dynamics, superovulatory response, and embryo recovery following superstimulatory treatment initiated at estradiol-17β induced follicular wave emergence and its comparison with conventional superstimulatory protocol in buffaloes. Materials and Methods: Six normal cycling pluriparous buffaloes, lactating, 90-180 days post-partum, and weighing between 500 and 660 kg were superstimulated twice with a withdrawal period of 35 days in between two treatments. In superstimulation protocol-1 (estradiol group) buffaloes were administered estradiol-17β (2 mg, i.m.) and eazibreed controlled internal drug release (CIDR) was inserted intravaginally (day=0) at the random stage of the estrous cycle. On the day 4, buffaloes were superstimulated using follicle stimulating hormone (FSH) 400 mg, divided into 10 tapering doses given at 12 hourly intervals. Prostaglandin F2α analogs (PGF2α) was administered at day 7.5 and day 8, and CIDR was removed with the second PGF2α injection. In superstimulation protocol - 2 (conventional group) buffaloes were superstimulated on the 10th day of the estrous cycle with same FSH dose regimen and similar timings for PGF2α injections. In both groups, half of the buffaloes were treated with luteinizing hormone (LH) 25 mg and other half with 100 ug buserelin; gonadotrophin releasing hormone (GnRH) analog at 12 h after the end of FSH treatment. All buffaloes in both protocols were inseminated twice at 12 and 24 h of LH/GnRH treatment. Daily ultrasonography was performed to record the size and number of follicles and superovulatory response. Results: Significantly higher number of small follicles (<8 mm) was present at the time of initiation of superstimulatory treatment in the estradiol group compared to the conventional group (12.5±0.80 vs. 7.3±1.21, respectively, p=0.019), however, the number of ovulatory size follicles (≥8 mm) did not differ significantly between the respective groups (15.5±1.24 vs. 12.2±1.30; p=0.054). Total embryos and transferable embryos recovered were non-significantly higher in the estradiol group compared to the conventional group (5.83±0.86 vs. 4.67±1.16, p=0.328, and 3.67±0.93 vs. 2.67±0.68, p=0.437, respectively). The significant higher proportion of transferable embryos were recovered in buffaloes treated with LH compared to GnRH (73.3% vs. 48.5%; p=0.044). Conclusion: The average number of ovulatory size follicles (>8 mm), corpora lutea, and transferable embryos was higher in buffaloes superstimulated at estradiol-induced follicular wave compared to the conventional protocol: Further the percentage of transferable embryos was significantly higher in buffaloes administered with LH compared to GnRH.

Effect of fish meal supplementation on production and biochemical alterations in dairy buffaloes during early postpartum period

Abstract Objective To assess the effects of supplementary feeding of fish meal on body condition score, milk production and associated blood biochemical alterations if any, in dairy buffaloes during early postpartum period. Methods Ten pluriparous buffaloes belonging to organized dairy farm were supplemented with 250 g fish meal (FM) daily from day of calving for 90 days postpartum and 5 buffaloes were kept as unsupplemented control. Heparinized venous blood samples were collected from each buffalo on day 0 and 15 days interval thereafter till 75 days postpartum. Body condition score and milk yield was recorded at fortnightly and weekly intervals respectively. Blood samples were analyzed for glucose, total protein, cholesterol, BUN and triglycerides. Results The fish meal supplementation did not affect the body condition score (2.90 ± 0.08 vs . 2.92 ± 0.10 and 2.31 ± 0.04 vs . 2.25 ± 0.09, at start and end of fish meal supplementation in supplemented and control buffaloes respectively) or the average milk production (3.30 ± 0.16 kg vs . 3.5 ± 0.17 kg in supplemented and control buffaloes respectively) significantly. The blood glucose and cholesterol concentrations increased during postpartum period (till 75 days postpartum) in both the groups and in a similar manner. Plasma blood urea nitrogen concentrations were significantly reduced in both groups in postpartum period ( P Conclusions The fish meal supplementation does not affect the observed biochemical parameters. From the study, it can be concluded that fish meal supplementation (250 g daily) do not alter either biochemical profiles or milk production and body condition of the buffaloes.