Effect of different antioxidant additives in semen diluent on cryopreservability (-196°C) of buffalo semen.

AIM: The aim of this study was to evaluate the effect of different antioxidant additives in standard tris-fructose-egg yolk-glycerol (TFYG) extender on the cryopreservability of buffalo semen. MATERIALS AND METHODS: Semen collection using artificial vagina, twice weekly for 5 weeks from three pedigreed health breeding bulls of Mehsani breed, aged between 6 and 8 years. Immediately after initial evaluation all 30 qualifying ejaculates (10/bull) were split into three aliquots and diluted at 34°C keeping the concentration of 100 million spermatozoa/ml with standard TFYG extender as control and TFYG having two antioxidant additives - Cysteine HCl at 1 mg/ml and ascorbic acid at 0.2 mg/ml to study their comparative performance. Semen filled in French Mini straws using IS-4 system and gradually cooled to 4°C and equilibrated for 4 h in cold handing cabinet. After completion of equilibration, straws were cryopreserved in LN2 by Programmable Bio-freezer. Semen was examined at post-dilution, post-equilibration, and post-thaw stages for sperm quality parameters, and at each stage plasma was separated for enzymatic analysis of aspartate aminotransferase (AST), lactate dehydrogenase (LDH), and alkaline phosphatase (AKP). RESULTS: The mean percentage of sperms in TFYG, TFYG + cysteine HCl and TFYG + ascorbic acid diluents at post-thaw stage in terms of progressive motility (52.83±0.52, 57.83±0.52, 57.83±0.52), livability (78.70±0.21, 82.33±0.23, 81.73±0.22), and abnormality (5.43±0.21, 5.03±0.17, 5.23±0.18) varied significantly (p<0.05) between control TFYG and TFYG having antioxidant additives. The mean U/L activities of AST (78.70±0.47, 72.80±0.48, 73.30±0.54), LDH (172.70±0.41, 155.78±0.42, 156.33±0.41), and AKP (103.61±0.34, 90.20±0.34, 91.03±0.34) in semen diluted with TFYG, TFYG + cysteine HCl and TFYG + ascorbic acid diluents at post-thaw stage, respectively, which showed significantly (p<0.05) higher leakage of enzymes in control TFYG than TFYG incorporated with additives. CONCLUSION: Incorporation of antioxidant additives such as cysteine HCl and ascorbic acid in standard TFYG diluents improves sperm quality parameters, reduces enzyme leakage, and ultimately advances cryopreservability of buffalo semen.

Is slow follicular growth the cause of silent estrus in water buffaloes?

The present experiment was conducted to study the growth profile of the ovulatory follicle in relation to the expression of estrus following administration of PGF(2alpha) to subestrus buffaloes. After detection of a mature corpus luteum by examination per rectum, confirmed by ultrasound scanning, subestrus buffaloes (n=20) were treated (Day 0) with single dose of Dinoprost tromethamin (25 mg, i.m.). Blood samples were collected at 0, 24 and 48 h after treatment for estimation of plasma progesterone concentration. Growth profile of the ovulatory follicle was monitored daily through ultrasound scanning starting from Day 0 until ovulation and the regression profile of CL was monitored at 0, 24 and 48 h of treatment. Estrus was detected by exposure to a fertile buffalo bull three times a day until expression of overt estrus or ovulation. Behavioral estrus was recorded in 14 animals and 6 animals ovulated silently. Sixteen animals including six animals with silent estrus ovulated from the dominant follicle present at treatment (Group A) and remaining four animals ovulated from the dominant follicle of succeeding follicular wave (Group B). The intervals from treatment to estrus (6.5+/-0.25 versus 3.2+/-0.27 days, P<0.001) and treatment to ovulation (7.5+/-0.25 versus 5.4+/-0.46 days, P<0.005) were significantly longer in animals of Group B compared with animals of Group A. Significant differences were observed in growth profile of the ovulatory follicle between animals of Groups A and B with respect to size of the follicle on Day 0 (9.8+/-0.7 versus 5.3+/-0.45 mm, P<0.001), daily growth rate (0.97+/-0.07 versus 1.6+/-0.2 mm/day, P<0.01) and increase in diameter (4.1+/-0.6 versus 7.8+/-0.7 mm, P<0.01). The animals with silent estrus (subgroup A-2) had significantly smaller diameter of the ovulatory follicle on Day 0 (7.7+/-0.4 versus 11.0+/-0.7 mm, P<0.005), its daily growth rate was significantly slower (0.7+/-0.02 versus 1.1+/-0.1 mm/day, P<0.01) and they recorded significantly longer interval from treatment to ovulation (7.3+/-0.56 versus 4.2+/-0.27 days, P<0.001) compared with the animals that showed overt estrus (subgroup A-1). The corpus luteum area (CL area) and plasma progesterone (P(4)) concentration declined continuously from 0 to 48 h after PGF(2alpha) treatment in the animals of both the Groups A and B. Non-significant differences were observed in mean CL area and plasma P(4) concentration at 0, 24 and 48 h post-treatment between animals of Groups A and B and also between animals of subgroups A-1 and A-2. The small size and the slow growth rate of the ovulatory follicle were identified as the possible cause of silent estrus in subestrus buffaloes after PGF(2alpha) treatment.

Is one-wave follicular growth during the estrous cycle a usual phenomenon in water buffaloes (Bubalus bubalis)?

The pattern of growth and regression of ovarian follicles was monitored once daily for one complete estrous cycle in eight individual water buffaloes by ultrasonographic scanning of the ovaries for an entire interovulatory interval of normal cycle length. One-wave follicular growth was observed in five animals and two-wave follicular growth in three buffaloes during the estrous cycle. The first follicular wave of a two-wave cycle emerged significantly earlier (P < 0.05) than the emergence of the solitary wave of a one-wave cycle. One- and two-wave cycles differed significantly (P < 0.05) with respect to the mean interovulatory interval (21.0 +/- 0.54 days versus 22.7 +/- 0.33 days) and the mean interestrus interval (20.8 +/- 0.58 days versus 22.3 +/- 0.66 days). The overall linear growth rate of the ovulatory follicle was significantly greater (P < 0.01) in a two-wave cycle compared to that of a one-wave cycle (1.17 +/- 0.33 mm/day versus 0.32 +/- 0.01 mm/day). In a one-wave pattern, the growth profile of the solitary dominant follicle was atypical, showing three distinct phases, i.e. growth phase, regression phase and regrowth phase culminating in ovulation. The level of plasma progesterone steadily increased from day 0 of estrous cycle, attained peak level on day 14 and declined thereafter. A slower growth rate of the dominant follicle was observed in the presence of higher plasma progesterone concentration. The present study shows that one-wave follicular growth is a normal phenomenon in suckled water buffaloes.