IVF study

Buffalo ovaries were collected from a local abattoir, Delhi regardless of the oestrous cycle stage and were transported within 2-3 h to the laboratory in physiological saline (0.9%, w/v NaCl) containing strepto-penicillin (50 mg/l). Ovaries were washed 3–4 times in normal saline and the cumulus-oocyte complexes (COCs) were aspirated from the follicles with the help of a vacuum aspiration unit (K-MAR-5200 IN, USA) in Hepes-buffered hamster embryo culture (HH) medium and the aspirated follicular fluid was placed in a dry bath at 37.5 °C for 35–40 min. The quality of COCs was observed and graded into grade A (> 5 layer cumulus layer) and B (3–5 cumulus layers) under the stereo-zoom microscope. The COCs of only grade A and B were selected for in vitro maturation (IVM) and in vitro culture (IVC). To perform IVM, the selected COCs were washed 3–4 times with maturation media (HEPES buffered TCM199 modified with 10% (v/v) fetal bovine serum (FBS), 0.005% (w/v) streptomycin, 0.01% (w/v) sodium pyruvate and 0.005% (w/v) glutamine supplemented with 5.0 μg/ml FSH and 10 μg/ml LH, 1 μg/ml estradiol 17-β and 50 ng/ml epidermal growth factor (EGF), 64 μg/ml cysteamine and 50 μl ITS). After washing the COCs were placed in a group of 15 in 100 μl of maturation medium drops. Three such drops were placed in a 35 mm culture dish which was then overlaid with mineral oil dressing. The dishes were cultured in duplicate for 24 h at 38.5 °C in a humidified atmosphere of 5% CO₂ in an incubator. The IVF was carried out in 100 μl droplets of BO medium supplemented with 1% (w/v) bovine serum albumin (BSA), 1.9 mg/ml caffeine sodium benzoate, 0.14 mg/ml sodium pyruvate and 0.01 mg/ml heparin. For the IVF, the control group didn’t contain anti-BuBD-129 antibody whereas, the three treatment groups comprised of three different concentrations of anti-BuBD-129 (0.5 mg/ml) antibody in the fertilization medium drops viz. the 1:15000, 1:10000 and 1:5000 dilution groups were prepared. The matured COCs were washed thrice in BO medium and placed in BO medium droplets supplemented with the anti-BuBD for treatment groups. The frozen buffalo semen was thawed simultaneously and subsequently processed for in vitro capacitation as per the procedure described earlier by Jain et al. [97]. Fifty μl of BO media was removed from the respective IVF medium drops and 50 μl of the sperm suspension (1 × 10⁶ spermatozoa/mL) was added to each fertilization drop with 15 COCs and was then incubated at 38.5 °C with 5% CO2 for 12 h. The presumptive zygotes were removed from the fertilization drops after 12 h of insemination and the adhered cumulus cells were mechanically removed by vortexing and were washed five times in a modified Charles and Rosenkrans 2 amino acid (mCR2aa) medium. Following washing, the 15 presumptive zygotes were co-cultured with the monolayer of granulosa cells in 100 μl drops of IVC-I medium (mCR2aa supplemented with 0.8% (w/v) BSA, 1 mM glucose, 0.33 mM pyruvate, 1 mM glutamine, 1 x MEM essential amino acid, 1x non-essential amino acid and 50 μg/ml gentamycin). After 48 h post insemination (hpi) zygotes were evaluated for evidence of cleavage. At 72 hpi, all the cleaved embryos were transferred to IVC-II medium (same as IVC-I with 10% FBS) and were maintained for 8 days post insemination at 38.5 °C with 5% CO₂. The culture medium was replaced regularly at an interval of 48 h. A total of four biological replicates were used for the IVF experiments. The data were analyzed on GraphPad prism7 (for Windows, GraphPad Software, La Jolla California USA, www.graphpad.com) to compare the observed differences in cleavage and blastocyst rates among the control and treatment groups.