Factors Affecting Successful in Vitro Maturation, fertilization and Culture of Buffalo follicular Oocytes

Abstract

Development of an efficient in vitro maturation, fertilization (IVM-IVF) and culture system of embryos appears to be the most economical and useful technique for the improvement of productive and reproductive performance in the buffalo. However, the poor recovery of oocytes and lack of proper conditions to support IVM-IVF are the major impediments in successful development of such system for the buffalo. Therefore, these studies were planned to investigate the recovery in vitro maturation, fertilization agriculture oocytes obtained from the varies of slaughtered buffaloes. The ovaries were collected immediately after the slaughter from a local abattoir in a thermos containing physiological saline solution, with added antibiotics at body temperature (37°C). For recovery of oocytes from the ovaries, three methods were studied i.e., aspiration, puncture and scoring. The scoring method yielded significantly (P<0.05) greater number (3.85 per ovary) of morphologically good oocytes than puncture (2.56/ovary) and aspiration methods (1.76/ovary). Maturation rates of three categories of buffalo follicular oocytes (Type A, B and C) were 78.77, 75.59 and 75.75 percent respectively. There was non-significant (P<0.05) difference among their maturation capabilities. Four types of serum protein supplements including estrus cow serum (ECS), estrus buffalo serum (EBS), pro-estrus buffalo serum (PrBS) and post-estrus buffalo serum (PiBS) added to TCM-199 and evaluated for their effect on in vitro maturation and fertilization of buffalo follicular oocytes. The maturation rates observed were 80.00, 82.08, 78.77 and 66.23 percent respectively for inedia containing ECS, EBS, PrBS and PiBS. The corresponding values for fertilization/cleavage rates were 54.54, 55.38, 52.80 and 36.76 percent. The medium containing PtBS gave significantly (P<0.05) lower maturation (66.23%) and fertilization/cleavage rates (36. 76%) than the other three media. No evidence of parthenogenesis was observed. Studies were under taken to determine the possible effects of different factors on I harvesting of follicular oocytes. The results showed that the effect of season, age of the buffalo and size of ovary on the harvesting of oocytes was non-significant, while the absence of corpus luteum (CL) from ovary had a significantly positive effect on harvesting of oocytes. The ovaries with CL yielded significantly lower number of (P<0.05) total and usable oocytes per ovary (3.94 ± 0.28 and 2.85 ± 0.35) than ovaries without CL (5.90 ± 0.39 and 4.52 ± 0.52). It was concluded that buffalo ovaries without CL be preferred for the recovery of good quantity and quality of the follicular oocytes. For the insemination of in vitro matured oocytes, semen preparation was carried out by swim up, percoll, Bracket & Oliphant and washing the spermatozoa with 2.9 percent sodium citrate solution. The sodium citrate washing proved to be a better method to prepare buffalo bull spermatozoa for in vitro fertilization which not only gave higher percentage of motile spermatozoa (53.06 ± 4.57) but also longer liveability (29.40 ± 1.06 hr). When the buffalo spermatozoa prepared by different methods of sperm preparation were allowed to penetrate the oocytes, the difference among fertilization/cleavage rate was non-significant. The effect of bull on fertilization rates was significant (P<0.05). Washing of the liquid semen with 2.9% sodium citrate solution was easier, economical and found better to prepare spermatozoa for IVF than the other conventional methods. The effect of different media on in vitro maturation, fertilization and embryonic V development (8-cell stage) of oocytes was investigated. The maturation media culture medium (TCM-199). Bovine synthetic follicular fluid (BSFF) and Ham's F-10 were equally good (81.06, 79.36 and 75.29% respectively) for in vitro maturation of buffalo oocytes while the Dulbecco's phosphate buffered saline (DPBS) proved relatively poor medium (59.11 %) for these 2 purposes. Similarly, the early embryo development of the oocytes matured in TCM-199, Ham's Fl O and BSFF were non significantly different (48.3 8, 44. 94 and 41.89% respectively), while it was significantly lower (25.86%) for oocytes matured in DPBS. The development of IVM-IVF produced 2-cell buffalo embryos to morula stage in undefined TCM-199 (6.52%), Ham's F-10 (5.68%) and CZB (3.12%) and defined (CRlaa) medium (4.41%) was quite low. However, after conditioning Ham's F-10, TCM-199 and CZB with buffalo oviduct epithelial cells (BOEC) the rate of development-of early embryo to the morula stage was improved. The development rates were increased to 14.12, 16.67 and 11. 11 percent after 24 hr conditioning respectively and to 18.67, 20.00 and 16.16 percent respectively after 48 hr conditioning. The difference between 24 hr and 48 hr conditioning was non-significant. Although the conditioning of the media with BOEC has increased the development rate to morula stage in the buffalo but in general the rate of IVF embryos was low as compare to that obtained in exotic dairy cattle. Greater research efforts are needed to optimize procedures for in vitro maturation, fertilization and embryo production particularly considering the distinctive physiological peculiarities of this species.