Pak. J. Pharm. Sci., Vol.28, No.1, January 2015, pp.95-101 95 Effect of gonadotrophins, oestradiol and insulin on cumulus expansion of Nili Ravi buffalo oocytes Beenish Shahid 1 , Samina Jalali 2 , Muhamad Ijaz Khan 2 and SA Shami 2 1 Department of Zoology University of Azad Jammu and Kashmir, Muzaffarabad, Pakistan 2 Department of Animal Sciences, Faculty of Biological Sciences, Quaid-i-Azam University, Islamabad, Pakistan Abstract: The objective of the present study was to investigate the cumulus expansions of Nili Ravi buffalo oocytes during cultured in TCM -199 supplemented with 2 µg/ml oestradiol (E 2 ), 0.05 IU/ml recombinant human follicle stimulating hormone (rhFSH), 2IU/ml human chorionic gonadotrophin (hCG), and 0.12 IU/ml insulin (I). The cumulus oocytes complexes (COCs) were collected from 2-8mm follicles from local abattoir ovaries. Supplementation of medium with single hormones showed significant (P<0.0001) increase in mean diameter of COCs with rhFSH except E 2 , hCG and insulin after 24 hours compared to the increase in the mean diameter of COCs matured in TCM-199 without any hormonal supplementation. With rhFSH even at 8th hour, significant increase (P<0.001) in cumulus expansion was observed. In combination of hormones the significant (P<0.0001) cumulus expansion was achieved in E 2 +rhFSH treatment group. The non significant (P>0.05) cumulus expansion was observed in treatment groups viz. E 2 +hCG, E 2 +Insulin, rhFSH+hCG, rhFSH+Insulin, hCG+Insulin, E 2 +rhFSH+hCG and E 2 +rhFSH+hCG+Insulin after 24 hours. In conclusion, supplementation of rhFSH alone and in combination with E 2 in TCM-199 has highly significant effect on cumulus expansion. Keywords: In vitro, buffalo, oocytes, gonadotrophins, insulin. INTRODUCTION In vitro production of buffalo embryos for research and commercial purpose has been an increasing interest (Mehmood et al., 2011). Because of poor superovulatory response the progress in the use of embryo transfer in the buffalo is very slow.The recovery of immature oocytes followed by in vitro maturation and fertilization is an attractive alternative to in vitro fertilization (Khalili et al., 2013). The culturing and maturing oocytes that have been harvested at an immature stateare defined as in vitro maturation of oocytes (Smith, 2001). As a result of the preovulatory surge of gonadotropins, in mammalian ovarian follicles the cumulus cells undergo a process of expansion while the residing oocytes undergo a process of resumption and complete meiotic maturation (Sutovsky et al., 1994; Sutovsky et al., 1995; Prchazka et al., 2000). During ovulation the expansion of cumulus cells maintains viability of the ovulated oocytes within the oviduct (Hess et al., 1999). The cumulus expansion is responsible for the creation of the proper micro environment for sperm motility and activation (Tesarik et al., 1988; Chen et al., 1993). In general, cumulus expansion and oocyte maturation is regulated by follicle- stimulating (FSH) hormone and (LH) hormone (Lee et al., 2007). To understand the mechanisms that regulate expansion of cumulus will thus lead to improvement of conditions for mammalian oocytes to be matured and fertilized in vitro (Prchazka et al., 2003). FSH can induce in vitro cumulus expansion in mouse, pig, rabbit and cattle (Eppig, 1979; Salustri et al., 1992). The 10µg/ml lectin concentrations showed a significant effect on cumulus expansion in buffalo oocytes (Pandey et al., 2009). For cytoplasmic maturation optimal cumulus expansion is very essential (Testart et al., 1983; Chen et al., 1993). The incidence of oocytes penetration is increased by cumulus expansion in bovine (Ball et al., 1983). The steroids in follicular fluid may play a role in in-vitro maturation of oocytes (Anisworth et al., 1980). Oocytes are been kept in meiotic arrest by the involvement of estradiol (E 2 ) and other steroids (McGaughey, 1977; Richter and MeGaughey, 1979; Smith and Tenney, 1980; Racowsky and McGaughey, 1982; Kaji et al., 1987; Barrett et al., 1996; Mingoti et al.,1995). The insulin (I) and insulin-like growth factor-I (IGF-I) proliferate the granulose cells and stimulate the progesterone production (Gong et al., 1993; Spicer et al., 1993). Therefore, it is expected that insulin and the growth factor IGF-I have some beneficial effects on bovine oocytes cultured in vitro. The cumulus expansion and oocyte fertilizability during in-vitro conditions can be improved by supplementation of maturation media with insulin (Lorenzo et al., 1994; Webb et al., 1996), but it was reported in some other studies that the supplementation of insulin in culture medium did not improve the fertilization rate or morula formation significantly (Stubbings et al., 1990). *Corresponding author: e-mail: beenishkhan124@gmail.com