27 Delayed puberty, poor expression of estrus, high incidence of early embryonic mortality, long inter-calving period and seasonal variation in fertility compromise the productivity of buffalo (Bubalus bubalis). Although some information is now available on the peripheral hormonal profiles of various hormones involved in reproduction (Mondal et al. 2007), very few studies have been conducted at the ovarian level in this species (Bhushan et al. 2004, 2005). Granulosa cells are the site of production of steroid hormones namely, estradiol-17–β and progesterone, growth factors like IGF-I, IGF-II, EGF and the inhibin family of hormones, i.e. inhibin, activin and follistatin, which are involved in maintenance and control of ovarian and hypophysial function through autocrine, paracrine and endocrine pathways. Nitric oxide (NO) is a highly reactive free radical, which is synthesized via the oxidation of L-arginine to NO and citrulline by nitric oxide synthases (NOS). It gained attention as a paracrine/autocrine regulator of granulosa cell steroidogenesis (Dixit and Parvizi 2001, Tamanini et al. 2003). It is reportedly present in follicular fluid in cattle, its concentrations being higher in follicular fluid from small than from large follicles (Basini et al. 1998). NO was found involved in various functions in granulosa cells from different species. Effects of NO on steroidogenesis by granulosa cells were studied in several farm animal species including cattle (Basini et al. 1998, 2001, Faes et al. 2009), pig (Masuda et al. 1997, 2001, Ponderato et al. 2000, Graselli et al. 2001) and horse (Pinto et al. 2002). However, there is no information available on the effects of NO on steroidogenesis by buffalo granulosa cells. The present study was, therefore, undertaken to determine if NO affects estradiol-17β and progesterone production by buffalo granulosa cells. MATERIALS AND METHODS Isolation of granulosa cells: Buffalo ovaries were collected immediately after slaughter in an abattoir and washed 3 times with chilled (4°-10°C) isotonic saline (0.9% NaCl) containing 100 IU/ml penicillin and 100 μg/ml streptomycin. After transport to the laboratory within 5–6 h in chilled saline, the ovaries were washed 3 times with saline at room temperature. Henceforth, the ovaries were kept at room temperature until the collection of granulosa cells was Present address: 1 Scientist (dr_shan@rediffmail.com), Project Directorate on Poultry, Rajendranagar, Hyderabad. 2 Principal Scientist (sujata.pandita@rediffmail.com), DCP Division; 3 Principal Scientist (prabhatpalta@yahoo.com), Animal Biotechnology Center. Indian Journal of Animal Sciences 83 (1): 27–31, January 2013/Article Inhibitory effects of nitric oxide on steroidogenesis by buffalo granulosa cells cultured in vitro M SHANMUGAM 1 , S PANDITA 2 and P PALTA 3 National Dairy Research Institute, Karnal, Haryana 132 001 India Received: 6 January 2011; Accepted: 28 August 2012 ABSTRACT This study examined the effects of S-nitroso-N-acetyl-penicillamine (SNAP), a nitric oxide (NO) donor and N w - nitro-L-arginine methyl ester hydrochloride (L-NAME), a nitric oxide synthase inhibitor on estradiol-17β and progesterone production by buffalo granulosa cells. Granulosa cells (3×10 5 ) from small ( <5 mm diameter) or large ( > 9 mm diameter) follicles were cultured for 24 h under completely serum-free conditions in DMEM: nutrient mixture F-12 Ham (1:1 ratio) supplemented with 10 –7 M androstenedione, 5 mg/ml human apo-transferrin, 0.1% BSA, in the presence or absence of FSH (8 ng/ml). Granulosa cells from large follicles produced higher estradiol-17β and progesterone than those from small follicles. SNAP reduced estradiol-17β and progesterone production at concentrations of 0.1 and 1 mM when used alone and in the presence of FSH by granulosa cells from follicles of both size categories. L-NAME (0.2, 1 and 5 mM) had, however, no effect on estradiol-17β or progesterone production, alone or in the presence of FSH. Our results demonstrated strong inhibitory effects of NO on estradiol-17β and progesterone production by buffalo granulosa cells from small and large follicles and indicated that it may be an autocrine/paracrine regulator of steroidogenesis by granulosa cells in buffalo. Key words: Buffalo, Granulosa cells, L-NAME, Nitric oxide, SNAP, Steroidogenesis