Use of Cystic Folucui.ar Fluid for Buffalo Oocyte Maturation in Vitro

Studies were conducted to examine the effect of substitution of serum with cystic follicular fluid in the IVM media and also to test the efficacy of cystic follicular fluid (buCFF) at 50 and 100 per cent levels for in vitro maturation of buffalo oocytes. Resutts indicated that buffalo cystic follicular fluid supplemented with PMSG possess the ability for induction of maturation of buffalo oocytes and could be successfully tried as buffalo oocyte maturation medium. Buffalo cystic follicular fluid as a whole maturation medium· also has the ability to induce the cumulus expansion which may be cost effective for the IVM of buffalo oocytes. INTRODUCTION Reproductive biotechnologies like embryo transfer technology (ETT), in vitro production (lVP) of embryos has made a tremendous advancement in the last decade. Since application of embryo transfer technology (ETT) in buffalo has had only a limited success due to poor superovulatory responses (Madan, 1996) IVP of embryos in buffalo is drawing growing interest in the rEj!cent times. The maximum rate of production of embryos in vitro depends largely on optimum in vitro maturation of oocytes both cytoplasmic and nuclear. Generally in buffalo IVM studies, oocytes are cultured in media supplemented with fetal. calf serum or hormones (Palta and Chauhan, 1998, Nandi et a( 2002a) which make the technology highly expensive. To make this technology cost effective and the cost of embryo within reach of farmers, substitution of costly media, hormones and fetal calf serum have been tried. Folliculadluids from normal follicles are being successfully incorporated in IVM media of buffato oocytes. The replacement level of media with follicular fluid was 40% (Chauhan eta/., 1997) and 100% (Gupta et aJ., 2001). Fluids contained in follicular cysts, are expected to contain varied amounts of steroids, gonadotrophin and growth factors . which may help in maturation of oocytes. In cattle, cystic follicular fluid supplemented with PMSG induced maturation of oocytes in vitro (Takagi eta1., 1998). Any evidence on the effect of cystic follicular fluid on in vitro maturation of buffalo oocytes is lacking. The aim of the present study is to examine .the effect of substitution of maturation medium with cystic follicular fluid in the IVM media and also to test its efficacy as a complete medium for the buffalo oocyte maturation. MATERIAL AND METHODS Buffalo ovaries collected from a local slaughter house were transported to the laboratory in 0.9% normal saline in a thermoflask within 1 hours of slaughter. Oocytes were aspirated from 2-6 mm follicles using an 18G hypodermic needle attached to a syringe. The aspiration medium consisted of Tissue Culture Medium (TCM)-199 supplemented with 10% steer serum and phosphate buffered saline (PBS) supplemented with 0.3% bovine serum albumin(BSA) at 1:1 ratio. Oocytes were graded by the morphological appearance of the cumulus cells and ooplasm under zoom stereomicroscope. Oocytes with more than two layers of compact cumulus cells and evenly granular homogenous ooplasm were used for the studies. Buffalo ovaries with cystic follicles over 30 mm in diameter and without a functional CL were collected immediately after slaugnter of the animals at the local abattoir and transported Vol. 37, No.1, 2003 41 in normal saline (0.9% NaCl) supplemented with gentamycin (50 Jlg/ml) to the laboratory within 1hr. Fluid was aspirated from cystic follicles of the ovaries using 18 gauge needle attached to a syringe. The cystic fluid was then poured in a conical centrifuge tube (15 ml) and centrifuged at 1500 rpm for 5 min to remove -the cellular debris. The supernatant was collected and was then heat inactivated at 50°C for 30 min. Steer serum (SS) was collected by clotting the blood of a castrated cattle bullock and was then heat inactivated at 50°C for 30 min. Both cystic follicular fluid and steer serum were filtered (0.22Jlml; sterilized and stored in 2ml aliquots at -20°C until use. Same batch of pooled cystic follicular fluid and steer serum were used for all the trials. In experiment 1, which aimed to determine the effect of substitution of steer serum with cystic follicular fluid in the IVM media, 7 to 12 oocytes were cultured in a single drop with each of the four diff~rent media . studied: i) TCM199 + SS (10%), ii) TCM199 + buCFF (10%), iii) TCM199 + SS (10%) + .PMSG (40IU/ml), iv) TCM199 + buCFF (10%) + PMSG (40IU/ml). In experiment 2, which was conducted to test whether cystic follicular fluid can be used at higher levels, oocytes were cultured in follOWing four media: i) TCM-199, ii) TCM199 + buCFF (50%), ii) TCM199 + buCFF (50%) + PMSG (40IU/ml), iii) buCFF + PMSG (40IU/ml), iv) buCFF (100%). Oocytes were cultured for 24 hr at 38.5°C in CO2 incubator (5% CO2 in air). Steer serum and PMSG were added at 10 % level and 40 I.U./ml as these concentrations were found to be optirrtilm for in vitro maturation of oocytes in an earlier study in this laboratory (Nandi et al, 2001). Maturation of oocytes were assessed on the basis of cumulus cell expansion as well as extrusion of first polar body in the perivitelline space after 24 hours of incubation (Nandi et al., 2002b). The statistical analysis was carried out in 8 replicates and comparison of each treatment by one way analysis of variance (ANOVA) and t-test (Snedecor and Cochran, 1980) with a probability level of' P<0.05·considered significant. RESULTS AND DISCUSSION Replacement of steer serum with buffalo cystic follicular fluid in the oocyte culture media without any exogenous gonadotrop'hin (PMSG) resulted in significant (P<0.05) increment in the oocyte maturation rate. In presence of PMSG, no significant difference in maturation rate was observed in oocytes cultured in media containing steer serum or cystic follicular fluid (Table 1). High rates of maturation were observed when oocytes were cultured in high~r levels of cystic follicular flUid. When oocytes were cultured in media containing 50% and 100% v/v cystic follicular flUid, addition of PMSG did not significantly increase the maturation rates of the oocytes. Total replacement of the basic oocyte culture' media (TCM-199) with buffalo cystic follicular flUid resulted in significant (P<0.05) increase in maturation rate of 86% (Table 2) without any hormonal supplementation. The follicular fluid is reported to be consisted of various growth factors, Follicle stimulating hormone, Leutinizing hormone and several other nutrients though it may contain oocyte maturation inhibitory factor. Cystic follicular flUids contain different concentrations of various sterOids like estradiol-1 713, progesterone, estrone, androstenedione. Estradiol-1713 is the major steroid in normal ovarian follicles, and its concentration is significantly lower in follicular cysts (Melven etal, 1963). Increased cumulus expansion and maturation rate of buffalo oocytes in our study may not be due to. presence of large concentrations of steroids as the role of steroids in oocytes maturation is still controversial (Nandi et al, 2002b). Addition of bovine follicular fluid at 60% v/v (Kim et al, 1996) or 42 INDIAN JOURNAL OF ANIMAL RESEARCH Table 1. Effects of substitution of steer serum with cystic follicular f1uld On in vitro maturation of buffalo oocytes Treatments Oocytes Oocytes Maturation rate, cultured matured (%±5EM) TCM199 + 55 (10%) B6 30 34.8±2.3" TCM199 + buCFF(1O%) 96 6365.6±3.1b . TCM199 + 55 (10%) + PMSG (401U/mI) 102 88 86.2±2.3' TCM199 + buCFF (10%) + PMSG (401U/mI) 101 81 81.1±3.3' Values with different superscripts in the same column differ significantly (P<0.05). TCM Tissue culture medium; 55 steer serum; PM5G Pregnant mare serum gonadotrophin; buCFF buffalo cystic follicular fluid. Table 2. Effect of use of higher levels of cystIC foUicular flUid on maturdtion of buffalo oocytes in vitro Treatments Oocytes Oocytes Maturation rate cultured matured (%±SEM) TCM-199 97 17 179±2.7", TCM199 + buCFF (50%) 95 11 74.73±3.461< TCM199 + buCFF (50%) + PMSG (401U 1m!) 93 79 84.94±2.641>< buCFF +.PMSG (401U 1m!) 94 77 80.85±2.86" buCFF 112 96 85.71±3.42' Values with different ~p(!~ript; i~thi~~e~oTu~ndiffur slgnlflc.;htlY(PThe authors thank Dr. Khub Singh,Director, National Institute of Animal Nutrition .and Physiology for providing the necessaryfacilities for carrying out this work. REFERENCESAyoub, M.A. and Hunte, RAG. (1993). J. Dairy Sci, 76: 95-100.Borromeo, V. eta/. (1996). Theriogenology, 46: 481-489.Chauhan, M.S. etal (1997). Theriogenology, 48: 461-469.Fenton, S.E. and Ax. A.L. (1990). Bioi. Reprod., 42( Suppl.l ): 89 (Abstract).Gupta, P.S.P. eta/. (2001). AsianAust J. Anim. Scl, 14: 693-696.Kim, K.S. (1996). Theriogenology, 45: 787-799.Madan, M.L. eta/. (1996). Anim. Reprod. 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