In vitro Maturation and Fertilization of Riverine Buffalo Follicular Oocytes in Media Supplemented with Oestrus Buffalo Serum and Hormones

Jamil H., H.A. Samad, N.U. Rehman, Z.I. Qureshi, L.A. Lodhi: In vitro Maturation and Fertilization of Riverine Buffalo Follicular Oocytes in Media Supplemented with Oestrus Buffalo Serum and Hormones. Acta Vet. Brno 2007, 76: 399-404. Effects of two maturation media (TCM-199 and Ham’s F-12) with and without the addition of oestrus buffalo serum (OBS) and hormones (FSH, LH, E2) on the maturation rate of buffalo follicular oocytes were evaluated. The results revealed a signifi cant (P < 0.05) increase in the maturation rate when the OBS and hormones were added to TCM-199 than in Ham’s F-12 medium. The percentage of maturation rates in TCM-199 + hormones + OBS, TCM-199 + hormones, TCM-199 + OBS and TCM-199 were 77.44, 55.17, 62.28 and 26.62 percent, respectively. While in Ham’s F-12 + OBS + hormone, Ham’F-12 + hormone, Ham’s F-12 + OBS and Ham’s F12 were 32.85, 27.52, 31.38 and 13.46 percent, respectively. A signifi cantly higher (P < 0.05) fertilization rate was recorded for modifi ed Ca2+ free Tyrode’s medium (63.72%) than in TALP (10.9%) and IVF-TL (32.18%). Thus, TCM-199 containing hormones and OBS appeared better for in vitro maturation, whereas modifi ed Ca2+ free tyrode’s medium was found to be more suitable for in vitro fertilization of buffalo follicular oocytes. Reproduction, Bubalus bubalis, TCM-199, Ham’s F-12, FSH, LH, E2 Riverine buffaloes (Bubalus bubalis) are hardy dairy animals, resistant to climate, stress and diseases. However, problems like delayed onset of reproductive maturity, seasonality of breeding, long calving interval, latent oestrus, low number of primordial follicles and poor superovulatory response have been attributed to poor reproductive performance of this species (Nandi et al. 2002). In vitro maturation (IVM) and in vitro fertilization (IVF) procedures performed on oocytes obtained from slaughter-house derived ovaries have recently provided a practical means for producing large number of bovine zygotes at low cost for research and commercial settings (Hansen 2006). Application of this technology in assisted reproduction of buffalo will not only improve productive and reproductive potential of the buffalo population but will also help rescue the precious germ plasma going to waste by indiscriminate slaughter of this animal. The acquisition of more insights into the buffalo culture requirements is critical to optimize the effi ciency of advanced reproductive strategies in this species. Lack of proper conditions to support in vitro culture of buffalo zygotes to a transferable stage embryo is the major impediment in the successful buffalo IVF system. The direct extrapolation of the methods from cattle to the buffalo resulted in poor fertility. Chauhan et al. (1997) reported that oocyte maturation and cleavage rates in buffalo are lower than those for cattle. There have been reports to enhance early development of in vitro produced embryos in domestic animals by addition of hormones, sera and somatic cells (Pawshe et al. 1996). To optimize in vitro embryo production of buffalo by improving the culture conditions, the present project was designed: 1) to assess the effect of addition of sera and hormones on in vitro maturation of buffalo oocytes and 2) to evaluate media in supporting fertilization of in vitro matured oocytes. ACTA VET. BRNO 2007, 76: 399-404; doi:10.2754/avb200776030399 Address for correspondence: H. Jamil Department of Animal Reproduction Faculty of Veterinary Science University of Agriculture Faisalabad 38040, Pakistan E-mail: [email protected] http://www.vfu.cz/acta-vet/actavet.htm Materials and Methods Collection of oocytes Ovaries from sexually mature buffaloes were collected within 30 min after slaughter from the local abattoir and transported to the laboratory in a vacuum fl ask containing sterilized phosphate buffered saline (PBS: at pH 7.35) supplemented with 100 i.u. penicillin G and 100 g/ml of streptomycin at the temperature maintained at 25 – 30 °C (Chauhan et al. 1997). In the laboratory extraneous tissue was removed and ovaries were washed with 70% ethanol, followed by three rinses in PBS. Cumulus oocyte complexes (COC’s) were recovered by scoring method (Samad et al. 1998). Oocytes possessing a full cumulus mass, unfragmented cytoplasm and intact zona were selected for further processing. In vitro maturation For in vitro maturation of COC’s, two types of culture media were used: a) Tissue Culture Medium 199 (TCM199 Sigma) and b) Ham’s Nutrient mixture F12 (Ham’s F-12 Sigma) either with or without the addition of Follicle Stimulating Hormone (FSH: 5μg/ml), Lutenizing Hormone (LH: 5μg/ml), Estradiol 17(1.5 g/ml) and gentamycin 10 g/ml (Sigma). To study the effect of oestrus buffalo serum (OBS) on the maturation rate of buffalo follicular oocytes, the media with and without the addition of OBS (20%) were prepared. The pH of all the media was adjusted to 7.3 7.4. Drops of 200 l of maturation media were prepared in a Petri dish covered with mineral oil and equilibrated in the incubator providing atmosphere containing 5% CO2 and 95% humidity. Selected oocytes were washed four times in fresh pre-warmed TL-Hepes medium and subjected to a fi nal wash with in vitro maturation medium before transferring to the drops. Batches of 10 20 oocytes were then transferred to the drops of TCM-199 and Ham’s F-12 in different containing; i) hormones; ii) hormones and OBS; iii) OBS without hormones and iv) without hormones and OBS. The drops in Petri dishes were covered with sterile mineral oil (Sigma) and were incubated at 39 °C in 5% CO2 in air for 24 h. The maturation of oocytes was evaluated under stereomicroscope. Oocytes having expanded cumulus cells with extrusion of the polar body were considered matured. Oocytes matured in TCM-199 containing hormones + OBS, hormones or OBS only were subjected further to in vitro fertilization. In vitro fertilization Fertilization media i.e. Tyrode albumin lactate pyruvate (TALP), modifi ed Ca2+ free Tyrode’s and IVFTL media were used to determine the fertilization rate of the in vitro matured buffalo follicular oocytes. For IVF, modifi ed Ca2+ free Tyrode, IVF-TL or TALP media’s adjusted to pH 7.8 were used. Fresh semen from a buffalo bull was used for the insemination of matured oocytes. For capacitation spermatozoa were prepared using swim up method described by Chohan and Hunter (2003) with modifi ed Ca2+ free Tyrode medium. The sperm concentration was adjusted to 1 × 106 for the insemination of oocytes, and 50 l drops of three types of fertilization media were prepared i) TALP ii) IVF-TALP and iii) Modifi ed Ca2+ free Tyrode medium. Batches of oocytes (5 10/drops) and sperm cells were co-cultured in the incubator at 39 °C with 5% CO2 and humidifi ed air for 24 h. Fertilization/cleavage rates in different media were recorded. Statistical analysis In a 4 × 2 factorial experiment, 702 COCs were randomly assigned to 8 treatment groups to evaluate the effect of maturation media and supplementation, whereas 198 IVM oocytes were randomly assigned to 3 treatment groups to determine the effect of media on the fertilization rate. The percentage of oocytes reaching the designated response variables in each replicate were determined and used in the analysis. The data thus obtained were subjected to analysis of variance to see the magnitude of variation among various treatment groups using M StatC (version 1.3) software. Results and Discussion The effect of two maturation media i.e. TCM-199 and Ham’s F-12 with the addition of oestrus buffalo serum (OBS) and hormones or OBS and hormones alone on the maturation rate of buffalo follicular oocytes is given in Table 1. The results revealed a signifi cant (P < 0.05) increase in maturation rate when the OBS and hormones were added separately or in combination to TCM-199 than in Ham’s F-12 medium. The percentage of maturation in TCM-199 with supplementation of OBS and hormones was signifi cantly higher (77.44 0.68) than in Ham’s F-12 (32.85 0.83). Similarly, supplementation of TCM-199 with hormones or OBS alone also signifi cantly improved the maturation rate of buffalo follicular oocytes than by Ham’s F-12 in these combinations. Proper maturation is essential for an oocyte to achieve full developmental competence for fertilization. A number of ultrastructural and molecular changes occurring during oocyte development are linked to the developmental competence of the gamete (Hyttel 1997). The culture medium employed for IVM of follicular oocytes not only affects the proportion 400