In vitro culture of buffalo ( Bubalus bubalis) preantral follicles

Abstract

Growth of buffalo preantral follicles in culture was studied to investigate the effect of size of preantral follicles, individual or group culture, long-term culture of preantral follicles for (40 days), addition of human follicle stimulating hormone (FSH), insulin–transferrin–selenium (ITS), growth factors (epidermal growth factor (EGF), fibroblast growth factor (FGF), vaso active intestinal polypeptide (VIP) in culture media, and substitution of pregnant mare serum gonadotrophin (PMSG) for FSH as gonadotrophin source in culture media. Preantral follicles were isolated mechanically from ovaries of matured, nonpregnant slaughtered buffaloes and cultured in droplets of culture media under mineral oil in a 35 mm petri dish in a CO 2 incubator (38–39 °C, 5% CO 2 in air, 90–95% relative humidity) for 15 days. Preantral follicle isolation and washing medium consisted of Minimum Essential Medium (MEM) supplemented with steer serum (10%), glutamine (2 mM), sodium pyruvate (0.23 mM), hypoxanthine (2 mM) and gentamycin (50 μg/ml), respectively. In Experiment 1, we placed isolated preantral follicles individually or in groups of 2–4 preantral follicles in 30 or 50 μl droplets, respectively, using two culture media: washing media and washing media+ ITS (1%)+ FSH (0.05 IU/ml), respectively. In Experiment 2, we grouped isolated preantral follicles were grouped into six different size classes: ≤36, 37–54, 55–72, 73–90, 90–108 and ≥109 μm. We cultured groups of 2–4 preantral follicles in washing media+ ITS (1%)+ FSH (0.05 IU/ml) in a CO 2 incubator for 15 days. In Experiment 3, we allocated groups of 2–4 preantral follicles to 10 treatments: (1) only washing media, (2) washing media+FSH (0.05 IU/ml), (3) washing media+ITS (1%), (4) washing media+ ITS (1%)+ FSH (50 IU/ml), (5) washing media+ ITS (1%)+ EGF (50 ng/ml), (6) washing media+ ITS (1%)+ FSH (0.05 IU/ ml)+ EGF (50 ng/ml), (7) washing media+ ITS (1%)+ FGF (50 ng/ml), (8) washing media+ ITS (1%)+ FSH (0.05 IU/ ml)+ FGF (50 ng/ml), (9) washing media+ ITS (1%)+ VIP (50 ng/ml), and (10) washing media+ ITS (1%)+ FSH (0.05 IU/ ml)+ VIP (50 ng/ml). In Experiment 4, based on the results of Experiment 3, we incubated preantral follicles from those treatments showing significantly ( P<0.05) higher growth up to 40 days. In Experiment 5, we allocated groups of 2–4 preantral follicles to two treatments: (1) washing media+PMSG (50 IU/ml), and (2) washing media+ ITS (1%)+ PMSG (50 IU/ml) and cultured in a CO 2 incubator for 15 days. The results indicated that the preantral follicles cultured in groups had a higher growth rate ( P<0.05) than those cultured as individuals. ITS, FSH, PMSG and growth factors significantly ( P<0.05) promoted the growth of the preantral follicles. Following 40 days of culture, follicular architecture was preserved in nearly 17% of the follicles though there was no antrum formation. The growth rate of preantral follicles was lower in buffalo than in cattle.