Isolation of preantral ovarian follicles in sheep and goats

Isolation of preantral ovarian follicles in sheep and goats

Theriogenoiogy 343 ISOLATION OF PREANTRAL OVARIAN FOLLICLES IN SHEEP AND GOATS P.K.Chelikani, D.Amamath, K.K.Reddy, K.S.Naidu, K.V.Rao and V.H.Rao E...

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Theriogenoiogy

343

ISOLATION OF PREANTRAL OVARIAN FOLLICLES IN SHEEP AND GOATS P.K.Chelikani, D.Amamath, K.K.Reddy, K.S.Naidu, K.V.Rao and V.H.Rao Embryo Transfer Lab, College Of Veterinary Science, Tirupati - 5 17 502, India In vitro maturation and fertilization of oocytes from ovarian preantral follicles (PF) could provide greatez access to female germplasm. This was achieved in mice but in cattle and pigs isolation of PF was only reported (Theriogenology, 47:73). We report here the first success in isolation of PF from ovaries of sheep and goats. For isolation of PF from ovaries of sheep and goats, five treatments were selected from a preliminary study: collagenase (Mu/ml), hyaluronidase (5Ou/ml), pronase (5u/ml), Hanks Balanced Salt Solution without calcium and magnesium (HBSS) and PBS. Four age groups were used (I 2yrs, I 3yrs, I 4yrs and I Syrs ) and three follicle types were selected to conduct an experiment with a factorial assessment of treatments. From the ovaries collected at slaughter, ovarian medulla was removed by dissection and the cortex was cut into pieces of -1mm3. All the cortical pieces from each ovary were separately but randomly incubated for 15 min at 380C in 5 ml of one of the treatment solutions and then 5 ml of PBS + 10% FCS was added before repeated aspiration through a Pasteur pipette. However, HBSS and PBS groups were homogenized at 400 rpm for 3 min. Subsequently the digest was filtered through a 200 ym nylon mesh and was allowed to remain for 15 min in a 100 mm square polystyrene dish before isolation of PF using an inverted phase contrast microscope. All the chemicals were from Sigma, USA and plastics from Nunc (Denmark.) The PF were classified as Type III: 40 - 60 pm diameter, single layer of cuboidal granulosa cells and a dark oocyte; Type II: < 40 l,irn diameter, a single layer of granulosa cells and a dark oocyte and Type I: I 20 pm diameter, indistinct granulosa cell layer but with a dark oocyte. In goats hyaluronidase (331f17), collagenase (285&20) and pronase (284*20) yielded similar numbers of PF which were significantly higher (p < 0.05) than those obtained with HBSS (221f19 ) or PBS (191f12). Among various possible interactions only treatment X follicle type was significant ( data not shown; p < 0.01). From sheep ovaries the highest number of PF was isolated with hyaluronidase (297!c38), but pronase (178f9), collagenase (175f14) and HBSS ( 147fl3) yielded lower and similar numbers of PF. But significantly @ < .O.M) more PF were isolated with all the other treatments than PBS (127f12). All possible interactions among the factors studied were significant (data not shown; p < 0.05) in this species. In both sheep (Y = - 48 x + 301) and goat (Y = - 23x + 312) regression of number of PF on the age (‘x’ in the equation) of the female was linear and significant @ < 0.05). Hyaluronidase is used for the first time for isloation of PF from ovaries of domestic animals. It has yielded highest number of PF and also maintained the best morphology. Significance of interactions and regression equations shall be discussed. This work was supported by a research grant of Acharya N.G. Ranga Agricultural University to VHR. DA was supported by the Department of Biotechnology, Government of India.