Developmental Capacity of Bovine Follicular Oocytes Collected from Ovaries of Pubertal Heifers Obtained through a Spay Device.

Abstract

Materials and Methods Collection of ovaries and oocyte maturationOvaries were obtained from 21 pubertal heifers of Japanese Black and Holstein breeds through a spay device. The heifers in this experiment were 8 to 12 months of age and were housed in closed stalls. Transvaginal recovery of ovaries from heifers was performed without anesthesia. The spay device (Kimberling-rupp spay device; Jorgensen Labo. Inc., Colo, USA) was inserted into the vagina, pushed through the vaginal wall up to each ovary, and the ovaries were collected. For the control, ovaries from adult cows of the same breed, which were more than 2 years old, were obtained at a local abattoir. The collected ovaries were placed in physiological saline (0.85%(w/v) NaCl) at 25 to 30 C. The ovaries were then transported to the laboratory within 2 h. Follicular oocytes were collected by aspiration of follicles 1 to 7 mm in diameter, and the follicular contents were mixed with modified Dulbecco' s phosphate buffered saline (mPBS) (Embryoteck; Nippon Zenyaku Co., Koriyama, Japan). After collection, the oocytes were washed twice with mPBS and once with the maturation medium (25 mM-Hepes TCM-199 with Earle's salts; Gibco, Grand Island, NY, USA) supplemented with 5% fetal bovine serum (FBS) and 0.01 A/ml FSH (Denka, Kawasaki, Japan). Then the oocytes surrounded with sufficient cumulus cells, were pooled and the number of the cumulus oocyte complexes were recorded. All the oocytes were cultured in 2.5 ml of maturation medium for 21 to 22 h at 38.5 C in 5% CO 2 in air.

Gene Expression Differences in Oocytes Derived From Adult and Prepubertal Japanese Black Cattle during In Vitro Maturation

“…Although pregnancies and birth of live calves have been reported after in vitro fertilization of oocytes from prepubertal and pubertal heifers, their developmental ability for in vitro cleavage, and blastocyst formation is still lower than adult cow oocytes (Armstrong et al. 1992; Otoi et al. 1995; Revel et al.…”

“…However, the composition of prepubertal vs adult bovine oocyte transcriptome and the identity of differentially expressed genes with potential regulatory roles during oocyte maturation and early embryonic development are not fully understood. Although pregnancies and birth of live calves have been reported after in vitro fertilization of oocytes from prepubertal and pubertal heifers, their developmental ability for in vitro cleavage, and blastocyst formation is still lower than adult cow oocytes (Armstrong et al 1992;Otoi et al 1995;Revel et al 1995). Several studies have demonstrated that one of the factors affecting the developmental potential is the intrinsic quality of the oocyte (De Sousa et al 1998;Sirard 2001;Rizos et al 2002a,b;Lonergan et al 2003a,b).…”