Offspring from One-Month-Old Lambs: Studies on the Developmental Capability of Prepubertal Oocytes1

Abstract

A wave of follicular growth in lamb ovaries occurs at about 4 weeks of age, generating a life-time peak in follicle numbers. In order to take advantage of the large number of oocytes available, and to substantially decrease the generation interval, embryos were derived from oocytes collected from 1-mo-old lambs. Animals were subjected to one of 3 regimes of hormonal stimulation: groups 1 and 2 were treated to obtain germinal vesicle-stage oocytes, and group 3 to produce mature metaphase II oocytes. Adult sheep stimulated by an appropriate dose of FSH served as control. The developmental ability of collected oocytes was evaluated by either in vivo or in vitro culture to the blastocyst stage after in vitro maturation and/or fertilization. Blastocysts were transferred immediately or after cryopreservation to suitable recipient sheep. In order to investigate the full developmental potential of these embryos, pregnancies were allowed to go to term. The results show significant differences (P < 0.001) between all experimental groups in blastocyst numbers produced. Embryos derived from group 1 animals produced the greatest number of blastocysts, under both in vivo (36. 7%), and in vitro (22.9%) culture systems. Group 2 gave lowest blastocyst production (5.0%), while group 3 yielded 13.2% blastocysts. The number of pregnant recipients carrying to term lamb-derived embryos was severely reduced for both in vivo- (2 of 9; 22.2%) and in vitro-cultured, fresh (3 of 10; 30.0%) and cryopreserved (1 of 6; 16.7%) lamb embryos. This study is the first report of the birth of live lambs derived from oocytes obtained from donors as young as 4 wk. Defects in the competence of lamb-derived embryos may account for the increased fetal loss during pregnancy and the occurrence of mummified fetuses delivered alongside normal healthy lambs.

Neonatal exposure to xenoestrogens impairs the ovarian response to gonadotropin treatment in lambs

“…This technique is used both in adults (multiple ovulation and embryo transfer (MOET)) (Wray & Goddard 1994) and in prepubertal females (juvenile in vitro embryo transfer (JIVET)) (Kelly et al 2005). It has long been known that follicles of 4-to 8-week-old lambs are particularly sensitive to gonadotropin administration using protocols developed for adult animals (Worthington & Kennedy 1979, Armstrong et al 1994, Ptak et al 1999. On the other hand, ovarian stimulation has proved to be a simple and useful tool to detect alterations in rodent reproductive organs and to study likely changes in the mechanisms of hormonal action induced by certain substances (Sekiguchi et al 2003).…”

Brilliant Cresyl Blue stain selects largest oocytes with highest mitochondrial activity, maturation-promoting factor activity and embryo developmental competence in prepubertal sheep

“…Fresh semen, obtained from three rams of proven fertility, was kept at room temperature (25 8C) for 90 min (Ptak et al 1999). Highly motile spermatozoa were selected by Ovipure density gradient (Nidacon EVB S.L., Barcelona, Spain) and fertilised with 1!10 6 spermatozoa/ml.…”

Embryo transfer and related technologies in sheep reproduction

“…Two years later the use of cysteamine, which increases the glutathione level in oocytes, was already adopted for in vitro embryo production using sheep [115] and lamb oocytes [83].…”