Morphology and function of ovarian follicles and oocytes following superstimulation treatments in heifer calves

Genetic improvement in cattle has focused in recent years on the large reproductive potential that resides in the ovaries of females at an early age. It is estimated that approximately 150,000 oocytes are present in primordial follicles in foetal ovaries at birth, and recruitment of follicles from the primordial pool has been initiated by the time of birth. Further, follicular growth can be superstimulated in heifer calves from around 4 weeks of age by treatment with gonadotrophins, and oocytes recovered and placed through in vitro maturation and fertilisation procedures to produce viable embryos.

The capacity to use embryos derived from heifer calves has the potential to reduce generation intervals and increase the rates of genetic gain in cattle. Studies on embryo production from heifer calves have reported inconsistent and unpredictable responses to superstimulation of follicle growth with FSH, similar to that observed in sexually mature heifers. Heifer calves that had a relatively large (>10mm) follicle on the ovary at the end of superstimulation, had a smaller number of total follicles compared with heifer calves that did not have a large follicle on the surface of the ovary. This observation led to the suggestion that follicular interrelationships may occur from an early age in heifers, and that a large follicle may suppress the development of other follicles. Nutrition appears to influence ovarian follicle status in peri-pubertal and pubertal heifers and possibly the response to superstimulation of follicular growth in older animals. There may be a role, therefore, for nutrition in ovarian follicle growth and responses to superstimulation in heifer calves.

In a number of studies oocytes obtained from heifer calves were reported to have a reduced developmental competency in vitro compared with oocytes obtained from ovaries of post-pubertal heifers.

In cattle, treatment with agonists of gonadotrophin hormone releasing hormone (GnRH) desensitises the anterior pituitary gland to GnRH which blocks pulsatile secretion of LH but allows continued basal LH secretion. Antagonists of GnRH prevent both pulsatile and basal secretion of LH. It is possible that treatment with GnRH agonists and antagonists might be used to regulate gonadotrophin secretion in heifer calves and prevent the development of large (functionally dominant) follicles. Subsequent initiation of superstimulation when a pool of small gonadotrophinresponsive follicles are present on the ovaries, and maturing these follicles in synchrony in vivo, may allow a pool of oocytes at similar stages of maturation to be collected for in vitro procedures.

The first two experiments in this thesis examined the requirement of LH for oocyte maturation by treating calves with gonadotrophin hormone releasing hormone (GnRH) agonist and antagonist before and during superstimulation with FSH. Simultaneous treatment with a GnRH agonist during superstimulation of ovarian follicle growth with FSH tended to increase the number of follicles stimulated to grow and significantly increased the number of Grade A and Grade B oocytes collected. In a second experiment, treatment with a GnRH antagonist tended to increase blastocyst development rate after in vitro fertilisation. It was concluded from these findings that exposure of oocytes to pulsatile secretion of LH, and/or a 'pre-ovulatory like' surge release of LH, is not an obligatory requirement for oocyte growth and development in heifer calves.

A third experiment examined the effects of nutrition and growth rate on maturation of the reproductive endocrine axis and the response of calves to superstimulation of ovarian follicle growth with FSH. Heifer calves were raised on two planes of nutrition (relatively low and high) and subsequently superstimulated with FSH. The nutritional treatments resulted in a significant difference in growth rate between the two groups of heifers. However, there were no apparent differences in ovarian follicular responses to stimulation with FSH, oocytes recovered, or in vitro developmental competency of oocytes, between the two groups of heifers.

In the fourth experiment, in vitro developmental competency was compared between oocytes obtained from heifer calves superstimulated with FSH, heifer calves that had not undergone superstimulation and post-pubertal heifers and cows that had not been stimulated with FSH. There were no differences in developmental competency between Grade A and Grade B oocytes derived from the three groups of animals. This finding demonstrated that oocytes obtained from pre -pubertal heifers do not have an intrinsic reduced capacity for in vitro development compared with oocytes obtained from post -pubertal heifers.

The ultimate test of viability of embryos derived from heifer calves is the transfer to recipients and the birth of calves. The aim of the fifth experiment therefore was to test the viability of embryos derived from 10 week-old heifer calves in which ovarian follicular growth was superstimulated with FSH. Transfer of blastocysts produced from oocytes obtained from heifer calves to recipient sexually mature heifers resulted in the birth of normal calves.

In summary, the competency of oocytes collected from heifer calves from an early age has been well established in the series of experiments undertaken in this thesis. The use of superstimulation protocols with heifer calves pre-treated with a GnRH agonist or antagonists increased the number of Grade A and Grade B oocytes, and tended to increase the development to blastocyst post-fertilisation. Grade A and Grade B oocytes collected from heifer calves form blastocysts at rates comparable to oocytes collected from mature cows, and establish pregnancies which result in the birth of calves.