Location: Range and Livestock Research
Project Number: 3030-31000-018-04-R
Project Type: Reimbursable Cooperative Agreement
Start Date: Sep 1, 2013
End Date: Aug 31, 2018
The long-term objective is to identify factors that enhance fertility via ovulation of an optimal follicle. We propose the central hypothesis that the decrease in pregnancy rate and late embryonic/fetal survival (days 28 to 70 post breeding) is due to a combination of decreased oocyte competence and inadequate preparation of the maternal environment for the establishment and maintenance of pregnancy when sub-optimal follicles are induced to ovulate or ovulate spontaneously. From preliminary studies we determined that the size of the dominant follicle at induction of ovulation, but not at spontaneous ovulation, affects the pregnancy rate and incidence of late embryonic/fetal mortality. Furthermore, in our previous NRI grant we have shown an effect of the physiological maturity of an ovulatory follicle at induced ovulation on fertilization rate and preparation of the maternal environment for pregnancy. The following whole animal to molecular objectives are designed to test the preceding hypothesis: 1) Determine the effect of ovulatory size on morphological (Objective 1 A) and biochemical (Objective 1 A&B) markers of oocyte competence, 2) Determine the effect of ovulatory follicle size on steroidogenic capacity (concentrations of estradiol and progesterone in circulation and follicular fluid), the follicular wall transcriptome (including microRNA profile), and the metabolome in follicular fluid of dominant follicles collected 20 hr after GnRH injection (GnRH at 48 hr after PG), and 3) Differentiate between effects of low or normal preovulatory estradiol and low or normal postovulatory progesterone on pregnancy establishment in postpartum beef cows. In summary, this proposal is aimed at identifying factors involved in ovulation of a fully competent oocyte, and development of a maternal environment conducive to pregnancy establishment and maintenance.
Objectives 1, 2, and 3 will be conducted in year 1, 2, and 3, respectively. Objective 1A is designed to determine the effect of ovulatory follicle size on markers of oocyte competence. Oocytes will be individually collected from ovulatory follicles of cows in estrus or large & small follicles induced to ovulate. Oocytes will be isolated from surrounding cumulus cells and in vitro fertilization and culture used to assess morphological development to the blastocyst stage. Objective 1B is designed to determine if the transcriptome of both oocytes and associated cumulus cells differ between cumulus/oocyte complexes collected from small or large dominant follicles using follicular aspiration. The cumulus cells will be isolated from oocytes and each will be pooled (10 per pool). RNA will be isolated from pools for investigation of differential gene expression between GnRH-induced large and small follicles and follicles from cows that exhibited estrus. Genes expressed in oocytes that will be examined specifically include follistatin, inhibin beta A, and inhibin beta B , and cumulus expressed genes such as tumor necrosis factor alpha-induced protein, cathepsins B, K, and S, hyaluronic acid synthases 2, and gremlin 1. All of the preceding genes have been shown to be associated with competence of bovine oocytes. Objective 2 is designed to determine the effect of dominant follicle diameter on steroidogenic capacity (concentrations of estradiol and progesterone in circulation and follicular fluid), the follicular wall transcriptome and the metabolome in follicular fluid of dominant follicles collected 20 hr after GnRH injection. Ovaries will be obtained from cows 20 h after onset of estrus or GnRH induced ovulation of a large or small follicle. RNA will be isolated and gene expression profiles compared between different groups known to have normal or lesser fertility, as will steroidogenic profiles and follicular fluid metabolites (metabolome). Objective 3 is designed to differentiate between effects of low or normal preovulatory estradiol and low or normal postovulatory progesterone on pregnancy establishment in postpartum beef cows. Induced ovulation of small or large follicles will be used to create low or normal estradiol profiles and low dose cloprostenol injections administered on d 3, 3.5, and 4 will be used to reduce progesterone synthesis for the ensuing cycle. Embryos will be transferred into cows on d 7 and blood samples and ultrasonography will be used to monitor pregnancy success.