When women with small ovarian reserves are subjected to assisted reproductive technologies (ART), high doses of gonadotropins are linked to high oocyte and embryo wastage and low live birth rates. We hypothesized that excessive FSH doses during superovulation are detrimental to ovulatory follicle function in individuals with a small ovarian reserve. To test this hypothesis, heifers with small ovarian reserves were injected twice daily for four days beginning on Day 1 of the estrous cycle with 35 IU, 70 IU, 140 IU, or 210 IU doses of Folltropin-V (FSH). Each heifer (n = 8) was superovulated using a Williams Latin Square Design. During each superovulation regimen, three prostaglandin F2α injections were given at 12 h intervals starting at the 7th FSH injection to regress the newly formed corpus luteum (CL). Human chorionic gonadotropin (hCG) was injected 12 h after the last (8th) FSH injection to induce ovulation. Daily ultrasonography and blood sampling were used to determine number and size of follicles and CL, uterine thickness, and circulating concentrations of estradiol, progesterone, and anti-Müllerian hormone (AMH). The highest doses of FSH did not increase AMH, progesterone, ovulatory-size follicle number, uterine thickness, or number of CL. However, estradiol production and ovulation rate were lower for heifers given high FSH doses compared to lower doses, indicating detrimental effects on ovulatory follicle function.

Cattle with an unknown antral follicle count (AFC) during follicular waves respond to different FSH doses during superovulation in a curvilinear fashion with the highest doses decreasing or not increasing ovulatory follicle number, number of transferable embryos, and number of corpora lutea (CL). These observations support the hypothesis that heifers treated with different FSH doses during superovulation will reach a superovulation maximum (SOVmax) and that doses exceeding SOVmax are excessive, resulting in a decrease in ovarian function. To test this hypothesis, we used eight 12-month-old Holstein heifers with a low AFC (<10 follicles ≥3mm in diameter, index for small ovarian reserve). These 8 heifers were subjected to 4 different superovulations with a 21-day intervals between each superovulation. During each superovulation, which began on Day 1 of a synchronized oestrous cycle before selection of a dominant follicle, 2 of the 8 heifers were treated (twice-daily FSH injections×4 days) with 20, 40, 80, or 120mg of Folltropin-V (Vetoquinol, Lure, France). At the end of the study, the same 8 heifers had been superovulated 4 times with each of the 4 FSH doses. To enhance ovulation rate in response to hCG, each superovulated heifer was injected with 3 different injections of prostaglandin F2α (PGF2α) spaced 12h apart to induce luteolysis of the original CL. Two PGF2α injections were given after each FSH injection on the last day of FSH treatment and the last PGF2α injection was given after the hCG (2500IU) injection, which was 12h after the last FSH injection. Daily ultrasonography was used to measure AFC and number of ovulatory follicles and CL during the study. Results of t-test analysis showed that at time of hCG, AFC was greater (P>0.05) for heifers treated with the 40-mg (mean±s.e.m., 27±3.7) v. 20-mg (17.9±2.5) FSH dose, but the response to higher FSH doses (27.9±4.4; 27±3.3) did not differ from the response to the 40-mg dose. Number of ovulatory follicles was greater (P<0.05) for heifers treated with the 40-mg (17.5±2.3) v. 20-mg (10.1±1.4) FSH dose, but the response to higher doses (18.9±3.3; 17.3±2.4) did not differ from the response to the 40-mg dose. At Day 7 post-hCG, CL number was greater (P<0.05) for heifers treated with the 40-mg (13.8±2.9) v. 20-mg (7.4±1.4) dose, but response to higher doses (9.9±3.2; 9.1±2.3) did not differ from the response to the 40-mg dose. Ovulation rate (ovulatory follicle number divided by CL number) was greater (P<0.05) for heifers treated with the 40-mg (79±0.08%) v. 80-mg (52±0.09%) or 120-mg (53±0.09%) dose, but similar to that of heifers treated with 20-mg (73±0.09%) FSH dose. These results support the conclusion that the 40-mg FSH dose achieves SOVmax and FSH doses greater than SOVmax in cattle with a low AFC and small ovarian reserve are excessive and detrimental to ovarian function. This project was supported by Agriculture and Food Research Initiative Competitive Grant no. 2017-67015-26084 from the USDA National Institute of Food and Agriculture (Washington, DC).