218 ULTRASOUND-GUIDED TRANSVAGINAL INJECTION OF A LOW DOSE OF FSH-LH INTO THE BOVINE OVARY AS AN ALTERNATIVE WAY TO STIMULATE FOLLICULAR GROWTH: PRELIMINARY RESULTS

Abstract

While some researchers claim a positive influence of FSH on the number of punctured follicles and retrieved oocytes in stimulated cows (Looney CR et al. 1994 Theriogenology 41, 67–72), others found comparable results between protocols with one stimulated v. two unstimulated ovum pick-up (OPU) sessions a week (Stubbings RB and Walton JS 1995 Theriogenology 43, 713–721). The use of FSH/LH causes explosive follicular growth and a substantial increase in ovarian blood supply when given at the superovulation dose. These drawbacks limit the repeated use of FSH stimulation prior to OPU in a twice per week puncture protocol. The aim of this experiment was first, to investigate the feasibility of injecting FSH directly into the ovary at a substantially lower dose and, second, to study the local effects of intra-ovarian FSH injection on follicular dynamics. Six donor cows with normal reproductive tracts were prepared as for transvaginal OPU (Bols et al. 1995 Theriogenology 43, 677–687). The ovary was visualized using a Multiple Angle 5 MHz ultrasound transducer (Pie Medical, Maastricht, The Netherlands), fitted next to a needle guidance system in an OPU handle. A vial of freeze-dried FSH/LH (Stimufol, 500 μg porcine FSH and 100 μg porcine LH, Ulg FMV, Liège, Belgium) was restored and diluted with saline to a solution containing 10 μg pFSH and 2 μg pLH per mL. A total dose of 160 μg pFSH and 32 μg pLH, divided over 8 injections of 1 mL diluted FSH/LH in each ovary, was given to 4 animals by transvaginal ultrasound-guided injection with a 19-gauge needle. The ovaries of one negative control cow were injected with 1 mL saline using the same protocol. The sixth cow received a classical FSH stimulation as prescribed by the manufacturer by means of i.m. injections at a total dose of 500 μg FSH and 100 μg LH as a positive control. During the first session, all follicles with a diameter of >5 mm were aspirated, while prior to each injection (morning and evening), ovarian activity was checked by ultrasound examination and taped on video for all cows. Intra-ovarian FSH injection was successful since, in most cases, a small echographically dense area was seen during and immediately following injection. This area moved around following displacement of the ovary, indicating intra-ovarian disposition of the FSH. Following the four-day stimulation treatment, the average (±SD) number of follicles with a diameter >5 mm was 5.5 ± 4.2 on the ovaries of intra-ovarian injected cows, 21 in the case of the positive control cow and only 1 follicle in the negative control. These results suggest that transvaginal, ultrasound-guided injection of a low FSH/LH dose directly into the ovary might be an alternative way for ovarian stimulation prior to OPU. Additional dose-titration experiments are ongoing.