127 OVARIAN STIMULATION IN CYNOMOLGUS MONKEYS BY A CONTROLLED RELEASE OF FOLLICULAR-STIMULATING HORMONE UTILIZING A MICRO-INFUSION PUMP

Abstract

We have established an indoor artificial breeding system for the cynomolgus monkey in an effort to increase the number of MII oocytes that are required for enhanced reproductive efficiency. A conventional ovarian stimulus method requires FSH to be administered to monkeys intramuscularly once a day for 9 days. Recently, a novel implantable and programmable micro-infusion pump (iPRECIO™, Primetech Corp, Tokyo, Japan) has been introduced for small laboratory animals to infuse fluids continuously for long periods of time in vivo. We adapted this micro-infusion pump to administer FSH to cynomolgus monkeys. In this study, we optimized the controlled-release program of FSH for the appropriate ovarian stimulation. First, laparoscopic evaluation was performed to identify animals that had small, underdeveloped follicles and gonadotropin-releasing hormone (0.9 mg animal^–1; Leuplin, Takeda, Osaka, Japan) was administered to all selected animals. Two weeks later, iPRECIO™ containing FSH (Gonapure, ASKA, Tokyo, Japan) was implanted subcutaneously and the continuous infusion was started at 15.0 IU kg^–1 per day. Five days after implantation, follicular development was evaluated by laparoscopy and the infusion rate was adjusted based on follicular profile (high level: reduced to 12.5 IU kg^–1 per day, n = 11; middle level: maintained at 15.0 IU kg^–1 per day, n = 47; low level: increased to 20.0 IU kg^–1 per day, n = 30). Four days later, hCG (400 IU kg^–1, IM) was administered and follicular aspiration was performed 40 h later. In the control group (n = 6), FSH (25.0 IU kg^–1 per day) was injected intramuscularly once a day for 9 days, followed by an hCG injection. Oocytes were collected and evaluated and MII oocytes were fertilized by intracytoplasmic sperm injection. Injected oocytes were cultured for 7 days in CRML-1066 medium supplemented with 20% bovine serum at 38°C, with 5% CO₂ and 5% O₂ in air and blastocyst development was evaluated. Data were analysed by a two-sided t-test. All animals treated with the controlled-release FSH using iPRECIO™ showed significantly higher MII maturation rates (mean: 59.4%, 22/37; P < 0.05) than those of the control group (MII rate: 46.3%, 19/41); however, there was no significant difference among the different FSH release programs. Blastocyst development rates of the test group were also significantly higher than those of the control group (test: 52.0%, control: 28.1%; P < 0.05); however, there was no significant difference among the different FSH programs. This controlled-release system did not require daily injections to the animal, which would be beneficial for decreasing stress. Further, the required dose of FSH using iPRECIO™ was much less than that of the conventional multiple-injection method. These results indicated that controlled release of FSH utilising an iPRECIO™ pump can be customized based on follicular profile and has financial and animal care advantages compared with the conventional method.