Microencapsulated rrBNGF as an alternative ovulation induction method in rabbits

Rabbits are an induced-ovulatory species such that exogenous hormone factors are needed to induce ovulation. Traditionally, intramuscular injections of gonadotropin-releasing hormone (GnRH) analogues are given at the time of artificial insemination (AI). To avoid the need for injections, the intravaginal delivery of molecules naturally present in seminal plasma has been explored. Here, we examined the possibility of using nerve growth factor (NGF) microencapsulated with chitosan to induce ovulation. First, the biological activity of these NGF microcapsules was assessed in pheochromocytoma of rat adrenal medulla cell (PC12) cultures, along with their effects on semen. Next, we examined the ability of the intravaginal NGF-chitosan delivery system administered at AI (NGFch-0) or 30 min before AI (NGFch-30) to elicit ovulation. To this end, progesterone concentrations on Day 7 post AI, pregnancy rates and prolificacy (kits born alive and stillbirths per doe) were determined in nulliparous and multiparous rabbit does and then compared amongst treatments: intravaginal NGFch-0 and NGFch-30, intramuscular injection of GnRH analogue, intravaginal empty-catheter (C-e) or intravaginal semen-containing catheter (C-s).

NGF-chitosan promoted similar PC12 differentiation to free NGF without impairing cell viability. The presence of the NGF-containing microcapsules did not interfere with semen motility, viability or capacitation status. In our in vivo experiments, nulliparous rabbits showed similar rates of ovulating females across treatments (GnRH 90%, NGFch-30 100%, NGFch-0 66.7%, C-e 83.3%), yet higher pregnancy rates were observed in response to GnRH and NGFch-30 (90% and 100%, respectively) than to NGFch-0 (60%). Prolificacy results in these does were similar across treatments. In multiparous does, GnRH treatment gave rise to the highest rate of ovulating female and pregnancy rates (100 and 90%, respectively). In contrast, the NGF-chitosan groups showed the lowest ovulating female and pregnancy rates (NGFch-30 50% and 25%, NGFch-0 41.7% and 21%, respectively). An intermediate ovulatory response was obtained in does stimulated with the catheter (C-e 70%, C-s 57.1%), and a pregnancy rate of 20% was obtained if the catheter contained diluted semen (C-s).

Intravaginal NGF-chitosan administered 30 min before AI induced ovulation at a similar rate to GnRH injection in nulliparous, but not multiparous, rabbit females. A better receptivity status of nulliparous females could be a determining factor for this response. However, mechanical stimulation gave rise to a high ovulation rate, so this could be masking or, in some cases, directly replacing the NGF-chitosan effect.