Development of methods for the consistent production of transferable embryos from oocytes collected from Mediterranean donors at prepubertal ages.

There is a growing demand for buffalo dairy products that cannot be met because of the limited number of females of high milk productivity available in Canada and the very long time it takes for these animals to achieve sexual maturity. This project targets the establishment of consistent methods to produce live offspring from prepubertal Mediterranean Buffalo females of 2 to 8 months of age, using in vitro embryo production technologies. It has been estimated that buffalo females are born with 10,000-20,000 germ cells potentially able to develop into mature oocytes. However, most of these cells are lost due to apoptosis during pre- and post-pubertal ages. Follicles and oocytes can grow at prepubertal age, but oocytes collected from prepubertal females have reduced developmental competence, i.e. the capacity to develop into a viable embryo following in vitro maturation (IVM), fertilization (IVF) and culture (IVC), compared to adult females. Oocyte competence is acquired during the final stages of follicle development and is the result of a complex interplay between the oocyte and the surrounding granulosa cells involving endocrine, paracrine and autocrine factors that regulate and are part of said interplay. We propose that the developmental competence of oocytes recovered from buffalo calves can be improved if proper conditions for full development and progression to maturation are created to compensate for the fact that the prepubertal hypothalamus-pituitary-ovary axis is not fully functional. Therefore, our first goal will be focused on creating an optimized environment to maximize acquisition of oocyte competence during follicle development. This includes the development of innovative priming protocols combining different hormones (FSH, eCG, hCG, progesterone, estradiol) and administration regimes (doses, frequency, treatment duration, interval to ovum pick-up), to maximize intra-follicular growth and cytoplasmic maturation prior to oocyte collection. Second, we will develop IVM conditions tailored for the special needs of prepubertal oocytes that will promote proper cytoplasmic maturation, resumption of meiosis and normal progression towards readiness for fertilization. For this, the IVM medium will be supplemented with compounds to stimulate oocyte-granulosa cells interplay and acquisition of oocyte competence (e.g. cytokines and growth factors). Third, since prepubertal oocytes were shown to have fewer and poorly arranged cortical granules (prone to polyspermy), we will test the efficiency of intracytoplasmic sperm injection (ICSI) as an alternative to IVF to prevent abnormal fertilization in prepubertal oocytes. Finally, to maximize impact of technology implementation at the commercial level, tailored protocols for embryo freezing will be developed, enabling producers to harvest embryos year-round followed by implantation in recipients at the time of the year that will result in milk production at peak market demands.