Effects of follicular fluid or progesterone on in vitro maturation of equine oocytes before intracytoplasmic sperm injection with non-sorted and sex-sorted spermatozoa.

In Expt 1, compact cumulus oocyte complexes (COCs) were matured in: (i) control medium (Hepes-buffered TCM-199 with 10% oestrous cow serum (OCS) + oestradiol, LH and FSH); (ii) Hepes-buffered TCM-199 with 20% follicular fluid; or (iii) control medium containing 250 ng progesterone ml(-1). Mature oocytes were collected by transvaginal aspiration as a positive control for the in vitro maturation (IVM) treatments. Oocytes were fertilized by ICSI and cultured in Menezo's B2 + 5% fetal calf serum (FCS). There were no significant differences among IVM treatments. In Expt 2, oocytes with expanded COCs were matured in Hepes-buffered TCM-199 with 10% OCS, oestradiol, LH and FSH with different concentrations of progesterone (0, 50, 250 and 1250 ng ml(-1)). Oocytes were fertilized by ICSI and cultured in a chemically defined medium. The medium containing 1250 ng progesterone ml(-1) resulted in fewer oocytes with a visible first polar body after maturation (P < 0.05), whereas the media containing 0 and 50 ng progesterone ml(-1) resulted in higher development rates to seven- to eight-cell embryos (P < 0.05), compared with media containing 250 or 1250 ng progesterone ml(-1). Six of the resulting morulae were transferred to recipient mares. In addition, oocytes (n=32) from Expt 2 were injected with sex-sorted spermatozoa, obtained by separating X- and Y-bearing spermatozoa with a Cytomation MoFlo flow cytometer/cell sorter. Two embryos resulting from ICSI with X-bearing spermatozoa were transferred to the oviduct of a recipient mare. No pregnancies were established after transfer of embryos in these experiments.