Thousands of offspring have now been produced via artificial insemination with spermatozoa sexed by flow cytometry and cell sorting. We are unaware of any other practical approach to sexing spermatozoa that maintains fertility. Accuracy of sexing usually is 85-95% in most species, but somewhat lower with human spermatozoa. Spermatozoa are sexed in series, one at a time, at routine rates of about 3000 live spermatozoa of each sex per second for most species, and nearly twice that rate under optimal conditions for some species. Owing to various constraints and statistical considerations, there appears to be an upper theoretical limit to sexing spermatozoa of about 10,000 live spermatozoa of each sex per second with current methodology. About a quarter of the spermatozoa processed are sexed; the rest are discarded in the process or lost due to logistical constraints. Spermatozoa undergo some damage during sorting, although much less in terms of viability than with routine cryopreservation; fertility is lower with sexed than control spermatozoa. Offspring from sexed spermatozoa appear to have no more abnormalities than do controls, and both groups grow and thrive similarly. Despite high costs and complex procedures, sexing spermatozoa, usually followed by cryopreservation, is being used commercially for cattle and horse production in several countries, and is used to produce girls to avoid X-chromosome-linked genetic diseases.

This study was conducted to evaluate the effects of roscovitine on suppression of meiosis, subsequent meiotic maturation, and cleavage rates after intracytoplasmic sperm injection of horse oocytes. Oocytes were classified as having compact or expanded cumuli (Com or Exp oocytes) and were divided into three culture groups: 30 h culture in maturation medium (30 h Mat); 54 h culture in maturation medium (54 h Mat), or 24 h culture in medium containing 66 micro mol roscovitine l(-1) and then 30 h culture in maturation medium (Ros+M). After maturation, oocytes were subjected to intracytoplasmic sperm injection and cultured in G1.2 medium for 96 h. Among oocytes fixed immediately after roscovitine culture, 26 of 31 (84%) Com oocytes and 16 of 28 (57%) Exp oocytes were at the germinal vesicle stage (P<0.05). After maturation culture, there were no differences in maturation rates or morphological cleavage rates among treatments. Among Com oocytes, significantly more embryos in the Ros+M treatment than in the 54 h Mat treatment had cleaved with > or = two normal nuclei (63 versus 36%; P<0.05); whereas among Exp oocytes, significantly more embryos in the 30 h Mat treatment than in the Ros+M treatment (63 versus 42%; P<0.05) had cleaved with > or = two normal nuclei. The average number of nuclei in embryos at 96 h was significantly higher (P<0.05) in Ros+M Com oocytes (13.5) than in any other Com or Exp group. These results demonstrate that roscovitine can reversibly maintain equine oocytes in the germinal vesicle stage for up to 24 h, and that such suppression may increase the developmental potential of Com, but not Exp, oocytes.