Advances in cooled semen technology

Abstract

In the horse industry, milk or milk-based extenders are used routinely for dilution and storage of semen cooled to 4–8 °C. Although artificial insemination (AI) with chilled and transported semen has been in use for several years, pregnancy rates are still low and variable related to variable semen quality of stallions. Over the years, a variety of extenders have been proposed for cooling, storage and transport of stallion semen. Fractionation of milk by microfiltration, ultrafiltration, diafiltration and freeze-drying techniques has allowed preparation of purified milk fractions in order to test them on stallion sperm survival. Finally, a high protective fraction, native phosphocaseinate (NPPC), was identified. A new extender, INRA96, based on modified Hanks’ salts, supplemented with NPPC was then developed for use with cooled/stored semen. Four experiments were conducted to compare INRA96 and milk-based extenders under various conditions of storage. The diluted semen was maintained under aerobic conditions when stored at 15 °C, and anaerobic conditions when stored at 4 °C. In experiment 1, split ejaculates from 13 stallions were diluted either in INRA96 extender then stored at 15 °C or diluted in Kenney or INRA82 extenders and then stored at 4 °C for 24 h, until insemination. In experiment 2, semen from two stallions was extended in INRA96 then inseminated immediately or stored at 15 °C for 3 days until insemination. In experiment 3, semen from three stallions was diluted in INRA96 then stored at 15 or 4 °C for 24 h until insemination, finally, in experiment 4, split ejaculates from four stallions were diluted in INRA96 or E– Z Mixin extenders then stored at 4 °C for 24 h until insemination. Experiment 1 demonstrated that at 15 °C, INRA96 extender significantly improved pregnancy rate per cycle compared to Kenney or INRA82 extenders at 4 °C after 24 h of storage (57%, n=178 versus 40%, n=171, respectively; P<0.01). Experiment 2 showed that semen stored at 15 °C for 3 days can achieve pregnancy at a fertility rate per cycle of 48% ( n=52) compared to 68% ( n=50, immediate insemination, P=0.06). Experiment 3 demonstrated that INRA96 extender can be as efficient at 15 °C (54%, n=37) as at 4 °C (54%, n=35) after 24 h of storage. Finally, experiment 4 showed that INRA96 extender used at 4 °C (59%, n=39) seems to improve fertility per cycle compared to E– Z Mixin at 4 °C (49%, n=39, P=0.25), but this result has to be confirmed. These results demonstrate that semen diluted in INRA96 extender and stored at 15 °C can be an alternative to semen diluted in milk-based extenders and stored at 4 °C for “poor cooler” stallions. Furthermore, INRA96 extender can be as efficient at 15 °C as at 4 °C, for preserving sperm motility and fertility.