Physiological Aspects of Stallion Semen Cryopreservation

The history of artificial insemination (AI) begins with a story about some Arabs stealing stallion semen from a rival tribe and bringing it home for breeding in a sponge. When AI began its modern development in Russia during the early 20th century by Ivanov, the main object was not cattle but horses as documented by Milovanov. In the late 1940s, Christopher Polge, working with chicken and the economically more important bull semen, pioneered development of frozen-thawed semen technology. This, however, was done by unknowingly using a bottle containing glycerol and allowing the semen to remain in glycerolated extender for 18 h (“glycerol equilibration time”). Thus, frozen semen technology was developed without much knowledge of the physiology of spermatozoa or the events leading to fertilization. Sperm motility was then, and largely remains, the main criterion upon which success or failure of the freezing procedure is evaluated in the laboratory. Sperm motility, however, proved to be of very little value in predicting the success of freezing swine semen. Polge had already shown in 1956 that the glycerol concentration in liquid semen was inversely related to its fertility. Glycerol concentrations of less than 2% were necessary to achieve good fertility also after freezing of boar semen, although maximum sperm motility was seen at a concentration of 7% glycerol. In 1957, Canadians Barker and Gandier reported the first foaling following insemination with frozen epididymal spermatozoa. During the next decade a dozen more successful reports with ejaculated semen followed from Germany, Japan, Russia, and the U.S. Sugar-containing diluents were most commonly used. Per cycle pregnancy rates varied between 13% and 44%, and the number of mares inseminated per trial varied between 5 and 116. Greater numbers were, however, used in the American Breeders Service (ABS) study utilizing 342 mares, and in China, where He Wen Ye reported respectable fertility in over 40,000 mares inseminated with semen diluted in sucrose-egg yolk and vapor frozen as pellets. All this happened before inseminations could be timed to ovulation by the use of ultrasonography. Moreover, wide individual variations in fertility of frozen semen among stallions may not yet have been recognized. A successfully preserved spermatozoon must be able to produce a viable embryo following an in vivo insemination. Many widely different mechanisms involved must be maintained. The spermatozoa must survive the uterine environment, be transported to the oviduct, be maintained there until the oocyte arrives, and be prepared to penetrate the