Commercial semen freezing: individual male variation in cryosurvival and the response of stallion sperm to customized freezing protocols.

One of the challenges for those attempting to cryopreserve stallion spermatozoa is dealing with the stallion to stallion variability in the cryosurvival of their semen. In the dairy industry, each bull stud, essentially utilizes a single cryopreservation technique, and bulls that produce sperm that do not cryopreserve well using that technique are replaced by other bulls. However, replacing stallions is unlikely to prove acceptable to the equine industry, where specific genotypes are desired. Instead, to increase the number of stallions that can be effectively utilized for cryopreserved semen production, it is likely that more than one method for cryopreserving sperm will be necessary. This manuscript reviews some of the processes involved in cryopreservation, how individual sperm physiology affects the ability to survive freezing and thawing, and how cryopreservation protocols can be customized to maximize sperm cryosurvival on an individual stallion basis.

The equine frozen semen industry.

Recent acceptance of frozen semen as a method to produce registered foals by two of the worlds largest breed associations, the American Quarter Horse and American Paint Horse, has stimulated new interest in frozen semen technology. This review will: (a) attempt to identify the major impediments to the development of the frozen semen industry, (b) suggest alternative methods for marketing and application of frozen semen, and (c) present the results of a recent study in our laboratory. The objective of which was to compare pregnancy rates of insemination with cooled and frozen semen. Major impediments to the development of the frozen semen industry include 1. Lower fertility with frozen semen as compared to cooled semen for many stallions. 2. Increased costs associated with management of mares for AI with frozen semen using current insemination protocols. 3. Unfavorable marketing practices for frozen semen. Reports of fertility with cooled transported semen in commercial breeding programs indicate seasonal pregnancy rates ranging from 60 to 90%. We compiled data from three commercial transported cooled semen programs in which semen from 16 stallions was used for insemination of 850 mares throughout North America by local veterinarians. During the 1999 and 2000 breeding seasons, first cycle and seasonal pregnancy rates of 59.4 and 74.7% were obtained. During that same period, first cycle and seasonal pregnancy rates of 51.3 and 75.6% were obtained following insemination of 876 mares with frozen semen from 106 different stallions processed by our laboratory and distributed through our commercial distribution program. First cycle and seasonal pregnancy rates were higher for mares bred outside of North America than for mares bred within North America (53.5 and 81.9 versus 49.4 and 65.6%, respectively). Seasonal pregnancy rates were higher presumably because of the better mare management employed for mares bred with exported semen and the fact that some of the domestic mares were switched to cooled semen insemination after a failed first cycle attempt with frozen semen. These data support the position that comparable seasonal pregnancy rates may be obtained using frozen and liquid cooled semen in a commercial setting.

Fertility of unfrozen and frozen stallion spermatozoa extended in EDTA-lactose-egg yolk and packaged in straws.

The fertility of frozen-thawed semen was compared with that obtained using fresh semen extended in skim milk. Semen for freezing was obtained in June from four stallions of unknown fertility; two ejaculates were collected 1 to 2 h apart every 3 or 4 d. The gel-free fraction of the ejaculate was mixed 1:1 with a glucose-EDTA solution (disodium ethylenediaminetetraacetic acid) and centrifuged at 650 x g for 15 min. The spermatozoa were resuspended in an EDTA-lactose-egg yolk extender containing 5% glycerol. Semen was frozen in .5-ml French straws containing 250 x 10(6) progressively motile spermatozoa before freezing. Only for 31% of the 54 ejaculates frozen was post-thaw spermatozoal motility greater than or equal to 50% of the percentage of progressively motile spermatozoa observed during evaluation of the neat semen. Spermatozoa in second ejaculates apparently were more susceptible to the adverse effects of dilution and centrifugation than spermatozoa in first ejaculates. Only samples containing greater than 30% progressively motile spermatozoa after freezing and thawing (at 38 C) were used for insemination. In June and July, 101 mares were inseminated daily with semen from one of three stallions beginning on d 2 and continuing through the end of estrus for one cycle. Mares were inseminated with semen in one straw or with 250 x 10(6) progressively motile spermatozoa extended in 10 ml of skim milk. Because of the poor survival of spermatozoa that had been frozen and thawed, mares inseminated with frozen-thawed semen received 100 to 130 x 10(6) progressively motile spermatozoa as compared with 250 x 10(6) progressively motile spermatozoa for mares inseminated with fresh semen. One cycle pregnancy rates, based on rectal palpation 50 to 60 d after ovulation, were 29% using frozen-thawed semen and 66% using fresh semen (P less than .05). Values for individual stallions were 19, 24 and 47% with frozen semen and 47, 61 and 67% with fresh semen. Routine use of frozen stallion semen is not recommended at this time.