In Vitro Evaluation of Frozen-Thawed Stallion Semen: A Review
Department of Clinical Veterinary Sciences, University of Helsinki, Finland
The article reviews methods used for in vitro evaluation of sperm, with particular emphasis on frozen-thawed stallion sperm. The techniques, limitations of the methods and correlations with fertility results are discussed. Very few studies have tried to find correlation between fertility of frozen stallion semen and laboratory tests. It is difficult and expensive to inseminate an adequate number of mares to achieve statistically significant differences. Significant, but low correlations have been demonstrated between the foaling rate and subjective motility of sperm incubated for 2 h and 4 h at 37°C and hypoosmotic swelling test after 0 and 3 h of incubation. Significant correlations have been reported between the pregnancy rate and viability of propidium iodide-stained sperm assessed by flow cytometry as well as for glass wool and Sephadex filtration tests. No correlations have been detected between fertility and motility immediately after thawing. In spite of that, motility estimation by light microscope is the most commonly used method to evaluate frozen-thawed stallion sperm. Computer assisted automatic sperm analyzers have replaced light microscopy in research projects, but so far nobody has been able to demonstrate a correlation between fertility of frozen stallion semen and any of the motility parameters obtained by these instruments.
Keywords: horse, sperm, cryopreservation, semen quality, motility, membrane integrity
Acta Veterinaria Scandinavica 2001, 42:199-217. Open Access Article.
There is considerable variation between individual stallions in how well their semen retains its fertilizing capacity after freezing and thawing. It has been estimated that only 20% of fertile stallions produce sperm that survive well the freezing and thawing processes . Although our knowledge and techniques have improved within the last 20 years, a considerable proportion of stallions are still not suitable for semen freezing. About 50% of examined French stallions showed poor freezability of sperm . However, according to , stallions which have passed the breeding health examination hardly ever are truly "poor freezers". It is only a matter of finding suitable freezing extenders and methods for individual stallions [54,50].
Development of freezing methods requires in vitro tests that correlate with in vivo fertility, but controlled breeding trials with an adequate number of horses are extremely expensive .  gives a good example: if we inseminate 10 mares, with the 95% confidence interval for the "true fertility" of 50%, the stallion’s "observed fertility" would be between 15% and 85%! Similarly, assuming a "true fertility" of 50%, the 95% confidence interval for the "observed fertility" based on 100 inseminations is 40% to 60%, and 47% to 53% if based on 1000 inseminations. It is hard to imagine that we could have hundreds of mares in frozen semen insemination trials.
With the increasing international trade and commercial use of frozen semen, the unacceptably poor pregnancy rates cause considerable frustration and economic losses in the equine breeding industry . The slow progress in the development of freezing techniques for equine semen is partly explained by the lack of reliable laboratory methods. Some in vitro methods work reasonably well in the assessment of fresh semen, the best example being motility evaluation. In spite of its limited applicability, motility is the most commonly used parameter in the evaluation of frozen-thawed semen, in both laboratories and stud-farms, because it is easily accessible and quick to perform. It is generally agreed that tests other than in vitro motility could be important for predicting fertility. Numerous promising assays have been reported in the literature but few have found their way into commercial semen freezing laboratories . A combination of laboratory tests should enable better assessment of the fertility potential of cryopreserved stallion semen .