Research Article: Fertility of frozen-thawed stallion semen cannot be predicted by the currently used laboratory methods

Date Published: August 17, 2006

Publisher: BioMed Central

Author(s): P Kuisma, M Andersson, E Koskinen, T Katila.


The aim of the project was to use current simple and practical laboratory tests and compare results with the foaling rates of mares inseminated with commercially produced frozen semen. In Exp. 1, semen was tested from 27 and in Exp. 2 from 23 stallions; 19 stallions participated in both experiments. The mean number of mares per stallion in both experiments was 37 (min. 7, max. 121). Sperm morphology was assessed and bacterial culture performed once per stallion. In Exp. 1, progressive motility after 0, 1, 2, 3, and 4 h of incubation using light microscopy, motility characteristics measured with an automatic sperm analyzer, plasma membrane integrity using carboxyfluorescein diacetate/propidium iodide (CFDA/PI) staining and light microscopy, plasma membrane integrity using PI staining and a fluorometer, plasma membrane integrity using a resazurin reduction test, and sperm concentration were evaluated. In Exp. 2, the same tests as in Exp. 1 and a hypo-osmotic swelling test (HOST) using both light microscopy and a fluorometer were performed immediately after thawing and after a 3-h incubation. Statistical analysis was done separately to all stallions and to those having ≥ 20 mares; in addition, stallions with foaling rates < 60 or ≥ 60% were compared. In Exp. 1, progressive motility for all stallions after a 2 – 4-h incubation correlated with the foaling rate (correlation coefficients 0.39 – 0.51), (p < 0.05). In stallions with > 20 mares, the artificial insemination dose showed a correlation coefficient of -0.58 (p < 0.05). In Exp. 2, the HOST immediately after thawing showed a negative correlation with foaling rate (p < 0.05). No single test was consistently reliable for predicting the fertilizing capacity of semen, since the 2 experiments yielded conflicting results, although the same stallions sometimes participated in both. This shows the difficulty of frozen semen quality control in commercially produced stallion semen, and on the other hand, the difficulty of conducting fertility trials in horses.

Partial Text

In many countries, artificial insemination (AI) has superseded natural mating as a breeding method for mares. Use of frozen semen, however, has not gained widespread use in horses, due to low pregnancy rates. In addition to semen quality, many other factors affect the outcome of AI, including the handling and freezing methods of semen, AI dose, timing of AI and management and fertility of the mares [1]. There is considerable variation between individual stallions in how their semen survives freezing and thawing. Otherwise fertile stallions can produce semen that results in very poor post-thaw pregnancy rates [2]. Tischner [3] estimated that approx. 20% of stallions are “good freezers”, another 20% are “bad freezers”, and the majority of stallions, 60%, produce semen that is affected adversely, but may be freezable using certain techniques. Unlike bulls, stallions are not selected for breeding on the basis of fertility or semen freezability [1]. Therefore, not much progress is to be expected in the use of frozen stallion semen. For prediction of fertility and for improving freezing methods, it is important to develop reliable techniques to assess the quality of semen in vitro. Many methods exist and are used, but not many studies have examined or showed the connection between laboratory test results and fertility of frozen-thawed stallion semen [4], [5]. The relationship between motility, the most frequently used test in horses, and fertility is far from clear [6], [7] and particularly for frozen semen it is not an exact measure of fertilizing potential [8]. In Malmgren’s review [9], conflicting correlations were reported between morphology – another commonly used test – and fertility of fresh stallion semen. It is often assumed that the condition of spermatozoa surviving after cryopreservation would be similar to the pre-freeze state. There is evidence that also the survivors have been affected [10]. Therefore, assessment and methods of examination applied for fresh semen may not be as useful for frozen semen.

Results of frozen semen evaluation tests and foaling rates of mares were compared in 2 experiments. In the first experiment, semen of 27 stallions was tested and in the second experiment semen of 23 stallions; 19 stallions participated in both experiments. Only stallions having foaling data from at least 7 mares were included; the data were also analyzed separately for stallions having ≥ 20 mares.

No single test was found to be a consistently reliable method for predicting the fertilizing capacity of semen. The results of Exp. 1 could not be repeated, although the stallions and methods used were partly similar. Differences in the outcome of these experiments could be explained also by different ejaculate batches.




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