Morphological and functional changes of stallion spermatozoa after cryopreservation during breeding and non-breeding season

The study compared quality and freezability of stallion semen during breedi ng and non-breeding seasons. Ejaculates were collected twice per week from four stallions during May (n = 24) and December (n = 24). The semen was mix ed with skim milk extender, centrifuged and resuspended in fresh extender. Aliquots of this sperm suspension were separated from extender and diluted in TALP medium for sperm evaluation or with cryoextender (type "Gent" or a combination of Triladyl(R) and skim milk). Samples of 0.5 mi were cryoprese rved in straws using a programmed freezer. Parameters of sperm quality were evaluated before and after freezing/thawing. These included percentages of motile spermatozoa and of morphological intact sperm. Typical injuries wer e demonstrated by scanning electron microscopy (S.E.M.). The acrosomal stat us was visualised using FITC-conjugated peanut agglutinin, and the acrosome reaction was induced by calcium ionophore A 23187. The chromatin stability was estimated by acridine orange test. In winter, the average percentages of motile and morphologically normal spe rm (67 and 74.3%. respectively) were higher than during the breeding season in May (59 and 65.9%: P < 0.05). After freezing/thawing the proportions of vital and intact sperm decreased significantly. The number of motile sperm declined to 15 and 18% in May and December (range 5-40%), and of morpholog ically intact sperm to 51% in both seasons. Results of S.E.M. showed typica l membrane ruptures in the acrosomal region and some sperm with abnormal ne cks. The proportion of frozen sperm with spontaneous acrosome reaction was higher during winter (86.5 versus 77.0%). suggesting a higher degree of mem brane reactivity. Percentages of spermatozoa with denaturated chromatin wer e minimal and showed minimal differences between fresh and frozen state, st allions or seasons. An additional decondensation treatment with papain and DTE revealed a slightly enhanced number of spermatozoa with denaturable DNA after cryopreservation, especially in December (5.4 +/- 1.3%). The influen ce of cryoextenders was not significant for most sperm parameters, but ther e was a high variability between the stallions. Altogether, the influence o f factors on the quality of spermatozoa has the following rank order: cryop reservation > stallion > season. Different cellular structures seem to have different susceptibilities to physicochemical stress. The cryopreservation of sperm during December results in survival rates similar to those measur ed during the breeding season, even more important for successful preservat ion is the selection of suitable semen donors. (C) 2001 Elsevier Science B. V. All rights reserved.