Seven experiments were conducted to study the effect of freezing extenders, antioxidants, motility stimulants, thawing temperature, incubation temperature and time, centrifugation and capacitation on sperm chromatin instability (CI) as well as the influence of sperm CI on pregnancy rates of heifers (n = 360) after AI with frozen semen. Semen was collected once a week from Blonde d'Aquitaine and Limousine bulls (n = 3/breed) via an artificial vagina and only individual ejaculates (n = 300) of >0.3 x 10(9) sperm/ml and >or= 70% progressive motility were used. Sperm CI was evaluated by nuclear DNA susceptibility to acid-induced denaturation using acridine orange fluorescence and by chromatin susceptibility to decondensation using quantitative transmission electron microscopy. Bioxcell extender was better than AndroMed and egg yolk extenders in terms of low incidence of sperm CI in one bull (p < 0.05). Neither antioxidants (EDTA-2Na, Na-pyruvate and albumin) nor motility stimulants (caffeine and blood serum) had any significant effect on sperm CI. Thawing of frozen semen at 45 degrees C for 30 s decreased (p < 0.025) CI in one bull. Incubation of frozen sperm at 25 and 39 degrees C for 240 min increased sperm CI percentages from 3.47 +/- 0.48 and 4.50 +/- 0.41% to 6.70 +/- 0.36 and 9.71 +/- 0.53%, respectively (p < 0.001). Although centrifugation and removal of extracellular milieu increased CI of cooled sperm, it decreased CI of frozen-thawed sperm (p < 0.025). Follicular fluid as a capacitating agent destabilized chromatin structure (p < 0.001). Sperm vulnerability to CI had a negative impact (r(2) = 0.37-0.77, p < 0.001) on fertility of frozen ejaculates. In conclusion, in vitro manipulation of bovine semen can influence incidence of sperm CI, whereas integrity of sperm chromatin contributes significantly to heifers' fertility. We would recommend selection of the appropriate extender and thawing temperature for each bull together with careful manipulation of frozen semen to minimize damage of sperm chromatin.
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