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ContentsTwenty ejaculates from _ve dairy AI!bulls were used to compare\ in a split!sample experiment\ the fertility ð45 day! non!return!rate "NRR# from more than 03999 AI# and sperm viability post!thaw of semen diluted with an egg yolk! "Tri! ladyl þ # or soybean!based "Biociphos!Plus þ # commercial extender[ The in vitro evaluations were divided in two experi! ments[ Experiment 0 "n 19# included post!thaw evaluations of motility "subjective and computerized#\ membrane integrity "CalceinAM:EthD!0\ SYBR!03:PI\ and osmotic resistance testÔ RT#\ and capacitation status "CTC:EthD!0#[ Experiment 1 "n 09# included evaluations of the capacitation!"CTC:EthD! 0# and acrosome status "FITC!PSA:EthD!0# during incubation with:without a challenge with solubilized zona pellucida pro! teins "SZP#[ No signi_cant di}erence in the fertility "58[0 2 9[7 versus 58[1 2 9[7# results was found between the two extenders[ In experiment 0\ the computerized motility evaluations post! thaw "CASA# showed higher values for Biociphos!Plus þ pro! cessed semen for the velocity patterns and lateral sperm head displacement[ After 5 h at room temperature "19Ð11>C# all the CASA motility patterns were signi_cantly higher for Biociphos! Plus þ [ The proportion of spermatozoa with intact membranes assessed by CalceinAM was signi_cantly higher in Biociphos! Plus þ "p ³ 9[990# compared to Triladyl þ \ but such di}erence was not seen when using SYBR!03 or the ORT!assay[ When using the CTC:EthD!0 assay\ a lower proportion of acrosome reacted "AR# spermatozoa post!thaw "p ³ 9[90# was found in Biociphos!Plus þ processed semen\ as well as a tendency "p ³ 9[96# for a higher number of uncapacitated spermatozoa[ In experiment 1\ the proportion of uncapacitated spermatozoa was signi_cantly higher for Biociphos!Plus þ when semen was incubated "27>C and 4) CO 1 # without SZP at both 9 "p ³ 9[990# and 29 min "p ³ 9[94#[ Concomitantly\ Triladyl þ showed a higher percentage of capacitated spermatozoa at 9 "p ³ 9[90#\ 29 "p ³ 9[94# and 019 min "p ³ 9[94#[ A higher "p ³ 9[94# incidence of AR!spermatozoa was seen in Triladyl þ at the beginning of the incubation with SZP[ No signi_cant di}erence between extenders was detected for the acrosome status by the FITC!PSA!assay[ Incubation with SZP induced acrosome reaction of capacitated spermatozoa in both extenders\ which was detected by CTC and FITC!PSA assays[ In conclusion\ fertility was not a}ected by Biociphos!Plus þ when 04 × 09 5 of spermatozoa per AI dose were inseminated[ The _nding that higher frequencies of spermatozoa seemed more membrane stable post!thaw\ when frozen in Biociphos! Plus þ \ might indicate that this extender better protects the sperm viability compared with Triladyl þ [

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The aim of this work was to study the effect of progesterone (P4) on capacitation and acrosome reaction (AR) of post-thaw bovine spermatozoa in vitro. Spermatozoa were incubated (0-180 min) in capacitation medium supplemented with 0, 0.1, 1.0 and 10.0 microg/ml of P4. At different time intervals aliquots were taken to determine sperm plasma membrane lipid destabilization, or capacitation (AR induced by lysophosphatidylcholine) in spermatozoa. The second experiment aimed to study the effects of P4, as potential inducer of AR in heparin-capacitated spermatozoa. The acrosomal status and viability of spermatozoa were evaluated under an epifluorescence microscope using Ethidium homodimer/peanut agglutinin fluorescein isothiocyanate staining method. Plasma membrane scrambling in spermatozoa was assessed by a flow cytometer, using merocyanine staining. The results show that P4 at the concentrations used had no negative effects on sperm viability. Progesterone significantly enhanced sperm capacitation (p < 0.001), but had no effect on plasma membrane lipid stability (p > 0.05) and did not significantly increase the AR of heparin-capacitated spermatozoa (p > 0.05). Progesterone displayed its effects in a dose-dependent manner with a maximum effect of 10 microg/ml P4 at 180 min of incubation. The results demonstrate that in cryopreserved bovine semen, P4 acts as capacitating, but not as an AR-inducing agent.

Contents In this study, the relations between fertility (56‐day non‐return rates, 56‐day NRR) after artificial insemination (AI) and bull sperm characteristics post‐thaw, after swim‐up and after co‐incubation with heparin (Hep) and hyaluronan (HA), respectively, were determined, attempting to determine if such a procedure could be of value to evaluate the potential fertilizing ability of frozen‐thawed AI bull spermatozoa. Spermatozoa from 20 semen batches derived from 20 Swedish Red and White AI bulls ranging widely in their field fertility after AI (55–79% 56‐day NRRs) were evaluated with regards to post‐thaw motility, membrane integrity, and migration through a simple swim‐up procedure. Sperm viability and capacitation status were evaluated by two different vital staining procedures and chlortetracycline hydrochloride staining. Sperm motility and membrane integrity post‐thaw (e.g. indicators of sperm viability) were significantly correlated (r = 0.53, p < 0.05 and r = 0.59, p < 0.01, respectively) with fertility. Heparin (5 µg/ml) significantly (p lt; 0.001) increased the frequencies of capacitation and acrosome‐reaction (AR) among swim‐up separated spermatozoa, whereas HA at a concentration of 50 ng/ml did not have any significant capacitating effect. The incidences of capacitated or AR‐spermatozoa following Hep‐treatment were not correlated with fertility. On the other hand, the percentage of viable spermatozoa was significantly (p < 0.001) lower in Hep‐treated samples than in control and HA‐treated samples and was significantly (r = 0.49, p < 0.05) correlated with fertility after AI (56‐day NRR). The results indicate that the percentage of viable spermatozoa after swim‐up separation and heparin‐exposure from a selected population of AI bulls were significantly and positively related to the AI fertility of the donors and thus could be used as a parameter to determine the fertilizing ability of frozen—thawed AI bull spermatozoa.