Abstract
     The treatment of sperm with cholesterol-loaded cyclodextrins (CLC) prior to cryopreservation enhances the percentage of sperm surviving the process in cold-shock sensitive species. It was recently reported that CLC improved also the quality of boar sperm (species highly sensitive to cold and osmotic shocks) when used at a concentration of 1 mg CLC/120 x 106 sperm. Nevertheless, it is unknown how this treatment affects the sperm functionality in vitro and the sperm fertilizing ability.
     The aim of this Thesis was to determine the effect of the treatment of boar sperm with CLC prior to cryopreservation, on different aspects of the sperm functionality in vitro (osmotic resistance, motility and integrity of the sperm plasma membrane, capacitation status and capacitation dynamics, ability to adhere to oviductal epithelial cells and chromatin integrity dynamics), and on the in vitro (ability to penetrate immature oocytes) and in vivo fertilizing abilities (in weaned sows hormonally treated with eCG-hCG and cervically inseminated 37 or 30 h after hCG injection).
     Treating boar sperm with CLC widened (P < 0.05) the osmotic resistance limits of the fresh sperm in both hypo- (50, 75 y 150 mOsm/kg) and hyper-osmotic sides (600 y 800 mOsm/kg). At least of 45% of the CLC-treated sperm maintained the sperm plasma membrane integrity (%LS) and the percentages of progressively motile (%PMS) and total motile sperm (%TMS) were maintained between 40% and 50% in these anisosmotic conditions. After freezing-thawing, the CLC-treated sperm exhibited similar quality (percentage of live sperm with intact acrosomes, %LIA; %TMS; %RMS) than control samples, but maintained their quality for longer (%LS, %TMS and %RMS, +1.5 h; P < 0.05) than the control sperm. In addition, the quality of the sperm stored at 16 ºC remained in acceptable values for 6 h (43% TMS and 30% PMS) and after 26 h the samples maintain 55% LS and 28% TMS. This aspect could be useful in the handling of frozen-thawed semen in the farms.
     The CLC-treated sperm showed a similar capacitation status and dynamics than control sperm after freezing-thawing. Moreover, this treatment did not affect to the chromatin integrity dynamics. However, the CLC-treated sperm showed a higher ability to adhere to oviductal epithelial cells in vitro (+5; P < 0.0001) than control sperm.
     Respect to the fertility, the CLC-treated sperm penetrated more effectively immature oocytes in vitro (+17%; P < 0.0001) and presented a higher number of sperm per oocyte (+2; P < 0.0001) than control sperm. Nevertheless, this treatment did not improve the fertilizing ability of the sperm in vivo (neither at 37 nor at 30 h of asynchrony), although when the timing of insemination with respect to the ovulation induction was reduced (from 37 to 30 h), an improvement in the farrowing rate was observed for the CLC-treated samples that matched the percentage obtained for the control samples (P > 0.05). Surprisingly, the control sperm fertilized equally well at both asynchronies.
     Although the data obtained are not conclusive, it is likely that this treatment modifies the sperm plasma membrane structure and its organization, and as a result its functionality. It is unknown how the membrane is exactly modified, but it seems to have some influence in the stages which take part in the fertilization process, and the CLC-treated sperm need different asynchronies (between ovulation and insemination) to those commonly used for frozen-thawed sperm.