Currently, considering cryopreservation of bull semen, there is no clear consensus over the comparability of cryoprotective efficacy of extenders with soybean lecithin and those based on egg yolk. The objective of this study was to prove the use of Low Density Lipoprotein (LDL) extracted from hen-egg yolk as an enhancing factor for soybean lecithin-based extenders. In total, 35 ejaculates of (seven bulls x five ejaculates per bull) were collected and cryopreserved at a commercial insemination centre. The effect of the LDL addition to the extenders AndroMed ® and Bioxcell ® was tested in a 6% (v/v) concentration on spermatozoa after thawing. Modified extender composition effects were assessed on sperm functional parameters motility, plasma membrane, mitochondrial membrane potential and acrosomal integrity after thawing by CASA, flow cytometry and fluorescent microscopy, respectively. Based on kinematic parameters determined from CASA, k-means cluster analysis was used to classify individual spermatozoon into specific subpopulations (fast, medium fast and slow). A subpopulation of fast spermatozoa was increased in the presence of LDL in both selected extenders (P < 0.05). Moreover, the positive effect of LDL on sperm motility was confirmed by decreasing the percentage of sperm in slow subpopulation (P < 0.05). The effect of LDL addition on the incidence of spermatozoa with intact plasma membrane was not demonstrated in any case of extender used (P > 0.05). The percentage of sperm with intact acrosome was improved when LDL was added to Bioxcell ® extender (P < 0.05). On the other hand, addition of LDL to AndroMed ® extender improved mitochondrial intactness after thawing (P < 0.05). In conclusion, our results showed that adding LDL to selected soybean lecithinbased extenders considerably ameliorated the functional parameters of spermatozoa after thawing and thus this lipoprotein could represent an improving agent for soybean lecithin-based extender for bull semen cryopreservation.

The objectives of this study were to determine the effects of sire, extender, and addition of Low Density Lipoprotein (LDL) to extenders used on the percentage rate of spermatozoa survival after cold shock. Two groups of extenders were compared: without LDL addition (control variants) and LDL enriched (experimental variants). Three extenders were used: AndroMed ® extenders, and 6-10% LDL addition into the Triladyl ® extender. In total, 12 samples of fresh semen were collected from 4 bulls during a period of 8 weeks. Bovine spermatozoa cold shock resistance (1 ± 1 °C, 10 min) was evaluated by the percentage rate of live sperm using eosin-nigrosine staining immediately and after heat incubation (37 ± 1 °C, 120 min). The results showed the effect of sire as important and individual differences between selected sires in their sperm resistance against cold shock were confirmed. AndroMed ® and Bioxcell ® were found to be providing better protection of bull semen to cold shock compared to Triladyl ® due to lower decline of live sperm proportion. Our results detected a positive effect of LDL addition on sperm resistance against cold shock, especially on lower decrease of live sperm percentage rate after 120 min of the heat test (P < 0.05). Further studies are needed to assess the optimal concentration of LDL in various kinds of extenders as well to state ideal time and temperature conditions for ensuring LDL reaction with sperm. , bull sperm, extender, LDL cholesterol, sperm survival, cold shock, eosin-nigrosine staining Reproduction

Krejcárková A., Folková P., Šimoník O., Šašková M., Krejčířová R., Drábek O., Rajmon R. (2017): Effects of zearalenone, α-zearalenol, and genistein on boar sperm motility in vitro. Czech J. Anim. Sci., 62, 435-445.Genistein (GEN) and zearalenone (ZEA), environmental oestrogens commonly present in feedstuff for pigs, are known for their effects on reproductive functions. The aim was to verify the in vitro effects of 0.5-20 µM concentrations of GEN, ZEA and its metabolite α-zearalenol (α-ZOL) on pig sperm motility. A dose-dependent increase of the immotile sperm amount against fast and medium-fast sperm clusters was observed with all three oestrogens from the lowest concentrations tested. Individual CASA (computer-assisted sperm analysis) parameters of motile sperms seemed to be less sensitive indicators. This should be considered especially in toxicological research on a sperm model. Background of inconsistencies in to date-published papers is discussed. The results shift the effective concentrations of ZEA, α-ZOL, and GEN to values achievable in vivo and raises the questions of risk assessment of these compounds in pig reproduction.

The aim of the study was to evaluate the effect of repeated semen collection and the substitution of normal egg yolk with clarified egg yolk to commercially produced semen extender on qualitative parameters of frozen-thawed canine semen. Two semen collections were scheduled in a 24-hour interval and in each of six dogs, three 1st and three 2nd collections were performed. The frozen-thawed sperm samples were prepared either with clarified or normal egg yolk and motility and viability were evaluated. The effect of the sequence of semen collection was demonstrated by significant differences in motility and also in viability of sperms both in native and frozen-thawed ejaculate. The percentage of viable sperms was significantly higher in samples from the 2nd compared to the 1st collection. This trend was the same also in motility except in native ejaculate. The addition of clarified egg yolk was beneficial for higher survival of sperms immediately after thawing and also after 30 min of incubation, compared to samples with normal egg yolk. Sperm motility evaluated after thawing was higher in samples with clarified egg yolk, without an apparent connection with semen collection sequence. The decrease of values of the qualitative parameters of sperms observed in the period of 30 min of incubation was significantly slowed down when clarified egg yolk was used. This was especially obvious in samples from the 2nd collection.

The aim of this study was to evaluate the eff ect of clarifi ed egg yolk addition to semen extender, and the semen collection sequence on the quality of frozen-thawed semen in dogs. Semen was collected from 6 dogs in a time interval of 24 hours. As parameter of the quality of frozen-thawed (F-T) semen, the motility by computer assisted sperm analysis (CASA) and plasma membrane integrity by hypoosmotic swelling test (HOS) were evaluated. All kinematic parameters of sperm motility were higher in F-T samples containing the whole in comparison to the clarifi ed egg yolk. The sequence of semen collection aff ected sperm movement characteristics of native as well as F-T semen, but it was not possible to determine whether the fresh semen from the 1 st or 2 nd collection is of higher quality. All motility parameters of sperms frozen with extender containing the whole egg yolk were signifi cantly higher in the case of the 2 nd collection. The situation was not so clear in the case of clarifi ed egg yolk addition, but the velocity values were higher in F-T samples from the 2 nd collection. In contrast to proven diff erences in motility, the eff ect of the addition of clarifi ed egg yolk and the sequence of semen collection were not projected at all on the quality of plasma membrane of canine sperms evaluated by HOS test.