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ABSTRACT:The objective was to determine effects of equilibration length and freezing curve type as well as their interactions on motility and live spermatozoa proportion in bull sperm after thawing. The ejaculates of 6 sires were repeatedly collected. Fresh semen was diluted with one extender and divided into 3 groups equilibrated for 30, 120, and 240 min. Subsequently, half straws of each group were frozen using standard 3-phase or 2-phase freezing curve differing in the rate of temperature decrease. Th e spermatozoa motility (M) was evaluated immediately after thawing and at 30, 60, 90, and 120 min of thermodynamic test (TDT). Live spermatozoa proportion was evaluated after thawing and at the end of TDT. Average of spermatozoa motility (AM), decrease of spermatozoa motility (MD), average proportion of live spermatozoa (ALS), and decrease of live spermatozoa proportion (DLS) through the TDT were calculated. Significant inter-sire differences in AM (0.45-17.0%; P < 0.05-0.01), MD (0.76-12.57%; P < 0.05-0.01), and ALS (0.99-23.8%; P < 0.01) were detected. The longest equilibration ensured the highest M during TDT and AM (+2.72 and +4.58%; P < 0.05-0.01), however higher MD (+4.06%; P < 0.01) compared to standard length as well. Straws freezed using 2-phase curve achieved higher M through TDT, AM (+7.3%; P < 0.01) as well as ALS (+11.77%; P < 0.01). The 2-phase curve presented higher M compared to the 3-phase freezing curve within all equilibration lengths. Significant differences in AM, MD,; P < 0.05-0.01) between equilibration length vs freezing curve interactions were determined. Results document the importance of equilibration length, freezing curve, and their interaction effect on live spermatozoa proportion and sperm motility after thawing as well as necessity of individual conditions for bulls semen processing and insemination doses production.

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

The objectives of this study were to determine and evaluate the eff ect of bull, diluter and addition of LDL in diff erent concentration on the percentage rate of spermatozoa survival a er cold shock. In total, four bulls were collected during a period of eight weeks. A total of 8 samples of fresh semen with required quality were processed. Three extenders were used for dilution of each sample; AndroMed®, Bioxcell® and Triladyl®, each in standard and LDL enriched variants. In the case of AndroMed® and Bioxcell®, 4, 6 and 8% of LDL were simply added. In Triladyl®, 6, 8 and 10% of LDL replaced the standard egg yolk component. Resistance of spermatozoa against cold shock (0 °C, 10 minutes) was evaluated by the percentage rate of live sperm using Eosin-Nigrosine staining immediately and 2 hours a er heat incubation (37 °C). The results showed the infl uence of bull individuality as an important factor. Among diluters used it is possible to recommend AndroMed® and Bioxcell® due to signifi cantly (P < 0.01) lower decline of live sperm proportion during the cold shock test than Triladyl® (−9.19, respectively −4.95%). The optimal LDL concentration increasing resistance of spermatozoa against cold shock was not determined, therefore subsequent research is necessary. bull semen, sperm survival, cold shock, extender, LDL cholesterol

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.