This study aimed to evaluate the effect of the ovarian status and steroid hormone concentration on the day of TAI on the reproductive performance of dairy cows subjected to estrus synchronization treatment and timed artificial insemination with sexed semen. Seventy-eight cyclic Holstein cows pre-treated with PGF2α-GnRH were divided in two groups—I (Preselect-OvSynch, n = 38) and II (OvSynch+PRID-7-day+eCG, n = 40)—and inseminated with sexed semen. The presence of preovulatory follicle (PF) with or without corpus luteum (CL), the PF diameter, the estradiol (E₂) and progesterone (P₄) concentrations on the day of TAI, the pregnancy rate (PR) and embryo loss were determined. On the day of TAI, 78.4% of all the pregnant cows presented a PF (mean size 1.80 ± 0.12 cm) without CL, low P₄ (0.59 ± 0.28 ng/mL) and high E₂ (12.35 ± 2.62 pg/mg) concentrations. The positive correlation between the size of the PF and the level of E₂ in the pregnant cows from group II was stronger than that of group I (R = 0.82 vs. R = 0.52, p < 0.05). The pregnancy rate on day 30 (57.5% vs. 36.8%) and day 60 (50% vs. 26.3%; p < 0.05) and the embryo losses (13% vs. 28.5%) showed better effects of treatment in group II. In conclusion, the ovarian status and the steroid hormone concentration on the day of TAI influence the pregnancy rates of dairy cows subjected to estrus synchronization and timed artificial insemination with sexed semen.

This study was aimed to assess the efficiency of coconut water extender with addition of soy lecithin and sucrose as nonpermeable cryoprotectants for canine semen vitrification, using a simple method that yields a high survival rate of spermatozoa for clinical use. Twelve ejaculates from 12 adult normozoospermic dogs were collected separately by digital manipulation and only the second semen fraction was used in this study. After evaluation of volume, concentration, viability, total and progressive motility, velocity parameters and morphology, semen was diluted with a coconut water extender (50% (v/v(volume per volume)) coconut water, 25% (v/v) distilled water and 25% (v/v) 5% anhydrous monosodium citrate solution) with addition of soy lecithin and fructose at 1% and 0.25M sucrose until final concentration of 100x10⁶ spermatozoa/ml. After equilibration at 5ºC for 60 minutes, semen was vitrified by “direct dropping method” into liquid nitrogen in spheres with a volume of 30 μl. After a week of storage the spheres were devitrified as three of them were dropped into 0.5 mL of CaniPlus AI medium (Minitüb, Germany), which was previously warmed in a water bath at 42ºC for 2 minutes and evaluated about the above mentioned parameters. It was found that vitrification resulted in a lower percentage of viable sperms, normal morphology, total and progressive motilities (p0.05) compared to fresh semen samples. In conclusion, our results demonstrate that vitrification with coconut water extender with addition of 1% soy lecithin and 0.25M sucrose as cryoprotectants, has an excellent potential for routine canine sperm cryopreservation.

The aim of the present study was to evaluate the effect of equilibration process on canine sperms after vitrification using coconut water extender with addition of soy lecithin and sucrose as nonpermeable cryoprotectants. Twelve ejaculates were collected separately by digital manipulation from 12 adult dogs. Only the second fraction of the ejaculate was used in this study, which was evaluated about volume, concetration, viability, total and progressive motility, kinetic parameters and morphology. After evaluation, semen was diluted with a coconut water extender (50% coconut water (v/v), 25% (v/v) distilled water and 25% (v/v) 5% anhydrous monosodium citrate solution) with addition of soy lecithin and fructose at 1% (v/v) and 0.25 M sucrose until final concentration of 100x106 spermatozoa/ml. Samples were divided into three aliquots and each of them was processed at different regimens: without equilibration, 5ºC for 30 minutes and 5ºC for 60 minutes and then vitrified by dropping 30 μl of sperm suspension directly into liquid nitrogen. Sperm pellets were devitrified at least one week later as three of them were dropped into 0.3 mL of CaniPlus AI (Minitüb, Germany), which was previously warmed in a water bath at 37ºC for 2 minutes. Sperm concentration and motility parameters were assayed using a computer-aided sperm analysis (CASA) system, viability-by supravital staining technique and morphology parameters were evaluated in Haemacolor® stained semen samples. In conclusion, our results demonstrate that when vitrification and coconut water extender with addition of 1% soy lecithin and 0.25 M sucrose as cryoprotectants were used, presence of equilibration time of 60 minutes returned the best canine sperm quality results.

The present study investigated the effect of sex-sorting by TLR7/8 ligand R848, cooling and cryopreservation on the motility and kinematic characteristics of ram spermatozoa for the first time. Four ejaculates per ram (n=2) were collected, pooled and after that were split to 8 equal parts. Four parts were used for sexing and four parts were analyzed as unsexed semen. The sperm sexing was done by application of the TLR7/8 ligand R848 (resiquimod). Diluted with freezing extender unsexed and sex-sorted semen were cooled in a refrigerator at temperature 5?C for 5 hours and after that freeze/thawed. Each part of semen at each step of the experiment was subjected to computer assisted semen analysis for evaluation of motility and speed parameters of spermatozoa. After sexing the significant reducing of kinematic parameters in both fractions were observed. The dilution of sexed spermatozoa with freezing extender before cooling reliably increased the progressive motility and speed parameters as VCL, VAP, and VSL in X- and Y-fractions and kept them during the cooling at relatively high level. Freezing adversely impaired the motility of sexed spermatozoa. The values of speed parameters of unsexed spermatozoa after thawing were significantly higher than those of X and Y spermatozoa. Dilution with an appropriate freezing extender and cooling are most suitable approaches for storing of sexed ram spermatozoa.

Background: Effect of the epigenetic factors on the male fertility is well proofed. Sperm acts as a carrier of genetic material, and its DNA methylome can affect maternal pregnancy rate and offspring phenotype. However, the research on the DNA methylation in the spermatozoids of livestock males, in particular rams, is still limited. To best of our knowledge the data about as a global as well as gene specific DNA methylation in ram spermatozoa from different breeds and ages are missed in the scientific literature. The present study was designed to analyze the relationship between methylation levels of the important for spermatogenesis gene SIRT1 in spermatozoa and fertilizing ability of sperm in rams from different breeds and ages. Materials, Methods & Results: The ejaculates of 16 rams from Lacaune, East Friesian and Assaf breeds at age between 18 to 96 months were evaluated. The kinematic parameters of 2 semen samples from each animal were estimated by CASA. The separated spermatozoa were used for DNA extraction followed by bisulfite conversion. The DNA methylation of SIRT1 was detected through quantitative methylation-specific PCR using 2 sets of primers designed specifically for bisulfite-converted DNA sequences to attach methylated and unmethylated sites. The breed and age effect on the gene SIRT1 methylation by ANOVA was estimated. Experimental females included 393 clinically healthy milk ewes (Lacaune, n = 131; East Friesian sheep, n = 100 and Assaf, n = 162) in breeding season. Reproductive performances (conception rate at lambing, lambing percentage and fecundity) of ewes, inseminated by sperm of the investigated rams, were statistically processed. ANOVA showed that the animal breed influences significantly on the level of DNA methylation of gene SIRT1 in ram spermatozoa (P = 0.002) An average value of DNA methylation of SIRT1 in ram sperm from Lacaune breed was significantly higher than in Assaf and East Friesian (81.21 ± 15.1% vs 36.7 ± 14.2% and 38.3 ± 18.6 respectively, P < 0.01). The highest percent of SIRT1 methylation was observed in old animals compared to the young and middle-age. Moderate and strong correlations (r from 0.44 to 0.71, P < 0.05) between the methylation level of the SIRT1 gene in rams' sperm and reproductive parameters of inseminated ewes in all breeds were established. Discussion: Our data are the first message about the effect of breed on the specificity of DNA methylation of gene SIRT1 in ram spermatozoa. These results demonstrated an existence of the sheep breeds with high and low level of DNA methylation of gene SIRT1 in ram sperm. Although the effect of age on the methylation level in sperm is still discussable, our results showed a moderate correlation between age and methylation level of SIRT1 in spermatozoa of rams. Taking into account that DNA methylation in sperm is stabilized with puberty onset and is a heritable epigenetic modification, it can be a promising marker of sperm quality in animal breeding. In all investigated breeds the rams with relatively high level of DNA methylation of gene SIRT1 in spermatozoa (50-68%) demonstrated a high conception rate at lambing (> 70%). In conclusion, the DNA methylation level of the SIRT1 gene in ram spermatozoa is determined by both the breed and the age of the animals and correlates with fertilizing ability of sperm. Keywords: SIRT1 methylation, ram spermatozoa, Lacuane, East Friesian, Assaf breeds.