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Background: Measurement of pregnancy-associated glycoproteins (PAGs) by radioimmunoassay or enzyme-linked immunosorbent assay has been commonly used for early pregnancy diagnosis in ruminants. However, an accurately pregnancy detection depends on test antibody, breed and number of embryos. Only few studies have conducted to detect or predict animals at risk of late embrionic mortality (LEM) and to use hormonal interventions for embryo losses reducing, but this area is still open in sheep. The current study aimed to evaluate the effect of gonadotropin releasing hormone (GnRH) administration on Day 4 after artificial insemination on the pregnancy-associated glycoproteins in dairy sheep with different reproductive status in relation to pregnancy and late embrionic mortality detection. Materials, Methods & Results: Sixty-five East Friesian sheep were divided in 2 groups - I (Control group, n = 35) and II (GnRH group, n = 30) and sublected to estrus synchronization and artificial insemination (AI). Group I was not treated and Group II received 50 µg GnRH on Day 4 after AI. PAGs in blood serum were measured by Alertys Ruminant Pregnancy test on Days 4, 12, 20, 25 and 35 after AI and ultrasound pregnancy test was conducted on Days 20, 25, 35 and 60. Reproductive status (non-pregnant, pregnant and animals with LEM) was determined by ultrasound and the results between different groups were compared. The PAGs mean values according to reproductive status and Day after AI were analysed. Accuracy, sensitivity and specificity of the PAGs test for pregnancy diagnosis on Day 35 were calculated. On Day 20 after AI the pregnat sheep (83.3%) in group I tended to be higher than those (77.1%) in group II with decrease of 25.7% and 20% for the same groups on Day 25. On day 25 LEM was recorded in 33.3% and 24% in group I and II, respectively. The ultrasound exams on Day 60 confirmed the results from Day 25 after AI. A total value of non-pegnant, pregnant animals and LEM was 20%, 56.9% and 28.8%, respectively. The mean values of PAGs in animals with the same reproductive status in group I and II no differed statistically between Days 4 and 35 after AI. On Day 25 the PAGs values in LEM groups (0.126 ± 0.072 and 0.179 ± 0.029) were higher than those (0.062 ± 0.038 and 0.083 ± 0.023) in the non-pregnant groups, but no significan difference was deternined. On Day 35 after AI the mean values of PAGs (0.414 ± 0.125 and 0.421 ± 0.121) for the pregnant groups were significantly (P < 0.05) higher than those (0.078 ± 0.053 and 0.093 ± 0.034) for the non-pregnant groups. The values of PAGs in LEM groups on Day 25 (0.099 ± 0.062 and 0.113 ± 0.058) were decreased and close to the mean values in non-pregnant sheep. A significant effect of the day after AI on the PAGs values was evidenced in pregnant animals only (control r = 0.97 and GnRH r = 0.98; P < 0.05). The PAGs started to rise rapidly between Days 25 and 35 after AI. On Day 35 the accuracy (98%), the sensitivity (97.3%) and the specificity (100%) of the PAG test for pregnancy diagnosis were similar to the values (100%) for ultrasound method. Discussion: The gonadotropin treatment on Day 4 after AI tended to improve the reproductive performance in the sheep, but no significant effect of GnRH on the PAGs values in the animals with different reproductive status was found. The PAGs profiles in non-pregnant and LEM sheep were close and distinguishing between non-pregnant and LEM ewes on Day 35 was impossible. The PAGs values in pregnant sheep showed significantly (P < 0.05) increase between Days 25 and 35 after AI with higher mean values in pregnant than non-pregnant groups on Day 35 (P < 0.05). Alertys Ruminant Pregnancy test was a reliable for pregnancy detection in sheep on Day 35 after artificial insemination. [ABSTRACT FROM AUTHOR]

The current study aimed to determine the reproductive performance and progesterone profile in East Friesian sheep after GnRH administration on day 4 post artificial insemination (AI). The experiment was carried out with 67 sheep subjected to estrus synchronization and artificial insemination. The animals were divided in two groups: I (control group, n = 30) and II (GnRH group, n = 37), treated by GnRH on day 4 after AI. Rams were introduced on day 12 and removed on day 20 after AI. Ultrasound pregnancy check was on days 20 and 60 after AI, and sheep were separated on the basis of their reproductive status (RS). Blood progesterone (P4) concentrations were measured by ELFA on days 4, 12 and 20 after AI, and a likehood for early embryonic mortality was estimated. The percentages of non-pregnant and pregnant sheep in both groups on day 20 were close; however, there were significantly (P < 0.05) more pregnant sheep in the GnRH group on day 60 compared with day 20. The investigated factors affected the P4 concentrations independently of each other. Analysis of the main effects of GnRH and RS showed a significant (P < 0.005) influence on the progesterone profile. The day after AI had a strong positive effect on the P4 concentration, with significant (P < 0.001) differences between mean P4 values measured during the different days. The estimated likehoods of early embryonic mortality (EEM) for both groups was 25.4%. In conclusion, GnRH injection on day 4 after artificial insemination and the introduction of ram on day 12 did not have a direct effect on the pregnancy rate, but led to improvement of the reproductive performance at the flock level. Gonadotropin releasing hormone treatment, reproductive status and day after AI affected the progesterone concentrations in East Friesian sheep irrespective of each other, and had a significant (P < 0.005) effect on the hormonal profile. The treatment by GnRH on day 4 after AI tended to reduce early embryo mortality, but future investigations are needed to clarify this effect. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

The application of the bovine serum albumin (BSA) column method for sexing of spermatozoa is cost effective and appropriate for small ruminants. The current study compares motility characteristics and kinematic parameters of fresh, chilled and sexed ram semen with the aim to select the high-quality ejaculates for semen sexing. Fresh, chilled and sexed semen from 4 East Friesian rams was analysed by CASA, and immotile sperm cells, motile sperm cells, progressive motility, non-progressive motility, VCL, VAP, VSL, STR, LIN and WOB were determined. Semen sexing was carried out in bovine serum albumin columns and incubation for 45 min at a temperature of 25°C. The average values of all semen parameters and the change of the most important indices in individual rams were estimated. Significant differences (P < 0.05) were detected between immotile and motile sperm cells, progressive and non-progressive motility, VCL and WOB of fresh and chilled semen. All investigated parameters between fresh and isolated upper and bottom layer spermatozoa, excluding STR and WOB, differed significantly (P < 0.05). The same dependency (P < 0.05) was detected for motile sperm, progressive motility, VCL, STR and LIN between chilled and sexed spermatozoa. In conclusion, the average values for motile sperm, progressive motility, VCL, STR and LIN of chilled and sexed ram spermatozoa are significantly (P < 0.05) affected by chilling and sexing in BSA columns at 25°C for 45 min. CASA analysis of motility and kinematic parameters of chilled semen can provide a correct choice of ejaculates with high quality for sexing. The individual features of rams have influence on the semen characteristics and should be take into consideration in selection of the donors for a production of the sexed semen. [ABSTRACT FROM AUTHOR]