In Vitro Gene Conservation Status and the Quality of the Genetic Resources of Native Hungarian Sheep Breeds.

Studies revealed a global loss of genetic resources for local sheep breeds. Therefore, the current study aimed to introduce and highlight the progress made on Hungary's existing gene conservation program (small Gene Bank). Furthermore, we evaluated breed (Tsigai, Cikta, and Racka), season, and individual variabilities (n = 24) of the pre-freeze and post-thaw semen stored in the Gene Bank to enhance the gene conservation of the breeds. The samples were cryopreserved manually, and post-thaw spermatozoa were analyzed for motility (CASA), viability, chromatin structure, and morphometry of the sperm nuclei. Ejaculate volume, spermatozoa concentration, subjective motility and standard motility, kinematic parameters, and spermatozoa's head area standard deviation of the post-thaw samples differed significantly among breeds (p < 0.05). Season affected ejaculate volume, total spermatozoa number/ejaculate, STR, BCF, and ALH. We observed a significant (p < 0.001; 0.05) breed and season interaction on concentration, total spermatozoa number/ejaculate, VCL, LIN, WOB, spermatozoa's head average perimeter and nucleus length (Tsigai and Cikta differed but were statistically the same as Racka). Similarly, season significantly (p < 0.05) affected the proportion of ejaculate suitable for freezing. There was a significant (p < 0.05) difference in kinematic parameters and viability among the rams across the breeds. The spermatozoa's head morphometry of the Tsigai and Cikta breeds differed significantly (p < 0.05) among the rams. There were individual and breed differences in many spermatozoa quality parameters. The stored samples are of good quality, with more than 40% having intact membranes and low abnormal chromatin condensation.

The Influence of Three Commercial Soy Lecithin-Based Semen Extenders and Two Spermatozoa Concentrations on the Quality of Pre-Freeze and Post-Thaw Ram Epididymal Spermatozoa.

There are limited studies on the factors affecting the success of ram epididymal spermatozoa (REPS) cryopreservation. On this note, the current study assessed the influence of three commercial soy lecithin-based semen extenders, AndroMed® (AND), BioXcell® (BIO), and OviXcell® (OVI), and two concentrations (400 × 106 vs. 200 × 106 spermatozoa/mL) on the pre-freeze and post-thaw quality of REPS. The REPS were retrieved from nine adult rams' testes and diluted with each of the three extenders to both concentrations. Straws were frozen manually. Standard motility (SMP) and kinematic parameters (KPs) were assessed via a CASA, while spermatozoa viability, morphology, and acrosomal integrity were assessed via the Kovács-Foote staining technique. The concentration did not significantly affect the pre-freeze and post-thaw SMP and KPs of REPS. BIO and OVI had significantly higher pre-freeze and post-thaw BCFs, post-thaw VAP, and the percentage of all intact heads than AND. In contrast, AND had a significantly lower percentage of REPS with tail defects than BIO and OVI. The 400 × 106 spermatozoa/mL concentration resulted in a significantly higher percentage of all intact heads than the 200 × 106 spermatozoa/mL concentration. Freezing significantly increased tail defects and decreased the percentage of REPS with distal cytoplasmic droplets. The cryopreservation of REPS at the 400 × 106 spermatozoa/mL concentration is recommended. All three extenders must be optimized to preserve the viability, membrane integrity, and better normal morphology of REPS; the reason for increased tail abnormality after the freezing/thawing process needs to be studied.

The impact of retrieval method and breed on the motility and kinematic parameters of fresh and post-thaw ram epididymal spermatozoa.

This study was conducted to develop ideal post-mortem gamete retrieval and conservation methods to establish a Hungarian ex-situ in vitro gene bank. Pairs of testes from German Mutton Merino (n = 7) and Hungarian Black Racka (n = 7) rams were collected at a slaughterhouse, transported to the laboratory and stored overnight (4-5 °C) before processing. Post mortem ram epididymal spermatozoa (REPS) were obtained from the cauda epididymidis by slice or incision methods. Fresh samples were extended to 200 × 106/mL cell concentration, filled into mini straws and equilibrated at 5 °C for 2 h. Freezing was performed manually in a Styrofoam box. The fresh and post-thaw total motility, progressive motility and kinematic parameters of REPS were assessed using the CASA technique. The collection method did not affect significantly the fresh and post-thaw motility and kinematic parameters. Merino had higher (P < 0.05) testicular weight. Racka had significantly better fresh and post-thaw linear movement but had statistically the same (P > 0.05) cryotolerance as Merino. In conclusion, both collection methods were found suitable for REPS retrieval. The REPS from Racka exhibited better linear movement values than those from the Merino breed. The cryotolerance of REPS of both breeds was comparable.

Microbial Profiling of Amniotic Fluid, Umbilical Blood and Placenta of the Foaling Mare.

The presence of a microbiome/microbiota in the placenta is hotly debated. In previous studies, the presence of bacteria in equine amniotic fluid and umbilical blood was independent of foal health. The objective of the present study was to determine if the same bacteria are present in the equine placenta as in amniotic fluid and umbilical blood. Samples were obtained from 24 parturient mares and foals. Placental bacterial DNA was extracted, and the microbiome was identified using 16S rRNA sequencing. All amniotic fluid samples contained some polymorphonucleocytes; bacteria were isolated from four samples. Aerobic or anaerobic growth was found in 18 and 3 umbilical blood samples, respectively. Serum amyloid A was <5 mg/L in all 24 samples, total WBC varied between 2900 and 10,700/µL, and fibrinogen varied between 0 and 5.16 g/L. In jugular blood, serum amyloid A was <5 mg/L in all 24 foals, total white blood count was 3200 to 8100/µL, and fibrinogen was 0.44 to 4.42 g/L. The diversity of bacterial microbiota was similar in all placental regions at the phylum level but differed at the genus level; the most abundant phyla were Proteobacteria (42-46.26%) and Actinobacteria (26.91-29.96%). In conclusion, bacteria were found in the fetal compartments and placenta of healthy equine pregnancies; however, we can neither prove nor disprove the hypothesis that the placenta has its own microbiome.

Andrological and cytogenetic investigations of an infertile Przewalski's stallion.

The case of an 8-year-old, sexually active but infertile Przewalski's stallion (Equus ferus przewalskii) was studied. Besides the infertility, the stallion also showed permanent problems with its body condition, being obviously weaker than all the other group members. The horse was kept in a separate place for two years with 12 mares in its harem group (six mares had foals earlier); however, none of the mares covered got pregnant. Andrological and cytogenetic investigations revealed underdeveloped testes, arrested spermatogenesis, azoospermia, and XY/XXY/X0 mosaicism. We classify the case as a mosaic Klinefelter syndrome, the first reported case in Przewalski's horse.

Assessment of equine alpha-fetoprotein levels in mares and newborn foals in the periparturient period.

Alpha-fetoprotein (AFP) is best known in human obstetrics for its association with fetal anomalies recognized in the 1970s. Although this fetal protein had been shown to be present in the sera of many mammalian species, its possible diagnostic role in the detection of abnormalities was evaluated only later, when a research laboratory published variable levels of AFP in different groups of mares with pregnancy problems (twins, conception failure, placentitis, embryonic loss), and subsequently differences were demonstrated in its serum levels between aborted and healthy mares. In this study, peri- and intrapartal AFP levels were measured in maternal serum, amniotic fluid, neonatal blood, and umbilical blood samples. The mean levels of AFP were lower in umbilical blood and amniotic fluid samples than in foal and maternal blood. Older mares had lower AFP levels correlated with their age in years. The time remaining until foaling had a significant, non-linear effect on AFP levels: an elevation could be detected in the last two weeks of pregnancy, followed by a decline after foaling. Also, AFP levels were found to be elevated in the hot summer months. There was a significant individual variation in AFP levels in the population studied.