The bovine kappa-casein (κ-CN) is a phospho-protein with 169 amino acids encoded by the CSN3 gene. The two most common gene variants in the HF breed are CSN3*A and CSN3*B while CSN3*E has been found with lower frequency. The aim of this study was to optimize a laboratory method for genotyping of these three alleles as well as to determine their genotype and allele frequencies in the HF cattle population in the Republic of North Macedonia. Genomic DNA was extracted from full blood from 250 cows. The target DNA sequence was amplified with newly designed pair of primers and the products were subjected to enzymatic restriction with HindIII and HaeIII endonucleases. Genotype determination was achieved in all animals. The primers successfully amplified a fragment of 458 bp and the digestion of this fragment with both endonucleases enabled differentiation of five different genotypes with the following observed frequencies: AA (0.39), AB (0.29), BB (0.16), AE (0.10), and BE (0.06). The estimated allele frequencies were: CSN3*A (0.584), CSN3*B (0.336) and CSN3*E (0.08). The observed genotype frequencies differed significantly (P<0.01) from those that would be expected under HW equilibrium, while the fixation index (F=0.17) indicated moderate heterozygosity deficiency. Nevertheless, the CSN3*B allele was present with relatively high frequency which should be used to positively select for its carriers, since increasing its frequency could help to improve the rheological properties of the milk intended for cheese production.
Motility patterns of spermatozoa are an indicator of their fertilizing capacity. Reduced glutathione (GSH) has been reported to induce positive effects on the biological quality of the frozen-thawed spermatozoa. The aim of this study was to investigate the effects of GSH addition to semen extender based on the following kinetic parameters: continuous line velocity (VSL), average path velocity (VAP), curvilinear velocity (VCL), amplitude of lateral head displacement (ALH), linearity (LIN), straightness (STR), beat cross frequency (BCF), total motility (tMOT) and progressive motility (pMOT), and to appoint them as indicators of its presence. A soybean-based extender was used for dilution of the semen samples, fractioned in two parts, one containing GSH (5 mMol/ml) and second without GSH. The ejaculates (n=48) were collected from two rams (January - May, 2013) which were classified in two groups according to the used extender: Group 1 (with GSH, n=24) and Group 2 (without GSH, n=24), and then frozen in liquid nitrogen on -196°C degrees in a programmable freezer. Assessment of the samples has been performed post-thawing (30 sec. at 37°C) on CASA equipment, acquiring kinetic parameters. Results showed that only VSL and BCF have a statistically significant difference between group1 and group 2 (102.98±15.13 vs. 88.47±20.63, t=2.77, p<0.01 and 32.01±2.68 vs. 89.47±2.92, t=3.13, p<0.01, respectively). The summary of the investigation concludes that none of the kinetic parameters could be appointed as indicators that confer the positive effect of GSH as an additive to soy-bean semen extender.
Reduced glutathione (GSH) and homologous ram seminal plasma (HSP), used as additives in cryopreserving (CP) media prior to freezing, showed conflicting results in retaining structural integrity and progressive motility in post-thawed ram spermatozoa. The aims of this research were: (1) to assess the effect of GSH and/or HSP supplementation via soybean-lecithin CP extender on cryopreserved ram spermatozoa viability, morphology and motility pattern; and (2) to assess the effect of incubation in the context of the previous aim. Quantitatively and qualitatively, homogenized and pooled ram ejaculates (N=10) were extended with one of the following extenders: control (C) – tris-based, GSH and HSP-free, experimental-1 (E1) – C + GSH 5 mM, experimental-2 (E2) – C + HSP 20 % and experimental-3 (E3) - GSH 5 mM + HSP 20 %. Following thawing, samples were taken at 0- and 3-hours from each group (n=10) and were assessed for spermatozoa viability, morphology, and motility pattern. C-0h samples yielded a spermatozoa population with low viability, altered head morphology and highly deviated motility pattern. E3-3h samples yielded spermatozoa with unaffected viability, head morphology and high progressive motility. In conclusion, E3 extender added to cryopreserved-thawed ram spermatozoa is most efficient in obtaining high viability, unaltered head morphology, and progressive motility.