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.
