The polymorphism of the bovine β-casein gene (CSN2) is of increasing interest due to its relevance for A2 milk production. This study genotyped 2773 Holstein-Friesian cows for five CSN2 alleles (A1, A2, A3, B, I) using both conventional DNA-based PCR and a newly evaluated direct PCR protocol. Eleven genotypes were detected, with A2/A2 (33.9%) and A1/A2 (30.3%) being the most common, resulting in an A2 allele frequency of 59.0%. Genetic diversity indices indicated moderate polymorphism and a significant deviation from Hardy–Weinberg equilibrium, consistent with ongoing selection for the A2 allele. Associations between CSN2 genotype and milk traits (305-day milk, fat, and protein yield; fat% and protein%) were evaluated using linear mixed-effects models including lactation number, age at calving, and calving year as covariates, and cow ID as a random intercept. Several genotype effects reached statistical significance (p < 0.05); however, all effect sizes were very small (partial η² < 0.01), indicating that any influence of CSN2 on production traits is negligible within this population and management context. These findings suggest that A2-oriented selection is unlikely to compromise productivity. The direct PCR genotyping method achieved 96–100% success and enabled substantially faster and more cost-efficient processing (approximately 80–90% reduction in reagent costs), providing a rapid and scalable approach for large herds.

The CSN2 gene encoding β-casein has gained increasing attention in dairy cattle breeding due to the global adoption of A2-oriented selection strategies. However, robust large-scale evidence assessing potential unintended effects on functional traits, particularly fertility, under intensive commercial conditions remains limited. This study evaluated whether selection for the CSN2 A2 β-casein variant is associated with biologically relevant differences in fertility traits in Holstein cows. Reproductive and genomic data from 7826 lactation records of 2773 Holstein cows collected between 2022 and 2025 in a large commercial dairy herd were analyzed. Fertility indicators included days open, number of services per conception, calving interval, first-service conception rate, and pregnancy by 100 days in milk. Mixed-effects models accounting for repeated lactations and cow- and sire-level clustering were applied, and predefined equivalence margins were used to distinguish statistical non-significance from biological irrelevance. Across all evaluated fertility traits, differences among CSN2 genotypes (A1A1, A1A2, and A2A2) were consistently small, biologically negligible, and well within predefined equivalence margins. Differences in days open were within ±2 days, and effect sizes for count and binary traits were close to unity. Parity and calving year significantly influenced reproductive performance, whereas no CSN2 genotype × parity interactions were detected. These findings indicate that selection for the CSN2 A2 β-casein variant does not compromise reproductive performance under intensive commercial management conditions. From a breeding and industry perspective, the results support the implementation of A2-oriented selection strategies without biologically meaningful adverse effects on fertility.

Reliable preimplantation embryo sexing is relevant in cattle breeding; however, PCR-based Y-marker detection may be sensitive to limited DNA input and sample-type-specific properties of embryo-derived material. In this observational study, we examined assay-output patterns in a low-input multiplex PCR workflow targeting a male-specific Y-chromosome marker (MSY) together with an autosomal internal control targeting cyclin-dependent kinase 1 (CDK1). A total of 141 bovine embryo-derived samples were analyzed across three sample categories: whole embryos, cleavage-stage blastomere biopsies, and blastocyst-stage trophectoderm biopsies. Assay evaluability differed across sample categories, being highest in blastocyst-stage biopsy samples (90.6%, 29/32), intermediate in whole embryos (82.9%, 58/70), and lowest in blastomere-biopsy samples (64.1%, 25/39). Among technically evaluable CDK1-positive samples, apparent MSY-positive classification also differed across categories, with rates of 55.2% (32/58) in whole embryos, 52.0% (13/25) in blastomere-biopsy samples, and 6.9% (2/29) in blastocyst-stage biopsy samples. In a same-lysate repeat-amplification subset, overall evaluability concordance was 92.5% and overall apparent MSY-based classification concordance was 88.2%, although subgroup sizes were limited. Because no independent reference method for true embryo sex was available and sample category was structurally confounded with developmental stage, semen type, sire, and partly PCR-date structure, the findings should be interpreted as assay-output observations rather than measures of biological sex ratio or diagnostic sexing accuracy. Overall, embryo-derived sample categories showed differing assay evaluability and apparent MSY-classification patterns within this low-input PCR setting, supporting the need for sample-type-specific validation when low-cell embryo-derived samples are used.

Maintaining udder health and milk quality is important in dairy goat production; however, the interpretation of somatic cell count (SCC) in goats is complicated by physiological and non-infectious factors. This study evaluated the associations among udder and teat morphological traits, SCC, bacteriological status, and udder surface temperature in clinically healthy Saanen dairy goats. Udder conformation was assessed using a linear scoring system in multiparous goats (n = 70). Composite milk samples were collected at three lactation stages for SCC and milk quality analysis, whereas separate half-udder milk samples were used for bacteriological examination. Udder surface temperature was measured using infrared thermography (IRT) at the final sampling time point. Stepwise multiple regression analysis was applied to identify udder and teat traits associated with log-transformed SCC. The results showed considerable variation in udder conformation, with the greatest deviations observed for teat placement. Fore udder attachment, udder cleft, teat length, and teat form were significantly associated with SCC, with stronger attachment, a more distinct udder cleft, shorter teats, and a more cylindrical teat shape being associated with lower SCC. Bacteriological examination detected mastitis-associated bacteria in 45.0% of half-udder samples, mainly coagulase-negative Staphylococcus spp. and Corynebacterium spp., while the proportion of obligate udder pathogens was highest in the >2,000,000 cells/mL SCC category. Bacteriologically positive udder halves showed slightly higher surface temperatures than bacteriologically negative udder halves (p < 0.05), whereas no significant differences in udder surface temperature were observed among SCC categories. These findings suggest that udder morphology is associated with SCC and may be useful in phenotypic udder health assessment. IRT may provide complementary information on bacteriological status under standardized conditions, but its diagnostic value in dairy goats requires further validation in larger, longitudinal studies.

This study presents a structured narrative review integrating methodological and decision-oriented perspectives. Milk proteins, particularly β-casein, have attracted increasing scientific and commercial attention due to their genetic variability and role in dairy production and product differentiation. Among β-casein variants, the A1 and A2 alleles of the CSN2 gene are of particular relevance, as their single-nucleotide difference has influenced breeding strategies and the expansion of A2-oriented dairy markets. Although multiple validated molecular genotyping approaches are available for CSN2 A1/A2 discrimination, guidance on their context-appropriate deployment in agricultural systems remains largely technique-centric. The present framework integrates analytical performance, sample complexity, and operational constraints to support the selection of fit-for-purpose methods across breeding, diagnostic, and dairy authentication contexts. Classical and advanced approaches, including polymerase chain reaction–restriction fragment length polymorphism (PCR-RFLP), allele-specific polymerase chain reaction (AS-PCR) and amplification refractory mutation system PCR (ARMS-PCR), high-resolution melting (HRM) analysis, sequencing-based methods, single nucleotide polymorphism (SNP) arrays, and digital polymerase chain reaction (dPCR), are comparatively evaluated not only in terms of sensitivity and throughput but also with respect to scalability, reproducibility, and decision risk. This framework provides a practical decision-support tool for aligning genotyping strategies with application-specific risk profiles, thereby improving reliability, transparency, and regulatory compliance in modern dairy systems.

The European wildcat (Felis silvestris) is threatened due to habitat loss and hybridisation with domestic cats (Felis catus), representing a significant conservation challenge in Hungary. We genotyped 118 field-collected cats from seven Hungarian sub-regions and 30 domestic cat references at 14 autosomal microsatellites, and sequenced a 669 bp mitochondrial ND5 fragment from the field samples. At K = 2, STRUCTURE assigned 84 field samples to the wildcat-associated cluster, 18 to the domestic-cat-associated cluster, and 16 (13.6%) to an intermediate membership interval. The combined non-wildcat proportion (28.8%) is not a hybridisation rate. Intermediate assignments fell to 10 at K = 3, demonstrating model dependence. Simulations recovered parental, F1 and F2 genotypes, but most second-generation and all third-generation backcrosses were assigned to a parental cluster. Genetic and geographic distances were significantly but weakly associated (Mantel r = 0.118, p = 0.005). Eight ND5 haplotypes were identified; dw4 predominated in the North Hungarian Mountains and w1 in Southern Transdanubia. These maternal patterns are compatible with historical lineage retention and/or introgression, but a single locus cannot date or distinguish these processes. The results support targeted genetic monitoring while highlighting the need for ancestry-informative SNP or genome-wide validation before management decisions are based on individual hybrid classifications.