Szilárd Bodó

Targeted CSN2 codon-67 genotyping supports A2-oriented breeding and herd management. We compared Kompetitive Allele-Specific PCR (KASP) with a locally implemented gel-based tetra-primer amplification refractory mutation system PCR (T-ARMS PCR) workflow in a balanced, non-population-representative Holstein–Friesian panel using re-audited Sanger-supported reference classifications. Both assays distinguish His67-associated (A1-type) from Pro67-associated (A2-type) β-casein classes rather than complete CSN2 alleles. The paired analysis included 96 independent samples. KASP produced 90 reference-concordant, four reference-discordant, and two no-call outcomes (93.8% all-record reference-concordant yield), whereas T-ARMS PCR produced 77 reference-concordant, six reference-discordant, and 13 ambiguous outcomes (80.2%). Concordance among callable/evaluable results was 95.7% and 92.8%, respectively. In the primary comparison, KASP showed a 13.5 percentage point higher yield (95% CI, 4.2–22.9 percentage points; p = 0.015), mainly because it generated fewer non-interpretable outcomes. All callable KASP discordances involved Pro67/Pro67 reference samples called His67/Pro67. Because no independent DNA dilution, new DNA extraction, repeat KASP run, or new Sanger sequencing was performed, their cause and reproducibility remain unresolved. Formal repeatability and between-run reproducibility were not systematically evaluated; therefore, this comparison is not a formal assay validation and does not define T-ARMS PCR performance beyond the tested local configuration. Discordant, ambiguous, or high-impact classifications require independent repeat testing or sequencing confirmation.

Heat stress negatively affects bovine fertility and embryonic development, while cryopreservation imposes additional cellular stress that may impair embryo survival. The present study investigated whether controlled heat treatment applied during the early morula stage could influence subsequent blastocyst development, molecular stress responses, and post-thaw survival of bovine embryos. In vitro-produced bovine embryos were exposed to controlled thermal treatment involving gradual elevation in temperature from 38.8 °C to 40.0 °C followed by subsequent cooling. Blastocyst formation, post-thaw survival after slow freezing, and the expression of stress-associated genes and microRNAs were evaluated. Heat treatment did not significantly affect blastocyst formation rates or post-thaw embryo survival compared to the control groups. However, significant alterations in the expression of stress-associated markers were observed. The expression of HSP90AA1 and HSPB11 was significantly reduced following heat treatment, while cryopreservation induced increased expression of these markers compared to embryos exposed only to heat treatment. Significant alterations were also detected in the expression of several stress-associated microRNAs. These findings suggest that controlled heat exposure during the morula stage may induce molecular changes associated with the embryonic response to heat stress without compromising developmental competence or cryosurvival. The results contribute to a better understanding of the molecular mechanisms underlying embryonic responses to thermal and cryogenic stress and may support future optimization of embryo production and cryopreservation strategies.

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.

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.

This study aimed to establish a model for investigating X chromosome inactivation using transgenic mouse strains expressing green fluorescent protein (GFP). The D4/XGFP-Tg (XGFP) strain carries the GFP transgene on the X chromosome; therefore, due to random X chromosome inactivation, female offspring from crosses between XGFP males and CD-1 females exhibit mosaic GFP expression. In contrast, the B5/EGFP-Tg (EGFP) strain harbours autosomal integration of the same reporter construct, resulting in uniform GFP expression in progenies. Analysis of CD-1 × XGFP attached blastocysts revealed strong GFP expression in giant trophoblast cells and primordial germ cells (PGCs) at E6.5, demonstrating paternal X-chromosome reactivation. In 14.5-day-old CD-1 × XGFP female embryos and CD-1 × EGFP embryos, intense CAG promoter-driven GFP signals were detected in the brain, heart, gonads, somites, and limbs. In line with random X-chromosome inactivation, only 56% of embryonic fibroblast cells, derived from CD-1 × XGFP female embryos, exhibited GFP expression. These findings validate that CD-1 × XGFP mice represent a valuable in vivo model for studying X chromosome inactivation during early embryonic development and PGC specification. Furthermore, CD-1 × XGFP embryonic fibroblasts represent a valuable in vitro model for investigating the molecular mechanisms governing X-chromosome activation and inactivation.

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.

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.

Az in vitro embrió-előállítás (IVEP) 40 éves töretlen fejlődésének köszönhetően napjainkra egyre fontosabb szerepet tölt be a világ szarvasmarha-tenyésztésében. A petesejtek kinyerése, laboratóriumi körülmények között történő termékenyítése, az embriók tenyésztése, a krioprezerváció, illetve a laboratóriumi környezet terén elért fejlődés több millió genetikailag magas értékű egyed előállítását tette lehetővé. Gyakorlati alkalmazási lehetőségei mellett az in vitro embrió-előállítás (IVEP) számos kutatás alapját biztosítja. Segítségével a korai vemhességi időszak modellezhető, az embriófejlődés környezeti tényezői tanulmányozhatók, így olyan kérdésekre kaphatunk választ, melyek a tudományos és gyakorlati szakemberek számára is fontosak. Egy megbízhatóan működő embrió előállító rendszer kialakítása során sok technológiai lépés optimalizálására van szükség. Az elmúlt év során a szerzőknek sikerült kialakítaniuk egy olyan, jól működő rendszert, melyben sikeresen folyik a szarvasmarha embriók előállítása, így oktatási, kutatási feladatok megvalósítására alkalmas. A közleményben a szerzők bemutatják a rendszer kialakítását egészen a kezdetektől, ennek sikereit és buktatóit egyaránt, valamint beszámolnak az itt végzett 31 IVEP program eredményességéről. A kialakított rendszerben átlagosan a termékenyített petesejtek 69%-a osztódik, és 23,4%-a fejlődik beültethető állapotú hólyagcsíráig, így alkalmas ovum pick-up (OPU) IVEP programok megvalósítására is.

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.

In cattle, initial pregnancy diagnosis takes place during the late embryonic/early fetal stage of gestation. From this point onward, pregnancy loss may occur in up to one fifth of pregnancies before the initial pregnancy diagnosis is confirmed. This means the early identification of risk factors is a key part of pregnancy diagnosis and herd management. The various factors responsible for pregnancy losses are classified into infectious and noninfectious. Among the noninfectious causes, several dam-related (circumstances of the individual pregnancy or milk production) and herd-related factors causing stress have been well established. In this review, we summarize the impacts of these noninfectious factors and predict associated risks of pregnancy loss.