Dairy production in Holstein cows in a semiarid environment is challenging due to heat stress. Under such conditions, genetic selection for heat tolerance appears to be a useful strategy. The objective was to validate molecular markers associated with milk production and thermotolerance traits in Holstein cows managed in a hot and humid environment. Lactating cows (n = 300) exposed to a heat stress environment were genotyped using a medium-density array including 53,218 SNPs. A genome-wide association study (GWAS) detected six SNPs associated with total milk yield (MY305) that surpassed multiple testing (p < 1.14 × 10⁻⁶). These SNPs were further validated in 216 Holstein cows from two independent populations that were genotyped using the TaqMan bi-allelic discrimination method and qPCR. In these cows, only the SNPs rs8193046, rs43410971, and rs382039214, within the genes TLR4, GRM8, and SMAD3, respectively, were associated (p < 0.05) with MY305, rectal temperature (RT), and respiratory rate. Interestingly, these variables improved as the number of favorable genotypes of the SNPs increased from 0 to 3. In addition, a regression analysis detected RT as a significant predictor (R² = 0.362) for MY305 in cows with >1 favorable genotype, suggesting this close relationship was influenced by genetic markers. In conclusion, SNPs in the genes TLR4, GRM8, and SMAD3 appear to be involved in the molecular mechanism that regulates milk production in cows under heat-stressed conditions. These SNPs are proposed as thermotolerance genetic markers for a selection program to improve the milk performance of lactating Holstein cows managed in a semiarid environment.

Background: Chlamydia abortus is the etiological agent of ovine enzootic abortion (OEA), a worldwide disease that significantly impacts to cattle, sheep, and goat production. This bacterium causes abortions during the last third of gestation and its zoonotic. The lack of prior symptoms before an abortive event complicates the clinical diagnosis of OEA and its differentiation from other infections, highlighting the need for an effective diagnostic tool. Some conventional diagnostic methods could be accurate, but in some cases require expensive equipment and highly trained personnel, limiting their field application. Under this context, the loop-mediated isothermal amplification (LAMP) emerges as an accessible and efficient alternative, capable of operating at a constant temperature and simplifying the interpretation of results visually through colorimetry. Objective: To validate a new LAMP assay using colorimetry for the visual detection of Chlamydia abortus in domestic ruminants. Method: Fifty-six DNA samples collected from ruminants with abortion history were used as biologic control of reference. LAMP specific primers were designed for the TARP gene of C. abortus. A colorimetric assay was validated for the LAMP test of C. abortus (crLAMP-Chla). The concordance (K), sensibility (Se) and specificity (Sp) parameters of crLAMP-Chla were calculated considering real time PCR as reference. Results: The crLAMP-Chla detected the pathogen from 15 minutes at 65 °C, even at low DNA concentrations. The K, Se and Sp values were 1, 100% and 100% (P=0.05) respectively. Conclusion: This methodology represents a significant advancement for the control of C. abortus in ruminants, providing an accessible and efficient diagnostic tool to improve public and animal health.

Background: Bovine trichomoniasis is a venereal disease caused by Tritrichomonas foetus (T. foetus) that significantly impairs the reproductive health of cattle. While bulls are asymptomatic carriers of this protozoan, cows infected with T. foetus exhibit embryonic deaths, abortions, reproductive tract infections or infertility. In México, the first report of T. foetus was made more than 60 years ago and, despite of the importance of trichomoniasis in cattle, efforts to control it through surveillance, diagnosis and adequate characterization have been scarce. Objective: To confirm the presence of T. foetus in animals from northwest México via molecular detection, sequencing, and phylogenetic analyses. Methods: Forty biological samples (smegma and mucopurulent vaginal secretion) were collected and analyzed by PCR technique to identify T. foetus using specific primers. Utilizing samples positive for T. foetus, sequencing and phylogenetic analyses were performed over the molecular structure of the generated amplicons to verify their homology and genetic relationships. Results: The PCR technique identified T. foetus in smegma and in clinical cases of metritis. Sequences obtained from PCR products revealed a 99.65% of similarity and a bootstrap of 99% with isolates of T. foetus reported in China, Spain, and USA. Based on the sequencing results, five new Mexican isolates of T. foetus were published in NCBI. Conclusion: This is the first report confirming the presence of T. foetus in México using sequencing and phylogenetic analyses from positive cattle samples. Our research effort represents the basis for developing alternative and accurate tests to diagnose T. foetus as part of the strategy to epidemiologically control bovine trichomoniasis in the country

This study aimed to identify and validate functional genetic markers associated with fertility and thermotolerance traits in dairy cows exposed to HS. Spring-calving Holstein cows (n=400) from three dairy farms located in the Yaqui Valley, Mexico, were genotyped using the BovineSNP50 panel. Reproductive traits such as days open (DO) and services per conception (SPC) were recorded following a fixed-time artificial insemination protocol. After quality controls 45,832 SNPs were included in a genome-wide association study, which identified 21 and 20 SNPs associated with DO and SPC (P < 0.05), respectively. Five SNPs common to both traits were selected for a marker-assisted association study using a mixed effects statistical model. This validation analysis was performed in an independent Holstein cow population (n=184) confirming associations of the SNPs rs136074864, rs109479159, rs43520457, and rs133770320 in the genes EXOC4, GLI3, GPR151, and TRHDE1, with reproductive and thermotolerance traits. Cows carrying favorable genotypes showed increased gene expression, higher serum AMH, progesterone and HSP70, and lower cortisol levels. Correlation analyses confirmed the physiological relevance of these genes. In conclusion, these results highlighted the key role of four candidate genes as molecular markers for improving reproductive efficiency and thermotolerance in Holstein cows managed under HS.

Heat stress (HS) is a major issue affecting productivity and physiological responses in dairy cattle. Advances in gene function research have made it possible to identify favorable genotypes for HS tolerance. This study evaluates the combined effects of temperature–humidity index (THI) and genomic load of favorable SNPs in the genes GRM8, SMAD3, and TLR4 on daily milk yield (DMY), rectal temperature (RT), and gene expression in Holstein cows (n = 160). Environmental conditions ranged from thermoneutral to severe HS, allowing the assessment of genotype by environment interactions. A subset of cows (n = 40) was selected based on genomic load to evaluate gene expression under opposite thermal conditions. Results showed that increasing THI was associated with reduced DMY and increased RT, with more pronounced effects in cows carrying fewer favorable genotypes. Slope analyses revealed that cows with higher genomic load exhibited a moderate decline in DMY and lower increase in RT, indicating enhanced resilience to HS. Significant THI × genomic load interactions were detected above a threshold of 79 units (p < 0.05). Gene expression analyses supported these findings, showing differential expressions of GRM8, SMAD3, and TLR4 under HS conditions. The functional and predictive value of these genes as markers of thermotolerance could help producers by supporting their application in genetic selection programs, thus providing another tool to combat HS and improve resilience and productivity in dairy systems.