The Anti-Müllerian Hormone as Endocrine and Molecular Marker Associated with Reproductive Performance in Holstein Dairy Cows Exposed to Heat Stress.

Anti-Müllerian hormone (AMH) is proposed as a biomarker for fertility in cattle, yet this associative relationship appears to be influenced by heat stress (HS). The objective was to test serum AMH and AMH-related single nucleotide polymorphisms (SNPs) as markers potentially predictive of reproductive traits in dairy cows experiencing HS. The study included 300 Holstein cows that were genotyped using BovineSNP50 (54,000 SNP). A genome-wide association study was then executed. Nine intragenic SNPs within the pathways that influence the AMH gene were found important with multiple comparisons adjustment tests (p < 1.09 × 10-6). A further validation study was performed in an independent Holstein cattle population, which was divided into moderate (MH; n = 152) and severe heat-stressed (SH; n = 128) groups and then subjected to a summer reproductive management program. Serum AMH was confirmed as a predictor of fertility measures (p < 0.05) in MH but not in the SH group. Cows were genotyped, which revealed four SNPs as predictive markers for serum AMH (p < 0.01), reproductive traits (p < 0.01), and additional physiological variables (p < 0.05). These SNPs were in the genes AMH, IGFBP1, LGR5, and TLR4. In conclusion, serum AMH concentrations and AMH polymorphisms are proposed as predictive markers that can be used in conjunction with genomic breeding value approaches to improve reproductive performance in Holstein cows exposed to summer HS conditions.

Blood Transcriptomic Analyses Reveal Functional Pathways Associated with Thermotolerance in Pregnant Ewes Exposed to Environmental Heat Stress.

Environmental heat stress triggers a series of compensatory mechanisms in sheep that are dependent on their genetic regulation of thermotolerance. Our objective was to identify genes and regulatory pathways associated with thermotolerance in ewes exposed to heat stress. We performed next-generation RNA sequencing on blood collected from 16 pregnant ewes, which were grouped as tolerant and non-tolerant to heat stress according to a physiological indicator. Additional samples were collected to measure complete blood count. A total of 358 differentially expressed genes were identified after applying selection criteria. Gene expression analysis detected 46 GO terms and 52 KEGG functional pathways. The top-three signaling pathways were p53, RIG-I-like receptor and FoxO, which suggested gene participation in biological processes such as apoptosis, cell signaling and immune response to external stressors. Network analysis revealed ATM, ISG15, IRF7, MDM4, DHX58 and TGFßR1 as over-expressed genes with high regulatory potential. A co-expression network involving the immune-related genes ISG15, IRF7 and DXH58 was detected in lymphocytes and monocytes, which was consistent with hematological findings. In conclusion, transcriptomic analysis revealed a non-viral immune mechanism involving apoptosis, which is induced by external stressors and appears to play an important role in the molecular regulation of heat stress tolerance in ewes.

Genetic Markers Associated with Milk Production and Thermotolerance in Holstein Dairy Cows Managed in a Heat-Stressed Environment.

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.

Molecular markers for thermo-tolerance are associated with reproductive and physiological traits in Pelibuey ewes raised in a semiarid environment.

Pelibuey sheep exhibit reproductive activity through the year, but warm weather lowers their fertility and demonstrates physiological limitations of environmental heat stress. Single nucleotide polymorphisms (SNPs) associated with heat stress tolerance in sheep have been reported previously. The objective was to validate the association of seven thermo-tolerance SNP markers with reproductive and physiological traits in Pelibuey ewes raised in a semiarid region. Pelibuey ewes were assigned to a cool (January 1st.- March 31st.; n = 101) or warm (April 1st.- August 31st.; n = 104) experimental group. All ewes were exposed to fertile rams and assessed for pregnancy diagnosis 90 days later; lambing day was reported at birth. These data served to calculate the reproductive traits of services per conception, prolificacy, days to estrus, days to conception, conception rate and lambing rate. Rectal temperature, rump/leg skin temperature and respiratory rate were measured and reported as physiological traits. Blood samples were collected and processed to extract DNA, which was genotyped using the TaqMan allelic discrimination method and qPCR. A mixed effects statistical model was used to validate associations between SNP genotypes and phenotypic traits. The SNPs rs421873172, rs417581105 and rs407804467 were confirmed as markers associated with reproductive and physiological traits (P < 0.05), and these SNPs were in the genes PAM, STAT1 and FBXO11, respectively. Interestingly, these SNP markers resulted as predictors for the evaluated traits but only in ewes from the warm group, which indicated their association with heat-stress tolerance. An additive SNP effect was confirmed with the highest contribution (P < 0.01) of the SNP rs417581105 for the evaluated traits. Reproductive performance improved (P < 0.05) and physiological parameters decreased in ewes carrying favorable SNP genotypes. In conclusion, three thermo-tolerance SNP markers were associated with improved reproductive and physiological traits in a prospective population of heat-stressed ewes raised in a semiarid environment.

Effect of calving season on the parameters and components of the lactation curve in Holstein dairy cows managed in a semi-desert climate.

The lactation curve in dairy cows is influenced by the calving season, which is highly dependent on the warm climate in semi-arid regions. Objective herein was to evaluate effects of calving season on the parameters and components of the lactation curve in Holstein cows. The study included 278,317 milk records collected from 1086 cows from the 1st to 5th calving and good body condition score. The cows were grouped according to the season in which they calved: winter-calved (CS1), summer-calved (CS2), and autumn-calved cows (CS3). Ambient temperature and humidity data were used to calculate the temperature-humidity index (THI). The NLIN procedure was used to estimate the parameters of the lactation curve that served to calculate the components. The mixed procedure was executed to analyze the fixed effect of calving season. Associations between lactation curve traits were tested using correlation and regression analyses. A univariate model was utilized to calculate heritability. Average THI values during the lactation period were 73.5, 68.5, and 69.5 units for CS1, CS2, and CS3 groups, respectively. Initial milk production and increasing rate to the maximum milk yield in CS1 and CS3 groups were higher (P < 0.05) than CS2 cows. However, persistency and total milk yield during the entire lactation period were superior (P < 0.05) for CS2 and CS3 cows compared to CS1 cows, probably due to the moderate heat stress during the lactation period in the CS1 group. In cows from CS2 and CS3 groups, total milk production at 305 days was moderately correlated with initial milk production (r = 0.47; P < 0.05), and highly correlated with milk yield at peak day (r = 0.91; P < 0.05) which resulted as reliable predictor for total milk yield during the entire lactation (R2 = 0.83). In conclusion, the THI prevailing during the different calving seasons appeared to be an important factor influencing the performance of the lactation curve.

Genome-wide association study of a thermo-tolerance indicator in pregnant ewes exposed to an artificial heat-stressed environment.

Environmental heat stress negatively influences sheep production in warm semi-arid regions. An animal's ability to tolerate warm weather is difficult to measure naturally due to environmental variability and genetic variation between animals. In this study we developed a thermo-tolerance indicator (TTI) to define heat stress tolerance in pregnant sheep in a controlled environment. Next, we performed a genome-wide association study (GWAS) to identify genomic regions and target genes associated with thermo-tolerance in sheep. Pregnant Columbia-Rambouillet crossbred ewes (n = 127) were heat-stressed inside a climate-controlled chamber for 57 days by increasing the temperature-humidity index to ≥30. Rectal temperature (RT) and feed intake (FI) data were collected daily and used for the predictive TTI analysis. After the tenth day of heat stress, the regression analyses revealed that FI was stable; however, when the ewe's RT exceeded 39.8 °C their FI was less than thermo-tolerant ewes. This average predicted temperature was used to classify each ewe as heat stress tolerant (≤39.8 °C) and non-heat stress tolerant (>39.8 °C). A GWAS analysis was performed and genomic regions were compared between heat stress tolerant and non-tolerant ewes. The single-marker genomic analysis detected 16 single nucleotide polymorphisms (SNP) associated with heat stress tolerance (P < 0.0001), whereas the multi-marker Bayesian analysis identified 8 overlapped 1-Mb chromosomal regions accounting for 11.39% of the genetic variation associated with tolerance to heat stress. Four intragenic SNP showed a remarkable contribution to thermo-tolerance, and these markers were within the genes FBXO11 (rs407804467), PHC3 (rs414179061), TSHR (rs418575898) and STAT1 (rs417581105). In conclusion, genomic regions harboring four intragenic SNP were associated with heat stress tolerance, and these candidate genes are proposed to influence heat tolerance in pregnant ewes subjected to an artificially induced warm climate. Moreover, these genetic markers could be suitable for use in further genetic selection programs in sheep managed in semi-arid regions.

Discovery and validation of candidate SNP markers associated to heat stress response in pregnant ewes managed inside a climate-controlled chamber.

Sheep production in desert environments during summer is challenging due to heat stress which reduces feed intake, growth, and fertility. Despite warm conditions, some ewes are able to maintain a normal performance suggesting the existence of genetic bases underlying heat tolerance. Our objective was to discover and validate genetic markers associated with thermo-tolerance in pregnant ewes exposed to warm environmental conditions. Using a well-defined model laboratory of heat stress in sheep, pregnant Columbia-Rambouillet crossbred ewes (n = 100) were examined. Following acclimation to the laboratory at thermo-neutral conditions, heat stress was induced in ewes by increasing the temperature-humidity index in a control environmental chamber during mid-gestation. Feed intake, water consumption, and rectal temperature were recorded daily and used to establish the heat stress tolerance index (HSTI) for each ewe. Rectal temperature was a predictor (P < 0.05) of feed intake, and the regression coefficient was used to classify the HSTI. In a subset of 24 ewes, a genome-wide association study (GWAS) was performed using the Illumina OvineSNP50 BeadChip. Single-marker analysis detected 3 intragenic SNPs associated with HSTI (P value = 10-5). Bayesian multi-marker approach discovered 26 chromosomal regions across the genome which accounted for 9.8% of the variation associated with HSTI. In an independent sheep population (n = 42), the three discovered SNPs were validated as molecular markers associated with thermo-tolerance phenotypic traits. These SNPs were located within the genes F13A1, PAM, and PRELID2. In conclusion, three SNPs appear to be novel molecular markers associated with heat stress tolerance in pregnant ewes providing new knowledge about genetic foundations of thermo-tolerance.