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Comprehension of the genetic basis of temperament has been improved by recent advances in the identification of genes and genetic variants. However, due to the complexity of the temperament traits, the elucidation of the genetic architecture of temperament is incomplete. A systematic review was performed following the Preferred Reporting Items for Systematic Reviews and Meta-Analyses (PRISMA) statement to analyze candidate genes related to bovine temperament, using bovine as the population, SNPs and genes as the exposure, and temperament test as the outcome, as principal search terms for population, exposure, and outcome (PEO) categories to define the scope of the search. The search results allowed the selection of 36 articles after removing duplicates and filtering by relevance. One hundred-two candidate genes associated with temperament traits were identified. The genes were further analyzed to construct an interaction network using the STRING database, resulting in 113 nodes and 346 interactions and the identification of 31 new candidate genes for temperament. Notably, the main genes identified were SST and members of the Kelch family. The candidate genes displayed interactions with pathways associated with different functions such as AMPA receptors, hormones, neuronal maintenance, protein signaling, neuronal regulation, serotonin synthesis, splicing, and ubiquitination activities. These new findings demonstrate the complexity of interconnected biological processes that regulate behavior and stress response in mammals. This insight now enables our targeted analysis of these newly identified temperament candidate genes in bovines.

Background: Temperament is an important production trait in cattle and multiple strategies had been developed to generate molecular markers to assist animal selection. As nonsynonymous single nucleotide polymorphisms are markers with the potential to affect gene functions, they could be useful to predict phenotypic effects. Genetic selection of less stress-responsive, temperamental animals is desirable from an economic and welfare point of view., Methods and Results: Two nonsynonymous single nucleotide polymorphisms identified in HTR1B and SLC18A2 candidate genes for temperament were analyzed in silico to determine their effects on protein structure. Those nsSNPs allowing changes in proteins were selected for a temperament association analysis in a Brahman population. Transversion effects on protein structure were evaluated in silico for each amino acid change model, revealing structural changes in the proteins of the HTR1B and SLC18A2 genes. The selected nsSNPs were genotyped in a Brahman population (n = 138), and their genotypic effects on three temperament traits were analyzed: exit velocity, pen score, and temperament score. Only the SNP rs209984404-HTR1B (C/A) showed a significant association (P = 0.0144) with pen score. The heterozygous genotype showed a pen score value 1.17 points lower than that of the homozygous CC genotype., Conclusion: The results showed that in silico analysis could direct the selection of nsSNPs with the potential to change the protein. Non-synonymous single nucleotide polymorphisms causing structural changes and reduced protein stability were identified. Only rs209984404-HTR1B shows that the allele affecting protein stability was associated with the genotype linked to docility in cattle., (© 2024. The Author(s).)

Knowledge of circadian rhythm clock gene expression outside the suprachiasmatic nucleus is increasing. The purpose of this study was to determine whether expression of circadian clock genes differed within or among the bovine stress axis tissues (e.g., amygdala, hypothalamus, pituitary, adrenal cortex, and adrenal medulla). Tissues were obtained at an abattoir from eight mature nonpregnant Brahman cows that had been maintained in the same pasture and nutritional conditions. Sample tissues were stored in RNase-free sterile cryovials at -80 °C until the total RNA was extracted, quantified, assessed, and sequenced (NovaSeq 6000 system; paired-end 150 bp cycles). The trimmed reads were then mapped to a Bos taurus ( B. taurus ) reference genome (Umd3.1). Further analysis used the edgeR package. Raw gene count tables were read into RStudio, and low-expression genes were filtered out using the criteria of three minimum reads per gene in at least five samples. Normalization factors were then calculated using the trimmed mean of M values method to produce normalized gene counts within each sample tissue. The normalized gene counts important for a circadian rhythm were analyzed within and between each tissue of the stress axis using the GLM and CORR procedures of the Statistical Analysis System (SAS). The relative expression profiles of circadian clock genes differed ( p < 0.01) within each tissue, with neuronal PAS domain protein 2 ( NPAS2 ) having greater expression in the amygdala ( p < 0.01) and period circadian regulator ( PER1 ) having greater expression in all other tissues ( p < 0.01). The expression among tissues also differed ( p < 0.01) for individual circadian clock genes, with circadian locomotor output cycles protein kaput ( CLOCK ) expression being greater within the adrenal tissues and nuclear receptor subfamily 1 group D member 1 ( NR1D1 ) expression being greater within the other tissues ( p < 0.01). Overall, the results indicate that within each tissue, the various circadian clock genes were differentially expressed, in addition to being differentially expressed among the stress tissues of mature Brahman cows. Future use of these findings may assist in improving livestock husbandry and welfare by understanding interactions of the environment, stress responsiveness, and peripheral circadian rhythms.

Physiological and psychological stressors have been associated with the attrition of telomeres, which are the protective caps of chromosomes. This study compares the telomere length (TL) in 4-year-old Brahman cows grouped by the first parity (n = 8) and the second parity (n = 11). The cows were bled via jugular venipuncture, weighed, and had their body condition scores recorded at Day -28 prior to calving and at Day + 7 and Day + 28 post-calving. The duration of labor (Dlabor) and parturition ease were recorded. The peripheral leukocytes were isolated, the leukocyte blood count with differential was recorded, and the genomic DNA was extracted. The relative quantity of telomere products, which is proportional to the average TL, was determined via multiplex quantitative PCR using the ratio (T/S ratio) of bovine telomere and β-globulin DNA. Standards of the bovine telomere (10 12 -10 7 dilution series) and β-globulin (10 9 -10 4 dilution series) genes were utilized to produce relative copy numbers. The samples were assayed in triplicate and were included if the triplicate C q difference was less than 0.25 cycles. The parity was the fixed effect, and the random effects included the sire and day repeated with the cow as the subject. Statistical significance was not observed in the leukocyte number or type ( p > 0.1). A reduction in the TL of approximately 9225 telomeric copies was found between Parity 1 and Parity 2 ( p = 0.02). A trend was found between the TL and Dlabor ( p = 0.06). The stress of parturition and raising the first calf of a cow's life may be responsible for TL attenuation. Parity may be considered a stressor of cow longevity.

The 3' untranslated region has an important role in gene regulation through microRNAs, and it has been estimated that microRNAs regulate up to 50% of coding genes in mammals. With the aim of allelic variant identification of 3' untranslated region microRNA seed sites, the 3' untranslated region was searched for seed sites of four temperament-associated genes ( CACNG4 , EXOC4 , NRXN3 , and SLC9A4 ). The microRNA seed sites were predicted in the four genes, and the CACNG4 gene had the greatest number with 12 predictions. To search for variants affecting the predicted microRNA seed sites, the four 3' untranslated regions were re-sequenced in a Brahman cattle population. Eleven single nucleotide polymorphisms were identified in the CACNG4 , and eleven in the SLC9A4 . Rs522648682:T>G of the CACNG4 gene was located at the predicted seed site for bta-miR-191 . Rs522648682:T>G evidenced an association with both exit velocity ( p = 0.0054) and temperament score ( p = 0.0097). The genotype TT had a lower mean exit velocity (2.93 ± 0.4 m/s) compared with the TG and GG genotypes (3.91 ± 0.46 m/s and 3.67 ± 0.46 m/s, respectively). The allele associated with the temperamental phenotype antagonizes the seed site, disrupting the bta-miR-191 recognition. The G allele of CACNG4 -rs522648682 has the potential to influence bovine temperament through a mechanism associated with unspecific recognition of bta-miR-191 .

Quantifying the natural inter-individual variation in DNA methylation patterns is important for identifying its contribution to phenotypic variation, but also for understanding how the environment affects variability, and for incorporation into statistical analyses. The inter-individual variation in DNA methylation patterns in female cattle and the effect that a prenatal stressor has on such variability have yet to be quantified. Thus, the objective of this study was to utilize methylation data from mature Brahman females to quantify the inter-individual variation in DNA methylation. Pregnant Brahman cows were transported for 2 h durations at days 60 ± 5; 80 ± 5; 100 ± 5; 120 ± 5; and 140 ± 5 of gestation. A non-transport group was maintained as a control. Leukocytes, amygdala, and anterior pituitary glands were harvested from eight cows born from the non-transport group (Control) and six from the transport group (PNS) at 5 years of age. The DNA harvested from the anterior pituitary contained the greatest variability in DNA methylation of cytosine-phosphate-guanine (mCpG) sites from both the PNS and Control groups, and the amygdala had the least. Numerous variable mCpG sites were associated with retrotransposable elements and highly repetitive regions of the genome. Some of the genomic features that had high variation in DNA methylation are involved in immune responses, signaling, responses to stimuli, and metabolic processes. The small overlap of highly variable CpG sites and features between tissues and leukocytes supports the role of variable DNA methylation in regulating tissue-specific gene expression. Many of the CpG sites that exhibited high variability in DNA methylation were common between the PNS and Control groups within a tissue, but there was little overlap in genomic features with high variability. The interaction between the prenatal environment and the genome could be responsible for the differences in location of the variable DNA methylation.

Prenatal stress can alter postnatal performance and temperament of cattle. These phenotypic effects may result from changes in gene expression caused by stress-induced epigenetic alterations. Specifically, shifts in gene expression caused by DNA methylation within the brain's amygdala can result in altered behavior because it regulates fear, stress response and aggression in mammals Thus, the objective of this experiment was to identify DNA methylation and gene expression differences in the amygdala tissue of 5-year-old prenatally stressed (PNS) Brahman cows compared to control cows. Pregnant Brahman cows ( n = 48) were transported for 2-h periods at 60 ± 5, 80 ± 5, 100 ± 5, 120 ± 5, and 140 ± 5 days of gestation. A non-transported group ( n = 48) were controls (Control). Amygdala tissue was harvested from 6 PNS and 8 Control cows at 5 years of age. Overall methylation of gene body regions, promoter regions, and cytosine-phosphate-guanine (CpG) islands were compared between the two groups. In total, 202 genes, 134 promoter regions, and 133 CpG islands exhibited differential methylation (FDR ≤ 0.15). Following comparison of gene expression in the amygdala between the PNS and Control cows, 2 differentially expressed genes were identified (FDR ≤ 0.15). The minimal differences observed could be the result of natural changes of DNA methylation and gene expression as an animal ages, or because this degree of transportation stress was not severe enough to cause lasting effects on the offspring. A younger age may be a more appropriate time to assess methylation and gene expression differences produced by prenatal stress., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2022 Baker, Earnhardt, Cilkiz, Collins, Littlejohn, Cardoso, Ghaffari, Long, Riggs, Randel, Welsh and Riley.)

As prenatal transportation stress altered behavior and adrenal glucocorticoid secretion of calves, we hypothesized that prenatal transportation stress would decrease ovarian reserve size and negatively impact female offspring fertility. The impact of prenatal transportation stress on ovarian follicle numbers in female offspring for three generations was studied. Brahman cows were transported for 2 h on day 60 ± 5, 80 ± 5, 100 ± 5, 120 ± 5, and 140 ± 5 of gestation. Ovaries were collected from offspring of transported or non-transported dams at multiple ages. Primordial, primary, secondary, and antral follicles were histologically analyzed. Antral follicle numbers were determined by ultrasound in a subset of offspring. Numbers of primordial, primary, secondary, and antral follicles were analyzed using the MIXED procedure, while the CORR procedure of SAS was used to determine the correlation between follicles observed by ultrasonography and histology. There were no differences (P > 0.05) in the number of primordial, primary, secondary, antral, or total follicles observed histologically due to treatment. Younger females had significantly greater numbers of follicles than older females (P < 0.0001). Antral follicles tended to be correlated with total histological ovarian follicles (P = 0.10). There was no difference in the number of antral follicles observed at ultrasound due to treatment (P = 0.3147), or generation (P = 0.6005) when controlling for age at observation. These results show that short-term transportation stress during early- to mid-gestation did not impact fertility as measured by ovarian follicle numbers in female Brahman offspring for three generations., (Copyright © 2022 Elsevier B.V. All rights reserved.)