Analysis of nonsynonymous SNPs in candidate genes that influence bovine temperament and evaluation of their effect in Brahman cattle.

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.

Differential Expression of Circadian Clock Genes in the Bovine Neuroendocrine Adrenal System.

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.

Comparison of Telomere Length in Age-Matched Primiparous and Multiparous Brahman Cows.

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 ß-globulin DNA. Standards of the bovine telomere (1012-107 dilution series) and ß-globulin (109-104 dilution series) genes were utilized to produce relative copy numbers. The samples were assayed in triplicate and were included if the triplicate Cq 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.

Genetic Variants and Their Putative Effects on microRNA-Seed Sites: Characterization of the 3' Untranslated Region of Genes Associated with Temperament.

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.

Inter-Individual Variation in DNA Methylation Patterns across Two Tissues and Leukocytes in Mature Brahman Cattle.

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.

DNA methylation patterns and gene expression from amygdala tissue of mature Brahman cows exposed to prenatal stress.

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.

Prenatal transportation stress did not impact ovarian follicle count for three generations of female Brahman offspring.

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.

Genome-wide DNA methylation alteration in prenatally stressed Brahman heifer calves with the advancement of age.

Possible phenotypic impairments associated with maternal stress during gestation in beef cattle may be explained by epigenetic effects. This study examined the impact of prenatal transportation stress on DNA methylation of lymphocytes of Brahman cows over the first 5 years of life. Methylation analysis through reduced representation bisulphite sequencing was conducted on DNA from lymphocytes from 28 paired samples from 6 prenatally stressed (PNS) and 8 control (Control) females obtained initially when they were 28 days of age and 5 years of age. There were 14,386 CpG (C = cytosine; p = phosphate; G = guanine) sites differentially methylated (P < 0.01) in 5-yr-old Control cows compared to their lymphocyte DNA at 28 days of age, this number was slightly decreased in 5-yr-old PNS with 13,378 CpG sites. Only 2,749 age-related differentially methylated CpG sites were seen within PNS females. There were 2,637 CpG sites differentially methylated (P < 0.01) in PNS cows relative to Controls at 5 years of age. There were differentially methylated genes in 5-yr-old cows that contributed similarly to altered gene pathways in both treatment groups. Canonical pathways altered in PNS compared to Control cows at 5 years of age were mostly related to development and growth, nervous system development and function, and immune response. Prenatal stress appeared to alter the epigenome in Brahman cows compared to Control at 5 years of age, which implies a persistent intervention in DNA methylation in lymphocytes, and may confer long-lasting effects on gene expression, and consequently relevant phenotypic changes.

Temperament influences mitochondrial capacity in skeletal muscle from 8 through 18 mo of age in Brahman heifers.

Temperamental cattle tend to yield carcasses of poorer quality, and Brahman cattle are reportedly more temperamental than non-indicus cattle breeds. A potential link between temperament and product quality may be mitochondrial activity. We hypothesized that mitochondrial measures would be greater in temperamental compared with calm heifers and that the relationships between temperament and mitochondria would persist as heifers age. Serum cortisol and skeletal muscle (longissimus thoracis [LT] and trapezius [TRAP]) mitochondrial profiles and antioxidant activities were quantified from the same calm (n = 6) and temperamental (n = 6) Brahman heifers at 8, 12, and 18 mo of age. Data were analyzed using a mixed model ANOVA in SAS (9.4) with repeated measures. Serum cortisol was greater in temperamental compared with calm heifers throughout the study (P = 0.02). Mitochondrial volume density (citrate synthase [CS] activity) increased over time (P < 0.0001) but was similar between temperament and muscle groups. Mitochondrial function (cytochrome c oxidase activity) was greatest in the temperamental LT at 8 mo of age (P ≤ 0.0006), greatest in the temperamental TRAP at 18 mo of age (P ≤ 0.003), and did not differ by temperament at 12 mo of age. Integrative (relative to tissue wet weight) mitochondrial oxidative phosphorylation capacity with complex I substrates (PCI), PCI plus complex II substrate (PCI+II), noncoupled electron transfer system capacity (ECI+II), and E with functional complex II only (ECII) were greater in the TRAP than LT for calm heifers at all ages (P ≤ 0.002), but were similar between muscle groups in temperamental heifers. Overall, calm heifers tended to have greater intrinsic (relative to CS activity) PCI and flux control of PCI+II (P ≤ 0.1) than temperamental heifers, indicating greater utilization of complex I paired with greater coupling efficiency in calm heifers. Within the LT, integrative PCI+II was greater (P = 0.05) and ECI+II tended to be greater (P = 0.06) in temperamental compared with calm heifers. From 8- to 18-mo old, glutathione peroxidase (GPx) activity decreased (P < 0.0001) and superoxide dismutase activity increased (P = 0.02), and both were similar between muscle groups. The activity of GPx was greater in temperamental compared with calm heifers at 8 (P = 0.004) but not at 12 or 18 mo of age. These results detail divergent skeletal muscle mitochondrial characteristics of live Brahman heifers according to temperament, which should be further investigated as a potential link between temperament and product quality.

Novel genes involved in the genetic architecture of temperament in Brahman cattle.

Cattle temperament is a complex and economically relevant trait. The objective of this study was to identify genomic regions and genes associated with cattle temperament. From a Brahman cattle population of 1,370 animals evaluated for temperament traits (Exit velocity-EV, Pen Score-PS, Temperament Score-TS), two groups of temperament-contrasting animals were identified based on their EV-average values ±1/2 standard deviation (SD). To be considered in the calm group, the EV of females ranged between 0.16-1.82 m/s (n = 50) and the EV of males ranged between 0.4-1.56 m/s (n = 48). Females were classified as temperamental if their EV ranged between 3.13-7.66 m/s (n = 46) and males were classified as temperamental if their EV ranged between 3.05-10.83 m/s (n = 45). Selected animals were genotyped using a total of 139,376 SNPs (GGP-HD-150K), evaluated for their association with EV. The Genome-Wide Association analysis (GWAS) identified fourteen SNPs: rs135340276, rs134895560, rs110190635, rs42949831, rs135982573, rs109393235, rs109531929, rs135087545, rs41839733, rs42486577, rs136661522, rs110882543, rs110864071, rs109722627, (P<8.1E-05), nine of them were located on intergenic regions, harboring seventeen genes, of which only ACER3, VRK2, FANCL and SLCO3A1 were considered candidate associated with bovine temperament due to their reported biological functions. Five SNPs were located at introns of the NRXN3, EXOC4, CACNG4 and SLC9A4 genes. The indicated candidate genes are implicated in a wide range of behavioural phenotypes and complex cognitive functions. The association of the fourteen SNPs on bovine temperament traits (EV, PS and TS) was evaluated; all these SNPs were significant for EV; only some were associated with PS and TS. Fourteen SNPs were associated with EV which allowed the identification of twenty-one candidate genes for Brahman temperament. From a functional point of view, the five intronic SNPs identified in this study, are candidates to address control of bovine temperament, further investigation will probe their role in expression of this trait.

Influence of prenatal transportation stress-induced differential DNA methylation on the physiological control of behavior and stress response in suckling Brahman bull calves.

The objective of this experiment was to examine potential differential methylation of DNA as a mechanism for altered behavioral and stress responses in prenatally stressed (PNS) compared with nonprenatally stressed (Control) young bull calves. Mature Brahman cows (n = 48) were transported for 2-h periods at 60 ± 5, 80 ± 5, 100 ± 5, 120 ± 5, and 140 ± 5 d of gestation (Transported group) or maintained as nontransported Controls (n = 48). From the offspring born to Transported and Control cows, a subset of 28-d-old intact bulls (n = 7 PNS; n = 7 Control) were evaluated for methylation of DNA of behavior and stress response-associated genes. Methylation of DNA from white blood cells was assessed via reduced representation bisulfite sequencing methods. Because increased methylation of DNA within gene promoter regions has been associated with decreased transcriptional activity of the corresponding gene, differentially methylated (P ≤ 0.05) CG sites (cytosine followed by a guanine nucleotide) located within promoter regions (n = 1,205) were used to predict (using Ingenuity Pathway Analysis software) alterations to canonical pathways in PNS compared with Control bull calves. Among differentially methylated genes (P ≤ 0.05) related to behavior and the stress response were OPRK1, OPRM1, PENK, POMC, NR3C2, TH, DRD1, DRD5, COMT, HTR6, HTR5A, GABRA4, GABRQ, and GAD2. Among altered (P < 0.05) signaling pathways related to behavior and the stress response were Opioid Signaling, Corticotropin-Releasing Hormone Signaling, Dopamine Receptor Signaling, Dopamine-DARPP32 Feedback in cAMP Signaling, Serotonin Receptor Signaling, and GABA Receptor Signaling. Alterations to behavior and stress response-related genes and canonical pathways supported previously observed elevations in temperament score and serum cortisol through weaning in the larger population of PNS calves from which bulls in this study were derived. Differential methylation of DNA and predicted alterations to behavior and stress response-related pathways in PNS compared with Control bull calves suggest epigenetic programming of behavior and the stress response in utero.

Influence of genetic markers on the feeding behavior of yearling bulls / Influencia de los marcadores genéticos en el comportamiento alimentario de toretes de un año / Influência de marcadores genéticos no comportamento alimentar de touros de um ano

Abstract Background: The search for gene and marker effects on economically important traits is aimed not only to understanding the genetic architecture of complex traits but also to applying the information to breeding schemes. Objective: To analyze the effect of two temperament-related SNPs (rs109576799 located in the DRD3 gene, and rs43696138 in the HTR2A gene) on feeding performance of Mexican beef cattle. Methods: One hundred and thirty-six young beef bulls were included in a centralized feed efficiency performance test based on residual feed intake (RFI), with 20 d for adaptation and 70 d of feed efficiency testing. In addition to feeding traits, temperament was assessed at the beginning of the trial using pen score (PS) and exit velocity (EV). All animals were genotyped with two markers located in the HTR2A and DRD3 genes, and an association analysis was conducted between these genotypes and the measured traits. Results: For Brangus breed, a significant association was obtained between average daily gain (ADG; p=0.019), and the rs43696138 marker, resulting in higher gains for homozygous genotype GG (1.69 ± 0.04 kg), when compared to the heterozygous genotype GA (1.54 ± 0.04 kg). Conclusion: The previously reported association of these markers with temperament was not confirmed in the evaluated breeds; however, the rs43696138 marker showed an effect on a feeding performance trait. Further studies are needed to determine the effect of this and other markers on both RFI and temperament.

Resumen Antecedentes: La búsqueda de efectos genéticos y marcadores de rasgos económicamente relevantes no solo se basa en el interés biológico de comprender la arquitectura genética de rasgos complejos, sino también en aplicar la información en los esquemas prácticos de mejoramiento. Objetivo: Analizar el efecto de dos SNPs relacionados con el temperamento (rs109576799 localizado en el gen DRD3, y rs43696138 localizado en el gen HTR2A) sobre la eficiencia alimenticia en el ganado bovino mexicano. Métodos: Ciento treinta y seis toretes de carne jóvenes fueron sometidos a una prueba de comportamiento alimenticio basada en el consumo residual de alimento (RFI), con 20 d de adaptación y 70 d de prueba para la eficiencia alimenticia. Además de los rasgos de comportamiento alimenticio, se evaluó el temperamento de los animales al inicio de la prueba, mediante la evaluacion de comportamiento en el corral (PS), y la velocidad de salida (EV). Todas las muestras se tipificaron con dos marcadores localizados en los genes DRD3 y HTR2A para posteriormente realizar un análisis de asociación de los genotipos con los rasgos evaluados. Resultados: En la raza Brangus, se observó una asociación significativa de la media de ganancia diaria de peso (ADG, p=0,019) con el marcador rs43696138, localizado en el gen HTR2A, resultando en mayores ganancias para el genotipo GG (1,69 ± 0,04 kg) en comparación con los toros heterocigóticos GA (1,54 ± 0,04 kg). Conclusión: No se confirmó la asociación de estos marcadores previamente reportados con el temperamento en las razas evaluadas; sin embargo, el marcador rs43696138 mostró efecto en un rasgo de comportamiento alimenticio. Se necesitan más estudios para determinar el efecto de éste y otros marcadores en el consumo residual de alimento (RFI) y el temperamento.

Resumo Antecedentes: A busca de efeitos genéticos e marcadores de características economicamente relevantes não se baseia apenas no interesse biológico de compreender a arquitetura genética de traços complexos, mas também na aplicação da informação nos esquemas práticos de melhoria. Objetivo: Analisar o efeito de dois SNPs relacionados ao temperamento (rs109576799 localizado no gene DRD3 e rs43696138 localizado no gene HTR2A) sobre a eficiência nutricional no gado mexicano. Métodos: Cento e trinta e seis touros jovens foram submetidos a um teste de comportamento alimentar com base na entrada de alimentação residual (RFI), com 20 d de adaptação e 70 d de teste para eficiência de alimentação. Além dos traços de comportamento alimentar, o temperamento dos animais foi avaliado no início do teste, através da avaliação do comportamento na caneta (PS) e da velocidade de saída (EV). Todas as amostras foram digitadas com dois marcadores localizados nos genes DRD3 e HTR2A para posteriormente realizar uma análise de associação dos genótipos com os traços avaliados. Resultados: Na raça Brangus, observou-se uma associação significativa do ganho diário médio (ADG, p = 0,019) com o marcador rs43696138, localizado no gene HTR2A, resultando em maiores ganhos para o genótipo GG (1,69 ± 0,04 kg), em comparação com os touros heterozigóticos GA (1,54 ± 0,04 kg). Conclusão: A associação destes marcadores previamente relatados com o temperamento nas raças avaliadas não foi confirmada; no entanto, o marcador rs43696138 mostrou um efeito sobre uma característica de comportamento alimentar. Mais estudos são necessários para determinar o efeito deste e outros marcadores com ingestão alimentar residual (RFI) e temperamento.

Influence of prenatal transportation stress on innate immune response to an endotoxin challenge in weaned Brahman bull calves†,‡.

The objective of this study was to assess the influence of prenatal stress (PNS) on innate immune responses to an endotoxin challenge in weaned bull calves. Altered innate immune response to lipopolysaccharide (LPS), as characterized by changes in a range of variables was hypothesized in PNS bull calves. Brahman cows (n = 96; 48 stressed by transportation at five stages of gestation and 48 Controls) produced 85 calves, from which 16 uncastrated male (bull) calves from each PNS and Control treatment were selected for an LPS challenge period. Rectal temperature (RT), sickness behavior score (SBS), serum concentrations of cortisol, interferon-gamma (IFN-γ), tumor necrosis factor-alpha (TNF-α), interleukin-6 (IL-6), and complete blood count (CBC) variables were assessed in response to intravenous LPS (0.25 µg/kg body weight) administration. Each reported variable increased or decreased following LPS administration. Prior to LPS, PNS bull calves exhibited increased TNF-α, IL-6, and monocyte counts, but decreased IFN-γ, eosinophils, and basophils (p < .05). Compared with Control, in response to LPS, PNS bull calves exhibited greater circulating concentrations of cortisol. PNS bull calves exhibited lower (p < .05) eosinophil and basophil counts at time 0 (time of LPS administration) but similar counts to Control bull calves 2 h after LPS. PNS bull calves exhibited a greater change from baseline for IFN-γ and monocytes in response to LPS administration. No other variables were influenced by prenatal treatment (p > .05). These findings suggest that PNS did not adversely affect basal or induced components of the innate immune response to an immunological challenge. Lay summary Our laboratory studied the influence of prenatal stress (i.e., transportation of pregnant cows) on immune function of bull calves at 8 months of age. This was accomplished by studying aspects of their innate immune response to an immunological challenge. Prenatal stress did not adversely affect basal or induced components of the innate immune response to an immunological challenge.

Prenatal transportation stress alters genome-wide DNA methylation in suckling Brahman bull calves.

The objective of this experiment was to identify genome-wide differential methylation of DNA in young prenatally stressed (PNS) bull calves. Mature Brahman cows (n = 48) were transported for 2-h periods at 60 ± 5, 80 ± 5, 100 ± 5, 120 ± 5, and 140 ± 5 d of gestation or maintained as nontransported Controls (n = 48). Methylation of DNA from white blood cells from a subset of 28-d-old intact male offspring (n = 7 PNS; n = 7 Control) was assessed via reduced representation bisulfite sequencing. Samples from PNS bulls contained 16,128 CG, 226 CHG, and 391 CHH (C = cytosine; G = guanine; H = either adenine, thymine, or cytosine) sites that were differentially methylated compared to samples from Controls. Of the CG sites, 7,407 were hypermethylated (at least 10% more methylated than Controls; P ≤ 0.05) and 8,721 were hypomethylated (at least 10% less methylated than Controls; P ≤ 0.05). Increased DNA methylation in gene promoter regions typically results in decreased transcriptional activity of the region. Therefore, differentially methylated CG sites located within promoter regions (n = 1,205) were used to predict (using Ingenuity Pathway Analysis software) alterations to canonical pathways in PNS compared with Control bull calves. In PNS bull calves, 113 pathways were altered (P ≤ 0.05) compared to Controls. Among these were pathways related to behavior, stress response, metabolism, immune function, and cell signaling. Genome-wide differential DNA methylation and predicted alterations to pathways in PNS compared with Control bull calves suggest epigenetic programming of biological systems in utero.

Use of random regression to estimate genetic parameters of temperament across an age continuum in a crossbred cattle population.

The objective was to estimate genetic parameters of temperament in beef cattle across an age continuum. The population consisted predominantly of Brahman-British crossbred cattle. Temperament was quantified by 1) pen score (PS), the reaction of a calf to a single experienced evaluator on a scale of 1 to 5 (1 = calm, 5 = excitable); 2) exit velocity (EV), the rate (m/s) at which a calf traveled 1.83 m upon exiting a squeeze chute; and 3) temperament score (TS), the numerical average of PS and EV. Covariates included days of age and proportion of Bos indicus in the calf and dam. Random regression models included the fixed effects determined from the repeated measures models, except for calf age. Likelihood ratio tests were used to determine the most appropriate random structures. In repeated measures models, the proportion of B. indicus in the calf was positively related with each calf temperament trait (0.41 ± 0.20, 0.85 ± 0.21, and 0.57 ± 0.18 for PS, EV, and TS, respectively; P < 0.01). There was an effect of contemporary group (combinations of season, year of birth, and management group) and dam age (P < 0.001) in all models. From repeated records analyses, estimates of heritability (h2) were 0.34 ± 0.04, 0.31 ± 0.04, and 0.39 ± 0.04, while estimates of permanent environmental variance as a proportion of the phenotypic variance (c2) were 0.30 ± 0.04, 0.31 ± 0.03, and 0.34 ± 0.04 for PS, EV, and TS, respectively. Quadratic additive genetic random regressions on Legendre polynomials of age were significant for all traits. Quadratic permanent environmental random regressions were significant for PS and TS, but linear permanent environmental random regressions were significant for EV. Random regression results suggested that these components change across the age dimension of these data. There appeared to be an increasing influence of permanent environmental effects and decreasing influence of additive genetic effects corresponding to increasing calf age for EV, and to a lesser extent for TS. Inherited temperament may be overcome by accumulating environmental stimuli with increases in age, especially after weaning.

Effects of LPA2R, LPA3R, or EP4R agonists on luteal or endometrial function in vivo or in vitro and sirtuin or EP1R, EP2R, EP3R or EP4R agonists on endometrial secretion of PGE and PGF2αin vitro.

In previous work, an EP2 prostanoid receptor (EP2R) agonist in vivo increased mRNA expression of luteal LH receptors (LHR), unoccupied and occupied luteal; LHR, and circulating progesterone, while an EP3R or FPR agonist decreased; mRNA expression of luteal LHR, unoccupied and occupied luteal LHR, and; circulating progesterone. An EP4R and lysophosphatidic acid (LPA) LPA2R and LPA3R agonists were reported to inhibit luteal function and sirtuins have been proposed to increase prostaglandin synthesis. The objectives were to determine; whether an EP4R, LPA2R, or LPA3R agonist affect ovine luteal function in vivo or; in vitro. In addition, whether sirtuin (SIRT)-1, 2, or 3; LPA2R or LPA3R; or EP1R, EP2R, EP3R, or EP4R agonists affect caruncular endometrial PGF2α or PGE (PGE1+PGE2) secretion in vitro. Day-10 nonpregnant ewes received a single injection of Vehicle (N = 5); an LPA2R (N = 5); LPA3R (N = 6); or EP4R (N = 5) agonist given into the interstitial tissue of the ovarian vascular pedicle adjacent to the luteal-containing ovary to determine effects on circulating progesterone, mRNA expression of luteal LHR, and luteal unoccupied and occupied LHR. In addition, agonists for LPA2R, LPA3R, EP1R, EP2R, EP3R, or EP4R or SIRT-1, SIRT-2, or SIRT-3 activators were incubated with caruncular endometrial slices in vitro to determine their effect on caruncular endometrial PGF2α, or PGE secretion. LPA2R, LPA3R, or an EP4R agonist in vivo did not affect (P ≥ 0.05) luteal weight, circulating progesterone, or occupied luteal LHR. However, an LPA2R or EP4R agonist, but; not LPA3R agonist, in vivo increased (P ≤ 0.05) mRNA expression of luteal LHR. An; LPA2R, LPA3R, or EP4R agonist increased (P ≤ 0.05) luteal unoccupied LHR, but; not occupied LHR. An LPA2R, LPA3R, or an EP4R agonist did not affect (P ≥ 0.05); luteal progesterone secretion in vitro. An LPA2R or LPA3R agonist did not affect (P ≥ 0.05) luteal PGF2α, or PGE secretion in vitro. However, an EP4R agonist tended to decrease (P < 0.066) luteal PGF2α secretion and increased (P ≤ 0.05) luteal PGE; secretion in vitro. EP1R, EP2R, EP3R, or an EP4R agonist did not affect (P ≥ 0.05); caruncular endometrial PGF2α secretion in vitro. However, EP1R, EP3R, or an EP4R agonist increased caruncular endometrial PGE secretion in vitro, while two different EP2R agonists did not affect (P ≥ 0.05) caruncular endometrial PGE; secretion. A SIRT-1 activator, but not SIRT-2 or SIRT-3 activators, increased (P ≤ 0.05) caruncular endometrial PGE secretion, while sirtuin 1, 2, or 3 activators did not affect (P ≥ 0.05) caruncular endometrial PGF2α secretion. In conclusion, receptors for EP4, LPA2, and LPA3 do not appear to be involved; in luteolysis, but EP4R and LPA2R might participate in preventing luteolysis by maintaining luteal mRNA expression for LHR and preventing loss of unoccupied luteal LHR. In addition, SIRT-1, EP1R, EP3R, and EP4R might be involved in; regulating caruncular endometrial PGE secretion, but not PGF2α secretion.

Salmonella Typhimurium and Multidirectional Communication in the Gut.

The mammalian digestive tract is home to trillions of microbes, including bacteria, archaea, protozoa, fungi, and viruses. In monogastric mammals the stomach and small intestine harbor diverse bacterial populations but are typically less populated than the colon. The gut bacterial community (microbiota hereafter) varies widely among different host species and individuals within a species. It is influenced by season of the year, age of the host, stress and disease. Ideally, the host and microbiota benefit each other. The host provides nutrients to the microbiota and the microbiota assists the host with digestion and nutrient metabolism. The resident microbiota competes with pathogens for space and nutrients and, through this competition, protects the host in a phenomenon called colonization resistance. The microbiota participates in development of the host immune system, particularly regulation of autoimmunity and mucosal immune response. The microbiota also shapes gut-brain communication and host responses to stress; and, indeed, the microbiota is a newly recognized endocrine organ within mammalian hosts. Salmonella enterica serovar Typhimurium (S. Typhimurium hereafter) is a food-borne pathogen which adapts to and alters the gastrointestinal (GI) environment. In the GI tract, S. Typhimurium competes with the microbiota for nutrients and overcomes colonization resistance to establish infection. To do this, S. Typhimurium uses multiple defense mechanisms to resist environmental stressors, like the acidic pH of the stomach, and virulence mechanisms which allow it to invade the intestinal epithelium and disseminate throughout the host. To coordinate gene expression and disrupt signaling within the microbiota and between host and microbiota, S. Typhimurium employs its own chemical signaling and may regulate host hormone metabolism. This review will discuss the multidirectional interaction between S. Typhimurium, host and microbiota as well as mechanisms that allow S. Typhimurium to succeed in the gut.

Sexually dimorphic innate immunological responses of pre-pubertal Brahman cattle following an intravenous lipopolysaccharide challenge.

This study was designed to characterize potential sexually dimorphic immunological responses following a lipopolysaccharide (LPS) challenge in beef cattle. Six female (heifers) and five male (bulls) Brahman calves (average age=253 ± 19.9 and 288 ± 47.9 days; average body weight=194 ± 11 kg and 247 ± 19 kg for heifers and bulls, respectively) were challenged with LPS (0.25 µg LPS/kg body weight). Following administration of LPS, all cattle displayed increased sickness behavior beginning at 0.5h, with heifers on average displaying less sickness behavior than bulls. A febrile response was observed in all animals following LPS administration, with a maximum response observed from 4 to 5.5h. The average rectal temperature response was greater in heifers than bulls. In all cattle there were elevated serum concentrations of cortisol from 0.5 to 8h, TNF-α from 1 to 2.5h, IL-6 from 2 to 8h, and IFN-γ from 2.5 to 7h after LPS challenge. Additionally, serum concentrations of TNF-α were greater in heifers than bulls from 1.5 to 2h after the LPS challenge. Concentrations of IFN-γ were also greater on average in bulls than heifers. Leukopenia occurred from 1 to 8h, with a decreased neutrophil to lymphocyte ratio for the first 5h among all calves. These data demonstrate the existence of a sexually dimorphic acute-phase response in pre-pubertal Brahman calves. Specifically, heifers may have a more robust acute response to LPS challenge, even though bulls display more signs of sickness.

Effects of intraluteal implants of prostaglandin E1 or E2 on angiogenic growth factors in luteal tissue of Angus and Brahman cows.

Previously, it was reported that intraluteal implants containing prostaglandin E1 or E2 (PGE1 and PGE2) in Angus or Brahman cows prevented luteolysis by preventing loss of mRNA expression for luteal LH receptors and luteal unoccupied and occupied LH receptors. In addition, intraluteal implants containing PGE1 or PGE2 upregulated mRNA expression for FP prostanoid receptors and downregulated mRNA expression for EP2 and EP4 prostanoid receptors. Luteal weight during the estrous cycle of Brahman cows was reported to be lesser than that of Angus cows but not during pregnancy. The objective of this experiment was to determine whether intraluteal implants containing PGE1 or PGE2 alter vascular endothelial growth factor (VEGF), fibroblast growth factor-2 (FGF-2), angiopoietin-1 (ANG-1), and angiopoietin-2 (ANG-2) protein in Brahman or Angus cows. On Day 13 of the estrous cycle, Angus cows received no intraluteal implant and corpora lutea were retrieved, or Angus and Brahman cows received intraluteal silastic implants containing vehicle, PGE1, or PGE2 on Day 13 and corpora lutea were retrieved on Day 19. Corpora lutea slices were analyzed for VEGF, FGF-2, ANG-1, and ANG-2 angiogenic proteins via Western blot. Day-13 Angus cow luteal tissue served as preluteolytic controls. Data for VEGF were not affected (P > 0.05) by day, breed, or treatment. PGE1 or PGE2 increased (P < 0.05) FGF-2 in luteal tissue of Angus cows compared with Day-13 and Day-19 Angus controls but decreased (P < 0.05) FGF-2 in luteal tissue of Brahman cows when compared w Day-13 or Day-19 Angus controls. There was no effect (P > 0.05) of PGE1 or PGE2 on ANG-1 in Angus luteal tissue when compared with Day-13 or Day-19 controls, but ANG-1 was decreased (P < 0.05) by PGE1 or PGE2 in Brahman cows when compared with Day-19 Brahman controls. ANG-2 was increased (P < 0.05) on Day 19 in Angus Vehicle controls when compared with Day-13 Angus controls, which was prevented (P < 0.05) by PGE1 but not by PGE2 in Angus cows. There was no effect (P > 0.05) of PGE1 or PGE2 on ANG-2 in Brahman cows. PGE1 or PGE2 may alter cow luteal FGF-2, ANG-1, or ANG-2 but not VEGF to prevent luteolysis; however, species or breed differences may exist.

Prostaglandin E1 or E2 inhibits an oxytocin-induced premature luteolysis in ewes when oxytocin is given early in the estrous cycle.

The objective of this study was to determine whether PGE1 or PGE2 prevents a premature luteolysis when oxytocin is given on Days 1 to 6 of the ovine estrous cycle. Oxytocin given into the jugular vein every 8 hours on Days 1 to 6 postestrus in ewes decreased (P ≤ 0.05) luteal weights on Day 8 postestrus. Plasma progesterone differed (P ≤ 0.05) among the treatment groups; toward the end of the experimental period, concentrations of circulating progesterone in the oxytocin-only treatment group decreased (P ≤ 0.05) when compared with the other treatment groups. Plasma progesterone concentrations in ewes receiving PGE1 or PGE1 + oxytocin were greater (P ≤ 0.05) than in vehicle controls or in ewes receiving PGE2 or PGE2 + oxytocin and was greater (P ≤ 0.05) in all treatment groups receiving PGE1 or PGE2 than in ewes treated only with oxytocin. Chronic intrauterine treatment with PGE1 or PGE2 also prevented (P ≤ 0.05) oxytocin decreases in luteal unoccupied and occupied LH receptors on Day 8 postestrus. Oxytocin given alone on Days 1 to 6 postestrus in ewes advanced (P ≤ 0.05) increases in PGF2α in inferior vena cava or uterine venous blood. PGE1 or PGE2 given alone did not affect (P ≥ 0.05) concentrations of PGF2α in inferior vena cava and uterine venous blood when compared with vehicle controls or oxytocin-induced PGF2α increases (P ≤ 0.05) in inferior vena cava or uterine venous blood. We concluded that PGE1 or PGE2 prevented oxytocin-induced premature luteolysis by preventing a loss of luteal unoccupied and occupied LH receptors.