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Environmental and occupational exposure to hexavalent chromium, Cr(VI), causes female reproductive failures and infertility. Cr(VI) is used in more than 50 industries and is a group A carcinogen, mutagenic and teratogenic, and a male and female reproductive toxicant. Our previous findings indicate that Cr(VI) causes follicular atresia, trophoblast cell apoptosis, and mitochondrial dysfunction in metaphase II (MII) oocytes. However, the integrated molecular mechanism of Cr(VI)-induced oocyte defects is not understood. The current study investigates the mechanism of Cr(VI) in causing meiotic disruption of MII oocytes, leading to oocyte incompetence in superovulated rats. Postnatal day (PND) 22 rats were treated with potassium dichromate (1 and 5 ppm) in drinking water from PND 22–29 and superovulated. MII oocytes were analyzed by immunofluorescence, and images were captured by confocal microscopy and quantified by Image-Pro Plus software, Version 10.0.5. Our data showed that Cr(VI) increased microtubule misalignment (~9 fold), led to missegregation of chromosomes and bulged and folded actin caps, increased oxidative DNA (~3 fold) and protein (~9–12 fold) damage, and increased DNA double-strand breaks (~5–10 fold) and DNA repair protein RAD51 (~3–6 fold). Cr(VI) also induced incomplete cytokinesis and delayed polar body extrusion. Our study indicates that exposure to environmentally relevant doses of Cr(VI) caused severe DNA damage, distorted oocyte cytoskeletal proteins, and caused oxidative DNA and protein damage, resulting in developmental arrest in MII oocytes. more...

Environmental and occupational exposure to hexavalent chromium, Cr(VI), causes female reproductive failures and infertility. Cr(VI) is used in more than 50 industries and is a group-A carcinogen, mutagenic and teratogenic, and a male and female reproductive toxicant. Our previous findings indicate that Cr(VI) causes follicular atresia, trophoblast cell apoptosis, and mitochondrial dysfunction in metaphase II (MII) oocytes. However, the integrated molecular mechanism of Cr(VI)-induced oocyte defects are not understood. The current study investigates the mechanism of Cr(VI) in causing meiotic disruption of MII oocytes, leading to infertility in superovulated rats. Postnatal day (PND)-22 rats were treated with potassium dichromate (1 and 5 ppm) in drinking water from PND 22-29 and superovulated. MII oocytes were analyzed by immunofluorescence, and images were captured by confocal microscopy and quantified by Image Pro Plus software. Our data showed that Cr(VI) increased microtubule misalignment, missegregation of chromosomes, a bulged and folded actin cap, oxidative DNA and protein damage, increased DNA double-strand break, and DNA repair protein RAD51. Cr(VI) also induced incomplete cytokinesis and delayed polar body extrusion. Our study indicates that exposure to environmentally relevant doses of Cr(VI) caused severe DNA damage, distorted oocyte cytoskeletal proteins, and caused oxidative DNA and protein damage leading to infertility. more...

Objective Our objective was to develop models to estimate the CP, NDF, lignin, and ash concentrations of bermudagrass [Cynodondactylon (L.) Pers.] forage daily throughout the grazing season and use them to estimate TDN daily. We hypothesized that the CP, NDF, lignin, ash, and TDN concentrations might change daily. Materials and Methods Coastal bermudagrass forage CP, NDF, lignin, and ash concentrations spanning several years were obtained from Overton, Texas. The forage samples used comprised hand-plucked plant parts intended to estimate animal-selected diets. We developed an empirical model for each nutritive variable with the day of the year (DOY) as a predictor. By incorporating these models into a pre-existing summative equation, we estimated the daily values of TDN. These nutritive value models were evaluated using the Willmott index, modeling efficiency, root mean squared error, and R2 as the measures of fit. Results and Discussion Bermudagrass CP concentration had a negative, curvilinear association with DOY, whereas lignin and NDF concentrations were positively, curvilinearly related to DOY. The ash concentration had a negative, linear association with DOY. The likely causal factors for these relationships were plant maturity and weather conditions, especially temperature. The Willmott index, modeling efficiency, and R2 values were greater than 0.87, 0.64, and 0.65, respectively. The root mean squared error and percent error values were less than 3.1 and 11%, respectively. These values indicated that the nutritive value models were reliable. Implications and Applications We showed that the CP, NDF, lignin, ash, and TDN values of bermudagrass forage changed daily. By incorporating these trends into a nutritive value prediction system, accurate estimates of daily TDN could be made. These nutritive value models could be a valuable asset in forage-animal modeling for more accurately studying the effects of various management strategies and environmental factors on forage nutritive value and animal performance. more...

Phenotypic selection during animal domestication has resulted in unwanted incorporation of deleterious mutations. In horses, the autosomal recessive condition known as Glycogen Branching Enzyme Deficiency (GBED) is the result of one of these deleterious mutations (102C > A), in the first exon of the GBE1 gene (GBE1102C>A). With recent advances in genome editing, this type of genetic mutation can be precisely repaired. In this study, we used the RNA-guided nuclease CRISPR-Cas9 (clustered regularly-interspaced short palindromic repeats/CRISPR-associated protein 9) to correct the GBE1102C>A mutation in a primary fibroblast cell line derived from a high genetic merit heterozygous stallion. To correct this mutation by homologous recombination (HR), we designed a series of single guide RNAs (sgRNAs) flanking the mutation and provided different single-stranded donor DNA templates. The distance between the Cas9-mediated double-stranded break (DSB) to the mutation site, rather than DSB efficiency, was the primary determinant for successful HR. This framework can be used for targeting other harmful diseases in animal populations. more...

Our research group has previously reported on grazing behavior, diet selection, and weight change in prenatally stressed and translocated Brahman heifers grazing native rangeland. Our current objective was to evaluate relationships between diet quality and metabolic indicators. Twelve yearling heifers were transported ~700 km from Overton, TX to Sonora, TX. Six heifers (283±10 kg) were born to dams subjected to transportation stress during mid-gestation (PNS) and 6 (279±17 kg) were born to non-stressed dams (CON). Heifers grazed a series of 24-ha native range pastures and were sampled (BW, BCS score, blood, and feces) at 2-wk intervals from May through September. Fecal samples collected from the ground or the rectum of each animal were stored at -20o C until processed for near infrared reflectance spectroscopy prediction of diet crude protein (CP) and digestible organic matter (DOM). Blood was obtained via coccygeal venipuncture and processed to yield serum. Serum analyses were obtained from the Texas A&M Veterinary Medical Diagnostic Laboratory. Relationships between constituents of interest were determined by linear regression. Significance was considered to be P < 0.05. Blood urea nitrogen (BUN; mg/dL) was greater (P = 0.04) in PNS (8.39±0.32) than CON (7.39±0.33). Positive correlations were found between CP and BUN (RSQ = 0.76, SE = 1.25 mg/dL; P < 0.001), albumin (RSQ = 0.57, SE = 0.06 mg/dL; P < 0.01), and average daily gain (RSQ = 0.63, SE = 0.47 kg; P < 0.01). Similar relationships were observed between DOM and glucose (RSQ = 0.64, SE = 3.91 mg/dL; P < 0.01), cholesterol (RSQ = 0.37, SE = 5.47 mg/dL; P < 0.05), and average daily gain (RSQ = 0.47, SE = 0.56 kg; P < 0.01). PNS affected circulating nitrogen concentration. Diet quality was related to weight gain and serum indicators of nutritional status. more...

Antiparasitic resistance has become a major concern in the livestock industry and can have a tremendous impact on calf performance. The objective of this study was to determine resistance to different anthelmintic treatments in Brahman and Brahman/Hereford (F1) calves at weaning. Purebred fall weaned Brahman calves (n=95) and spring weaned F1 calves (n=45) were assigned to one of six treatment groups at weaning: Control (received no anthelmintic), received a benzimidazole (Synanthic 22.5% or Safeguard), a macrocyclic lactone (Dectomax or LongRange), or a combination of the two (LongRange and Synanthic). Fecal samples were collected on d 0, 14 or 28 (dependent on treatment), and 42 to determine fecal egg count (eggs/g; EPG). Body weights were collected at all time points. Data were analyzed as an ANOVA using the GLM procedure, or as an ANOVA with repeated measures using the MIXED procedure in SAS. There tended to be an effect of treatment (P = 0.0865), and there was an effect of breed (P < 0.0001), time (P < 0.0001), treatment by breed (P = 0.0458), treatment by time (P = 0.0067), and treatment by breed by time (P = 0.0214) on EPG. Overall, Brahmans had increased EPG compared with F1s (P < 0.0001; 178.83±11.73 vs. 79.81±17.08). Benzimidazoles had a greater reduction in EPG (99.29%; P = 0.0011) compared with macrocyclic lactones (45.36%). There was no effect of treatment (P = 0.5184), or treatment by time (P = 1.0) on body weight. Body weight increased similarly among all treatment groups throughout the study. Furthermore, there was no difference in ADG during the study among treatments (P = 0.73). These data indicate that antiparasitic resistance occurs in all breeds, and resistance varies between benzimidazoles and macrocyclic lactones. Furthermore, there were differences between breeds and/or seasons among treatments even within a single operation. more...

Longitudinal studies of gene expression where sampling certain organ systems is precluded may become more feasible if a more accessible, representative biological sample could be identified. The objective of this study was to determine whether peripheral blood leukocytes (PBL) could be an effective surrogate for assessing gene expression differences within neuroendocrine tissues that regulate stress responses. Specifically, leukocyte samples and stress axis tissues, including the paraventricular region of the hypothalamus, anterior pituitary gland, adrenal cortex, and adrenal medulla were isolated from humanely slaughtered 5-yr-old Brahman cows (n = 8). RNA was extracted from each sample and gene expressions were calculated using bioinformatic approaches of RNA-Seq analysis. Differentially expressed genes (DEG) between adrenal axis tissue RNA and PBL RNA were identified using the edgeR Package from Bioconductor, R. More than 3,000 DEGs were identified in each tissue relative to the PBLs. Overall, higher gene expression levels were observed in the hypothalamus vs PBL, while the remaining tissues had reduced expression compared to PBL (Table 1). Pearson correlation values were calculated from the mean normalized gene count in the PBL and tissues for genes (n = 17) important for stress response and metabolism to assess PBL as a possible surrogate for gene expression analyses (Table 2). Only two genes, oxytocin and 11β-hydroxylase, were positively correlated (P < 0.05) between PBL and specific tissue gene expression. As typically observed among different cell types, gene expression differences exist between the PBL and various tissues of the HPA axis. Therefore, it remains to be determined if gene expression in the surrogate PBL will shift in a direction similar to that of specific adrenal axis tissue of mature Brahman cows exposed to stressors during key developmental periods. more...

Prenatal transportation stress (PNS) results in calves that are more temperamental and have greater circulating concentrations of cortisol compared to control calves. The objective of this experiment was to evaluate whether PNS alters the number of pituitary corticotrophs in mature Brahman cows. We hypothesized that the increased circulating cortisol concentrations previously characterized in this bovine model is associated with developmental changes in the anterior pituitary leading to an increased number of corticotrophs. Pregnant Brahman cows (n = 48) were transported in trailers for 2-hour periods at 60±5, 80±5, 100±5, 120±5, and 140±5 days of gestation. Non-transported pregnant cows (n = 48) were designated as the Control group. Control and PNS offspring heifers were managed together under the same environmental conditions. At approximately 5 yr of age, randomly selected non-pregnant cows (Control, n = 8; PNS, n = 6) were humanely harvested and the whole pituitaries were collected. Pituitaries were weighed, fixed in paraformaldehyde, serially dehydrated with graded ethanol, embedded in paraffin blocks, and cut into 5-μm sections. Immunohistochemistry was performed to detect cells expressing adrenocorticotropic hormone (ACTH) as a marker for corticotrophs. Three comparable sections from the midsagittal plane from each animal were processed using an ovine ACTH-specific antibody (Dr. A.F. Parlow, NIDDK). Five fields of view were analyzed per section (15 fields per animal). Anterior pituitary gland weight did not differ (P > 0.10) between groups (Control = 2.11 ± 0.12 g; PNS = 2.10 ± 0.15 g). The mean number of ACTH-positive cells between control (531±56 cells/section) and PNS cows (477±49 cells/section) also did not differ (P > 0.10). In conclusion, the number of pituitary corticotrophs in mature Brahman cows was not affected by prenatal transportation stress and is likely not a mechanism mediating the increased circulating cortisol concentrations seen in this bovine model of fetal programming. more...

Telomeres are comprised of G-rich nucleotide sequences (5’-TTAGGG-3’) at the chromosome termini that are responsible for protecting chromosomes; however, attrition of these sequences has been observed due to aging and stress. The purpose of this study was to compare telomere length (TL) in control and prenatally stressed (PNS) calves. Mature Brahman cows inseminated to a single Brahman sire in 2018 were assigned to be either Control (n=35; not transported) or PNS (n=37; 2 h of transportation at 60, 80, 100, 120, and 140±5 d of gestation). Jugular blood samples were obtained by venipuncture from 10 Control (5 bulls and 5 heifers) and 10 PNS (5 bulls and 5 heifers) calves at 25±2 d of age. Peripheral leukocytes were isolated and genomic DNA was extracted utilizing silicone membrane spin column kits. The relative quantity of telomere products, which is proportional to the average TL, was determined by multiplex quantitative PCR analysis using the ratio of bovine telomere and β-globulin DNA (T/S ratio). An absolute standard of bovine telomere (1012–107 dilution series) and β-globulin (109-104 dilution series) genes was utilized to produce relative copy number. All samples were processed in triplicate and samples were included if the triplicate Cq difference was less than 0.25 cycles. Data were analyzed using ANOVA. TL differed by sex (P< 0.03; bulls 11,959±211; heifers 12,850±366) and treatment (P< 0.02; Control 12,895±278; PNS 11,913±303). There was not an interaction of sex and treatment (P >0.43) although TL of Control heifers (13,482±396) exceeded the TL of PNS bulls (11,609±348). These observations of fewer copies of telomere sequences suggest an influence of sex on TL within 1 month of age; and, that prenatal transportation stress may increase attrition of telomeres in cattle. Support: USDA NIFA Award No. 2019-67015-29573 more...

The objective of this study was to evaluate whether prenatal transportation stress (PNS) affects the immune response of Brahman calves after vaccination against Bovine Viral Diarrhea Virus (BVDV). Mature Brahman cows inseminated to a single Brahman sire in 2018 were assigned to either Control (n=35; not transported) or PNS (n=37; 2h of transportation at 60, 80, 100, 120, and 140±5 d of gestation). Of the calves born in 2019, 11 Control and 12 PNS calves were studied for vaccine treatment (VAX). Temperament score, body weight, and blood samples were obtained from calves prior to weaning (D-28), prior to first BoviShield vaccine (D+35), and after second BoviShield vaccine (D+98). Serum cortisol was determined by RIA. Humoral immune response was evaluated by virus neutralization (VN) assays using serum collected at respective timepoints post-vaccination. Cell-mediated immune (CMI) response was evaluated using a novel PrimeFlow RNA assay that incorporates cell surface marker staining with intracellular RNA expression of cytokines on blood samples obtained at D+35 and D+98. Data were analyzed using GLM procedures of SAS. Only total WBC and absolute lymphocytes were increased with PNS (P< 0.05), while lymphocytes were also decreased with VAX (P< 0.05). There was no PNS effect on the VN or CMI results, serum cortisol concentration, or temperament score. VAX tended (P=0.06) to reduce serum cortisol, as well as resulted in an increase in VN titers (P< 0.01) and frequency of interferon-γ mRNA expression in the CD4+ population (P< 0.01), but not in CD25+ population (P=0.26). Interaction between sampling day and VAX resulted in more drastic increase (P< 0.05) from D+35 to D+98 in VN titers, CD4+ population frequency of interferon-γ mRNA expression, and CD25+ population mean fluorescent intensity. Vaccine treatment positively affected humoral and cell-mediated immune responses; however prenatal transportation stress did not negatively impact response of Brahman calves to immunization against BVDV. more...

Mitochondrial activity correlates with meat product quality post-harvest but little is known about the relationships between live animal mitochondria and product quality at slaughter. To test the hypothesis that live animal skeletal muscle mitochondrial markers would be correlated with longissimus Warner-Bratzler shear force (WBSF) post-harvest, longissimus thoracis (LT) and trapezius (TRAP) samples were collected from Brahman and Angus steers (n = 36 per breed) during the grazing growth phase (GP) and after 120 days on feed (FP). Samples were analyzed for mitochondrial enzyme activities via colorimetry and for oxidative phosphorylation (P) and electron transfer (E) capacities via high-resolution respirometry. Relationships between mitochondrial variables and WBSF were determined using PROC CORR in SAS (v9.4). Within Angus in GP, LT mitochondrial volume density as measured by citrate synthase activity (CS) was positively correlated with WBSF (P=0.009). In FP, Angus LT intrinsic (relative to CS) P with complex I (PCI) and maximal E with complexes I and II (ECI+II) were positively correlated with WBSF (P≤0.05), while the contribution of maximal P with complexes I and II (PCI+II) to maximal E (flux control ratio, FCRPCI+II) was negatively correlated with WBSF (P=0.03). Also in FP, Angus TRAP integrative (relative to mg tissue) PCI, PCI+II, and ECI+II and FCRPCI were negatively correlated with WBSF (P≤0.02), while the FCR for electron transfer through complex II only (FCRECII) was positively correlated with WBSF (P=0.007). In Brahman, no mitochondrial markers correlated with WBSF during either GP or FP. Post-harvest meat tenderness is related to live animal skeletal muscle mitochondrial markers when evaluated close to slaughter in Angus but not in Brahman steers. more...

Maternal undernutrition during pregnancy followed by ad libitum access to nutrients during postnatal life induces postnatal metabolic disruptions in multiple species. Therefore, an experiment was conducted to evaluate postnatal growth, metabolism, and development of beef heifers exposed to late gestation maternal nutrient restriction. Pregnancies were generated via transfer of in vitro embryos produced using X-bearing sperm from a single Angus sire. Pregnant dams were randomly assigned to receive either 100% (control; n = 9) or 70% (restricted; n = 9) of their total energy requirements from gestational day 158 to parturition. From post-natal day (PND) 301 until slaughter (PND485), heifers were individually fed ad libitum in a Calan gate facility. Calves from restricted dams were lighter than controls at birth (PPP>0.10). To assess pancreatic function, glucose tolerance tests were performed on PND315 and PND482 and a diet effect was seen with glucose area under the curve being greater (PPPP more...

We previously determined that prenatal stress (PNS) differentially affected methylation of DNA from leukocytes of 28-d-old calves. Specifically, COX14 (cytochrome c oxidase (COX) assembly factor) and CKMT1B (mitochondrial creatine kinase U-type) were hypomethylated and COA5 (COX assembly factor 5), COX5A (COX subunit 5A), NRF1 (nuclear respiratory factor 1), and GSST1 (glutathione S-transferase theta-1) were hypermethylated in PNS compared to non-PNS calves (P ≤ 0.05). Our current objective was to test the hypothesis that PNS exhibit impaired mitochondrial function and greater oxidative stress than non-PNS calves. Blood and longissimus dorsi muscle samples were collected from yearling Brahman calves whose mothers were stressed by 2 h transportation at 60, 80, 100, 120, and 140 days of gestation (PNS; 8 bulls, 6 heifers) and non-PNS calves (4 bulls, 6 heifers). Serum was evaluated for the stress hormone, cortisol, and muscle damage marker, creatine kinase; muscle was analyzed for mitochondrial volume density and function by citrate synthase (CS) and COX activities, respectively, concentration of malondialdehyde, a lipid peroxidation marker, and activity of the antioxidant, superoxide dismutase (SOD). Data were analyzed using mixed linear models with treatment and sex as fixed effects. Serum cortisol was numerically higher in PNS than non-PNS calves but was not statistically different. Muscle CS and COX activities relative to protein were greater in PNS than non-PNS calves (P ≤ 0.03), but COX relative to CS activity was similar between groups. Activity of COX was greater in bulls than heifers (P = 0.03), but no other measure was affected by sex. All other measures were unaffected by PNS. Prenatal stress did not affect markers of muscle damage and oxidative stress in yearling Brahman calves at rest but mitochondrial volume density and function were greater in PNS calves. Acute stressors induce oxidative stress, so implications of differences in mitochondria in PNS calves following a stressor should be investigated. more...

The effect of an ionophore on the ruminal metabolome of temperate and tropically-adapted cattle was evaluated. For 21 d, five Angus, 5 Brahman, and 5 BrahmanXAngus F1 steers (264 kg BW) with 5 steers per pen within breedtype, were fed Tifton bermudagrass hay ad libitum, at the conclusion of which rumen fluid (100-to-250 mL) was collected and stored at -80°C (PRE). During a subsequent 21-d period, all steers were fed Tifton hay ad libitum plus a supplement (908 g corn, 217 g soybean meal, 60 g dried molasses) with monensin (220 mg·steer−1·d−1) individually, at the conclusion of which rumen fluid (100-to-250 mL) was collected and stored at -80°C (POST). Rumen fluid samples were processed for an untargeted analysis by LC/MS on Compound Discoverer v.3.1. Approximately 2,043 compounds were detected with 825 being annotated by ChemSpider and other databases, of which several were associated with four neurotransmitters; acetylcholine (ACh), dopamine, γ-aminobutyric acid (GABA), and serotonin. After monensin, several ACh-related molecules increased (P < 0.003) in Angus (POST:PRE ratio=7.906) but ACh decreased (P < 0.043) in the F1 (ratio= 0.391) steers. Dopamine and dopamine-related molecules decreased (P < 0.01) in Angus (ratio= 0.149) and F1 steers (ratio= 0.206) after monensin. No changes for rumen presence of ACh or dopamine occurred in Brahman steers after monensin. Multiple molecules related to GABA increased in Angus (P < 0.03) and Brahman (P < 0.02) steers. Serotonin-related molecules such as the metabolite 5-hydroxyindoleacetic acid (5-HIAA; ratio= 4.389) increased (P < 0.005) only in Brahman steers. The addition of monensin to the diet differentially altered the presence of neurotransmitter-related molecules in the rumen fluid of temperate and tropically adapted beef cattle. Further research regarding physiological roles of ruminal neurotransmitters and modulation by ionophores that affect ruminal microbiota is warranted. Support: USDA FAH more...

The objective of this experiment was to evaluate whether prenatal transportation stress (PNS) affects the weight of endocrine and immune tissues of calves. Mature Brahman cows inseminated to a single Brahman sire in 2018 were assigned to be either Control (n = 35; not transported) or PNS (n = 37; 2 h of transportation at 60, 80, 100, 120, and 140 ± 5 d of gestation). Of the calves born in 2019, 16 Control (8 bulls and 8 heifers) and 16 PNS (8 bulls and 8 heifers) calves were studied. Pen score, body weight, and blood samples were obtained from calves at 25 ± 2 d of age. At that time, calves were euthanized by barbiturate overdose in order to collect tissues (brain, pituitary and adrenal glands, spleen and thymus). Tissues were trimmed and weighed. Serum cortisol was determined by RIA. Data were analyzed using ANOVA, GLM, and CORR procedures of SAS with body weight at sample collection as a covariate. Whole pituitary weight tended (P = 0.08) to be greater in females compared to males, whereas anterior pituitary weight tended (P = 0.06) to be greater in PNS. Whole brain weight was greater (P < 0.01) in males. The interaction of treatment and sex on whole brain weight of PNS tended (P = 0.09) to fall between that of the Control females and Control males. Whole pituitary weight was positively correlated with total adrenal weight (r = 0.32; P = 0.08). Total thymus weight positively correlated with whole brain and total adrenal weight (r = 0.43 and 0.41, respectively; P < 0.05). Serum cortisol was negatively correlated with spleen weight and total immune tissue weight (r = -0.37 and -0.38, respectively; P < 0.05). Results suggest next steps should include a closer look at function of immune tissues by studying PNS effect on thymus response to immunization. more...

In forage-animal nutrition modeling, diet energy is estimated mainly from the forage total digestible nutrients (TDN). As digestibility trials are expensive, TDN is usually estimated using summative equations. Early summative equations assumed a fixed coefficient to compute digestible fiber using the lignin-to-neutral detergent fiber (NDF) ratio. Subsequently, a structural coefficient (φ) was added to the summative equations to reflect an association between lignin and cell wall components. Additional modifications to the summative equations assumed a constant φ value, and they have been used as a standard method by many commercial laboratories and scientists. For feeds with nutritive values that do not change much over time, a constant φ value may suffice. However, for forages with nutritive values that keep changing during the grazing season owing to changes in weather and plant maturity, a constant φ value may add a systematic bias to prediction because it is associated with the variable lignin-to-NDF ratio. In this study, we developed a model to estimate φ as a function of the day of the year by using the daily TDN values of bermudagrass [Cynodon dactylon (L.) Pers.], a popular warm-season perennial grass in the southern United States. The variable φ model was evaluated by using it in the TDN equation and comparing the estimated values with the observed ones obtained from several locations. Values of the various measures of fit used—the Willmott index (WI), the modeling efficiency (ME), R2, root mean square error (RMSE), and percent error (PE)—showed that using the variable φ vis-à-vis the constant φ improved the TDN equation significantly. The WI, ME, R2, RMSE, and PE values of 0.94, 0.80, 0.80, 2.5, and 4.7, respectively, indicated that the TDN equation with the variable φ model was able to mimic the observed values of TDN satisfactorily. Unlike the constant φ, the variable φ predicted more closely the forage nutritive value throughout the grazing season. The variable φ model may be useful to forage-beef modeling in accurately reflecting the impacts of plant maturity and weather on daily forage nutritive value and animal performance. more...

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. more...

The availability of tools to accurately replicate the clinical phenotype of rare human diseases is a key step toward improved understanding of disease progression and the development of more effective therapeutics. We successfully generated the first large animal model of a rare human bone disease, hypophosphatasia (HPP) using CRISPR/Cas9 to introduce a single point mutation in the tissue nonspecific alkaline phosphatase (TNSALP) gene (ALPL) (1077 C > G) in sheep. HPP is a rare inherited disorder of mineral metabolism that affects bone and tooth development, and is associated with muscle weakness. Compared to wild-type (WT) controls, HPP sheep have reduced serum alkaline phosphatase activity, decreased tail vertebral bone size, and metaphyseal flaring, consistent with the mineralization deficits observed in human HPP patients. Computed tomography revealed short roots and thin dentin in incisors, and reduced mandibular bone in HPP vs. WT sheep, accurately replicating odonto-HPP. Skeletal muscle biopsies revealed aberrant fiber size and disorganized mitochondrial cristae structure in HPP vs. WT sheep. These genetically engineered sheep accurately phenocopy human HPP and provide a novel large animal platform for the longitudinal study of HPP progression, as well as other rare human bone diseases. more...

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. more...

DNA methylation (DNAm) patterns are tissue specific and aid in tissue specific gene expression changes. The use of DNAm patterns from peripheral blood leukocytes (PBL) as a surrogate for patterns in other tissues is common, especially in longitudinal studies when sampling of tissues is not plausible. Thus, the objective of this study was to investigate the suitability of using DNAm patterns of PBL as a surrogate for the DNAm patterns in neuroendocrine tissues responsible for stress responses and energy metabolism. Samples from the paraventricular region of the hypothalamus, anterior pituitary gland, adrenal cortex, and the adrenal medulla were harvested from 5-yr-old Brahman cows (n = 8) and DNA was extracted from each sample. Methylation was assessed using reduced representation sodium bisulfite sequencing and differentially methylated regions (DMR) between the PBL DNA and tissue DNA were identified using EdgeR from Bioconductor, R. Analysis revealed over 15,000 DMRs located within promoter regions of genes in each tissue, with the majority of the sites having increased methylation in the PBL (Table 1). To further evaluate the use of PBL DNA as a surrogate, Pearson correlation values were calculated for genes (n = 20) pertinent to each respective tissue using the mean methylation of the specific gene in the PBL and in the tissue (Table 2). Three correlations were significant (P ≤ 0.05), two of which were negative. The sizable differences indicate that DNA methylation patterns from PBL do not compare well to patterns from hypothalamic, pituitary, adrenal cortex, and adrenal medulla tissues from 5-yr-old Brahman cows. This is especially the case for the majority of the specific genes examined in this study. Whether DNAm in the surrogate PBL will shift in a direction similar to that of specific tissues of Brahman cows exposed to stressful stimuli during developmental periods remains to be determined. more...

The primary objective of this investigation was to determine whether circadian clock genes were differentially expressed within or among bovine hypothalamic paraventricular nucleus (PVN), anterior pituitary gland (AP), adrenocortical (AC) and adrenomedullary (AM) tissues. The PVN, AP, AC, and AM were isolated from 5-yr-old Brahman cows (n = 8) harvested humanely at an abattoir between 0800-1100 h. Expression of target genes in each sample was evaluated via RNA-sequencing analyses. Gene counts were normalized using the trimmed mean of M values (TMM) method in the edgeR Package from Bioconductor, R. The normalized gene counts of genes important for circadian rhythm were statistically analyzed using the GLM Procedure of SAS. The genes analyzed were circadian locomotor output cycles protein kaput (CLOCK), cryptochrome circadian regulator 1 and 2 (CRY1 and CRY2), aryl hydrocarbon receptor nuclear translocator like (ARNTL), period circadian regulator 1 and 2 (PER1 and PER2), neuronal PAS domain protein 2 (NPAS2), and nuclear receptor subfamily 1 group D member 1 (NR1D1). Overall, relative expression profiles of clock genes differed (P < 0.01) within each tissue with PER1 having greater expression in all tissues (P < 0.01). Within the PVN expression of CLOCK, CRY1, ARNTL, and PER2 was less than that of CRY2, NPAS2, and NR1D1 (P < 0.01). In the AP, with the exception of PER1, no other clock gene differed in degree of expression. In the AC, expression of CLOCK and NPAS2 was greater than CRY1, ARNTL, PER2, and NR1D1 (P < 0.05), whereas CRY2 expression exceeded only CRY1 (P < 0.05). Within the AM, CLOCK and CRY2 expression was greater than CRY1 and ARNTL (P < 0.05). Overall, clock gene expression among tissues differed (P < 0.01) for each individual clock gene. The AC and AM had similar clock gene expression, except expression of CRY2 and PER2 was greater in AM (P < 0.05). The AC and AM had greater expression of CLOCK than the PVN and AP (P < 0.01), with PVN having greater expression than AP (P < 0.01). The AP had greater expression of NPAS2, followed by PVN, with the least expression in the AC and AM (P < 0.01). Both PVN and AP had greater CRY1 and NR1D1 expression than AC or AM (P < 0.01). The AP had greater PER1 expression than PVN, AC, and AM (P < 0.01), whereas PVN, AC, and AM had greater ARNTL expression than AP (P < 0.05). Both AP and AM had greater expression of PER2 than PVN or AC (P < 0.01). The PVN had greater expression of CRY2 than the AP, AC, and AM (P < 0.01). These results indicated that within each tissue the various clock genes were expressed in different quantities. Also, the clock genes were expressed differentially among the tissues of the bovine neuroendocrine adrenal system. Temporal relationships of these genes with the primary endocrine products of these tissues should be investigated to define the roles of peripheral clock genes in regulation of metabolism and health. more...

Temperament impacts skeletal muscle mitochondria in Brahman heifers, but this has not been investigated in steers or between cattle breeds. We hypothesized mitochondrial measures would be greater in Angus than Brahman, temperamental than calm steers, and the trapezius (TRAP) than the longissimus thoracis (LT) muscle. Samples from calm (n = 13 per breed), intermediate (n = 12 per breed), and temperamental (n=13 per breed) Angus and Brahman steers (mean±SD 10.0±0.8 mo) were evaluated for mitochondrial enzyme activities via colorimetry. Calm and temperamental LT samples were evaluated for oxidative phosphorylation (P) and electron transfer (E) capacities by high-resolution respirometry. Data were analyzed using linear models with fixed effects of breed, muscle, temperament, and all interactions. Brahman tended to have greater mitochondrial volume density (citrate synthase activity; CS) than Angus (P = 0.08), while intrinsic (relative to CS) mitochondrial function (cytochrome c oxidase activity) was greater in Angus than Brahman (P = 0.001) and greater in TRAP than LT (P = 0.008). Angus exhibited greater integrative (per mg tissue) and intrinsic P with complex I (PCI), P with complexes I+II (PCI+II), maximum noncoupled E, and E with complex II (ECII; P ≤ 0.04) and tended to have greater intrinsic leak (P = 0.1) than Brahman. Contribution of PCI to total E was greater in Angus than Brahman (P = 0.01), while contribution of ECII to total E was greater in Brahman than Angus (P = 0.05). A trend for the interaction of breed and temperament (P = 0.07) indicated calm Angus had the greatest intrinsic ECII (P ≤ 0.03) while intrinsic ECII was similar between temperamental Angus and calm and temperamental Brahman. Integrative PCI+II and ECII, and the contribution of PCI and PCI+II to overall E tended to be greater in temperamental than calm steers (P ≤ 0.09), while intrinsic ECII tended to be greater in calm than temperamental steers (P = 0.07). The impact of these mitochondrial differences on meat quality measures remains to be determined. more...

The utility of genetic engineering has increased significantly in recent years, particularly with the wide acceptance of CRISPR-Cas9 genome editing technology. The wide application of genome editing techniques provides a unique opportunity for musculoskeletal investigators to consider the examination of rare (and other) disease phenotypes as well as gene function in domestic animals. It is particularly important to consider developing domestic animal models of bone disorders in which the bone remodeling process parallels that observed in humans and since rodent models have not fully recapitulated the phenotype of the homologous human mutations. In contrast to the use of larger animal models, rodent experiments are less costly and time-consuming, making genetically engineered small animal models (primarily rodents) widely attractive for studying bone metabolism. However, many important issues not fully addressed in small animal models remain, such as Haversian remodeling, metaphyseal fracture healing and the development and evaluation of orthopedic implants. Once generated, a specific bone phenotype in a domestic large animal model has the potential to uncover new mechanistic insights into bone remodeling, specific details of muscle-bone-tendon and ligament interactions, and provide an opportunity for improved treatment and/or targeted therapeutic interventions that are mechanism-based. In this perspective, we interrogate the modeling of bone phenotypes in domestic animals and evaluate the role of these species in the growth of the musculoskeletal field, considering the high current preference for rodent models. Whatever the eventual outcome, it is clear that recent biotechnology developments in gene editing and the growing list of well-annotated genomes is bringing significant changes to the way bone phenotypes are developed, studied and evaluated. more...

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. more...

Beef cows in the southeastern United States are likely maintained on pasture during mid-gestation, a critical period for fetal development of traits important to performance of the growing/finishing calf. Climate projections are for increased warming with highly variable precipitation in this region. Forage quantity and quality are affected by temperature and precipitation. Thus, in order to obtain a better understanding of downstream effects from cow nutrition during gestation on subsequent performance of growing cattle, we analyzed 579 records of crossbred steers and heifers born (fall and winter) to cows grazing typical forages in Overton, Texas. Cows were involved in a long-term stocking rate study and were assigned to either high (H; 4 cow/calf ha-1) or low (L; 2 cow/calf ha-1) treatment groups. Cow body condition scores (BCS; 1–9) were collected at breeding and weaning. Calves produced were pastured from weaning to approximately 1 to 1.5 yr of age, then shipped to a commercial feedlot and subsequently slaughtered at a commercial facility. Calf data included birth weight, weaning weight (~240 d), yearling weight, on-feed weight, final pre-harvest weight, hot carcass weight (all weights in kg), dressing percentage, marbling score, yield grade, backfat thickness (cm), ribeye area (cm2), and kidney-pelvic-heart fat (%). Differences (P < 0.05) between treatment groups were determined by a general linear model in SAS with stocking rate as the main effect. Mean separation was accomplished using Tukey’s test. Cow BCS (H; 4.27 ± 0.06 vs L; 5.64 ± 0.07, P < 0.0001), final pre-harvest weight (H; 594.80 ± 6.18 kg vs L; 619.85 ± 6.12 kg, < 0.005), hot carcass weight (H; 366.60 ± 3.89 kg vs L; 383.17 ± 3.74, P < 0.003), and ribeye area (H; 90.52 ± 0.90 cm2 vs L; 93.74 ± 1.03 cm2, P < 0.03) were all lower in H than L. Calf weaning weight followed a similar trend (H; 289.24 ± 2.58 kg vs L; 296.56 ± 3.08 kg, P < 0.07). Diminished nutritional provision to the fetus during mid-gestation was apparently manifest as epigenetic effects on offspring performance. more...

This study evaluated the possible effects of sire and dam on age at first calving in Brahman heifers. A total of 570 heifers born between the years 2001 and 2017 were exposed as yearlings to fertile bulls through time of pregnancy determination. A calving code was determined by calculating the mean (993 d) and standard deviation (187 d) of heifer age at first calving. Heifers considered to calve early (≤899 d; calving code = 1) or late (≥1087 d; calving code = 3) were at least half a standard deviation (94 d) away from the mean. All other heifers were considered to have a normal age at first calving (900 to 1086 d; calving code = 2). There were 35 sires with 5 or more daughters, resulting in a total of 543 heifers evaluated for the effect of sire. There were 323 daughters of evaluated heifers, which were evaluated for the effect of dam calving code. Also evaluated was the effect of dam and sire calving code on age at first calving of female offspring (n = 287). Variables analyzed included dam and sire of the heifer, age at first calving, and calving code. Data were analyzed using the GLM procedures of SAS and proportions were tested using Chi-square. Sire calving code did affect (P < 0.01) age at first calving and calving code in heifers, but dam calving code did not affect daughter calving code and age at first calving (Table 1). The proportion of daughters calving early compared to sire calving code significantly differed from the expected proportions (P < 0.01), whereas the proportions of daughters calving early for dam calving code did not differ (Table 2). A selection effort to produce Brahman heifers capable of calving early will not be effective from the dam side, but can be effective on the sire side. more...

l-carnitine is a potent antioxidant used for in vitro culture systems. Controversial results have been reported using l-carnitine in culture medium at different stages of in vitro bovine embryo production. Cumulus-oocyte complexes (n = 843) were in vitro-fertilized and cultured and added (treatment group) or not added (control group) with l-carnitine. At day three of culture, each group was subdivided into two subgroups receiving no l-carnitine (group 1), 3.8 mM l-carnitine added during in vitro maturation (group 2), 1.5 mM added during the in vitro culture (group 3), and 3.8 mM and 1.5 mM added during the maturation and culture, respectively (group 4). At day 8, blastocyst embryos were examined for mitochondrial activity, the presence of lipid droplets, total cell number, gene expression, and cryotolerance by vitrification. The data were analyzed with a one-way analysis of variance. l-carnitine added in the late in vitro culture significantly reduced mitochondrial activity and lipid content, and upregulated ifn-&tau, and ptgs2 gene expression compared to controls (p &lt, 0.05). l-carnitine supplementation did not significantly affect the embryo rate production or survival rate after vitrification and warming (p &gt, 0.05). l-carnitine supplementation significantly improved embryo potential to develop viable pregnancies in agreement with a study reporting improved pregnancy rates. more...

The objective of this experiment was to examine differential DNA methylation as a mechanism of altered temperament and stress response in young Prenatally Stressed (PNS) compared with Control 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 non-transported 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 methods. Increased DNA methylation of gene promoter regions typically results in decreased transcriptional activity of the corresponding gene. Therefore, 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 the altered signaling pathways related to behavior and stress response were: opioid signaling, GABA receptor signaling, dopamine-DARPP32 feedback in cAMP signaling, corticotropin-releasing hormone signaling, dopamine receptor signaling, serotonin receptor signaling, and alpha-adrenergic signaling. Among differentially methylated genes (P ≤ 0.05) related to behavior and stress response were OPRK1, OPRM1, PENK, POMC, NR3C2, TH, ADRA1A, DRD1, DRD5, COMT, HTR6, HTR5A, GABRA4, GABRQ, GAD2, SLC6A11, and SLC6A12. Alterations to behavior and stress response related genes and canonical pathways supported previously observed elevations in temperament and increased circulating concentrations of cortisol through weaning in the larger population of PNS calves from which bulls in this study were derived (J. Anim. Sci. 94:2:602–609). Differential DNA methylation and predicted alterations to behavior and stress response related pathways in Prenatally Stressed compared with Control bull calves suggest epigenetic programming of behavior and stress response in utero. more...

The most highly regulated and abundant fatty acid in animal tissue is oleic acid (18:1n9). Oleic acid is synthesized by the Δ9 desaturase, stearoyl-CoA desaturase-1 (SCD1), which is responsible for the synthesis of the putative cytokine palmitoleic acid (16:1n7) and 18:2 cis-9, trans-11 conjugated linoleic acid. Owing to the importance of SCD1 in lipid metabolism, we generated porcine swine kidney (SK6) transgenic cell lines for sustained overexpression or knockdown of porcine stearoyl-CoA desaturase-1 (pSCD1) in an inducible manner by utilizing a lentiviral expression system. We successfully validated these cell culture models for expression and functionality of pSCD1 by documenting that the pSCD-transduced cells overexpressed pSCD1 protein and mRNA. Additionally, the pSCD1-transduced cells increased the conversion of palmitate (16:0) to palmitoleic acid nearly fourfold. The lentiviral vectors utilized in this study can be further used to generate transgenic animals to document the effects of the overexpression of SCD1 on obesity and steatosis. more...

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. more...

Endometriosis is a debilitating, estrogen-dependent, progesterone-resistant, inflammatory gynecological disease of reproductive age women. Two major clinical symptoms of endometriosis are chronic intolerable pelvic pain and subfertility or infertility, which profoundly affect the quality of life in women. Current hormonal therapies to induce a hypoestrogenic state are unsuccessful because of undesirable side effects, reproductive health concerns, and failure to prevent recurrence of disease. There is a fundamental need to identify nonestrogen or nonsteroidal targets for the treatment of endometriosis. Peritoneal fluid concentrations of prostaglandin E2 (PGE2) are higher in women with endometriosis, and this increased PGE2 plays important role in survival and growth of endometriosis lesions. The objective of the present study was to determine the effects of pharmacological inhibition of PGE2 receptors, EP2 and EP4, on molecular and cellular aspects of the pathogenesis of endometriosis and associated clinical symptoms. Using human fluorescent endometriotic cell lines and chimeric mouse model as preclinical testing platform, our results, to our knowledge for the first time, indicate that selective inhibition of EP2/EP4: (i) decreases growth and survival of endometriosis lesions; (ii) decreases angiogenesis and innervation of endometriosis lesions; (iii) suppresses proinflammatory state of dorsal root ganglia neurons to decrease pelvic pain; (iv) decreases proinflammatory, estrogen-dominant, and progesterone-resistant molecular environment of the endometrium and endometriosis lesions; and (v) restores endometrial functional receptivity through multiple mechanisms. Our novel findings provide a molecular and preclinical basis to formulate long-term nonestrogen or nonsteroidal therapy for endometriosis. more...

Embryo culture and assisted reproductive technologies have been associated with a disproportionately high number of epigenetic abnormalities in the resulting offspring. However, the mechanisms by which these techniques influence the epigenome remain poorly defined. In this study, we evaluated the capacity of oxygen concentration to influence the transcriptional control of a selection of key enzymes regulating chromatin structure. In mouse embryonic stem cells, oxygen concentrations modulated the transcriptional regulation of the TET family of enzymes, as well as the de novo methyltransferase Dnmt3a. These transcriptional changes were associated with alterations in the control of multiple imprinted genes, including H19, Igf2, Igf2r, and Peg3. Similarly, exposure of in vitro produced bovine embryos to atmospheric oxygen concentrations was associated with disruptions in the transcriptional regulation of TET1, TET3, and DNMT3a, along with the DNA methyltransferase co-factor HELLS. In addition, exposure to high oxygen was associated with alterations in the abundance of transcripts encoding members of the Polycomb repressor complex (EED and EZH2), the histone methyltransferase SETDB1 and multiple histone demethylases (KDM1A, KDM4B, and KDM4C). These disruptions were accompanied by a reduction in embryo viability and suppression of the pluripotency genes NANOG and SOX2. These experiments demonstrate that oxygen has the capacity to modulate the transcriptional control of chromatin modifying genes involved in the establishment and maintenance of both pluripotency and genomic imprinting. more...

Natural, organic, and grass-fed beef provides alternative products for consumers. During 2 consecutive yr, Bonsmara steers (n = 130) grazed winter pasture at 2 environments [semiarid, Uvalde (UVL), and humid, Overton (OVT)] and received pasture only (PAS) or pasture plus 0.8% BW/steer per day corn (SUPL) to quantify growth and carcass traits. Each year, 8 steers each from UVL and OVT were slaughtered off pasture and after a 90-d feedlot (FED) period. From initiation to termination (mid-May) of stocking on Maton rye (Secale cereale L.) plus TAM-90 annual ryegrass (Lolium multiflorum Lam.) at OVT and irrigated TAM-90 at UVL, ADG was greater (P 0.05) FED ADG. Growth in BW was linear (P 0.05) by SUPL. Tenderness was enhanced (P more...

Muscle mitochondrial capacity and animal temperament have been linked to beef tenderness. We tested the hypotheses that mitochondrial characteristics would be: 1) greatest in temperamental animals; 2) higher in the trapezius than the longissimus dorsi (LD); and, 3) stable as the heifer ages. Samples from both muscle types were collected from the same calm (n=6) and temperamental (n=6) Brahman heifers at 8, 12, and 18 mo of age. Temperament was determined at weaning using pen score and exit velocity. Mitochondrial density and function were measured by colorimetry and mitochondrial capacity was measured utilizing high-resolution respirometry. Data were analyzed using PROC MIXED in SAS (9.4) with repeated measures. Mitochondrial density (citrate synthase activity, CS) increased over time (P < 0.0001) but was similar between temperament and muscle groups. At 12 mo, temperamental heifers had lower mitochondrial function (cytochrome c oxidase activity) compared to calm in the LD (P=0.05) but had similar function between muscle and temperament groups by 18 mo. Integrated (relative to tissue weight) mitochondrial oxidative phosphorylation capacity with complex I substrates (PCI; P=0.012), PCI with complex II substrates (PCI+II; P=0.008), electron transport system capacity (ECI+II; P=0.002), and ECII (P=0.013) were higher in trapezius than LD for calm heifers at all ages, but were similar between muscle groups in temperamental heifers. While unaffected by temperament, intrinsic (relative to CS activity) PCI (P < 0.0001), PCI+II (P=0.0004), ECI+II (P < 0.0001), and ECII (P=0.001) were higher in the trapezius than the LD. Fractional control of CI+II increased from 12 to 18 mo in the trapezius of calm (P=0.035) and the LD of temperamental heifers (P=0.038) but was unchanged by age in the LD of calm and trapezius of temperamental heifers. Calm, but not temperamental, heifers had higher integrative mitochondrial capacity in the trapezius compared to the LD muscle from 8 through 18 mo of age. more...

Nutritional insults during gestation are known to impact postnatal life, evident through altered growth and performance, and metabolic, immune, and reproductive dysfunction. In a previous study, we observed reduced pancreas weights and lower insulin concentrations in fetuses originating from dams nutrient restricted during late gestation. The present study investigated the effects of late gestation maternal nutrient restriction on postnatal growth of beef heifers. Embryos were produced in vitro utilizing Angus-type oocytes and sexed semen from one Angus sire for X-bearing sperm. Resulting embryos were transferred into virgin dams similar in age, body condition and frame score. Pregnant dams were moved into individual rationing facilities on gestation day (GD) 130 for 28-d acclimation and were randomly assigned to dietary treatments beginning on GD 158, receiving either 70% nutrient requirements (n=9; restricted) or 100% nutrient requirements (n=9; control). Individual feed intakes were adjusted twice monthly based on predicted pregnancy requirements using mean metabolic body weight. Dam BWs were recorded twice monthly, and rib eye area (REA) and last rib back fat (LRBF) were recorded monthly. Maternal BW (P0.01) and REA (PP=0.15). At calving, all dams were maintained on high-quality improved pasture to meet nutritional demands for lactation. Restricted calves were lighter than control calves (P0.05) at birth, postnatal day (PND) 35 (P0.01) and PND 70 (P=0.05). There was no difference (P>0.11) in calf BW at PND 105, 140, 175, 210, 245, 315, 350, 385, 420, or 455. At weaning (PND210) there was no difference in calf REA (P=0.45) or LRBF (P=0.39). These data support previous observations of reduced birth weight followed by compensatory growth in nutritionally restricted calves. Ongoing analyses will determine respective age at attainment of puberty and organ and carcass characteristics. more...

The effect of monensin on the ruminal and fecal metabolome and microbiome of temperate and tropically-adapted cattle (264±6 kg BW) consuming Tifton bermudagrass hay plus a supplement was evaluated. During Period 1, 10 Angus, 10 Brahman, and 10 BrahmanXAngus steers were fed Tifton bermudagrass hay ad libitum for a 21-d period with 5 steers per pen within breedtype at the conclusion of which rumen fluid (100-to-250 mL) and fecal samples were collected and stored at -80C. During Period 2 all steers were fed Tifton bermudagrass hay ad libitum plus 1.185 kg of supplement (908 g corn, 217 g soybean meal, 60 g dried molasses) with or without monensin (220 mg·steer−1·d−1) individually for 21 d. Rumen fluid and fecal samples were stored at -80C until DNA was extracted and total prokaryotes, methanogens and fungi determined by qPCR. Rumen and fecal samples were extracted to quantify short chain fatty acids (SCFA) and monensin content by GC and LCMS, respectively. After 21 d of consumption of bermudagrass hay, neither rumen nor fecal prokaryotic copy number, fungal copy number, nor methanogen copy number differed among breedtypes. The rumen, but not fecal, fungi:bacterial ratio was least for Angus (0.77) and similar between Brahman (1.31) and BrahmanXAngus (1.21) steers (P < 0.0032). Ruminal SCFA concentrations were similar among breeds as was the acetate:propionate ratio (Angus: 3.52; Brahman: 3.64; BrahmanXAngus: 3.43). Monensin was not detected in control rumen fluid but averaged 363±24 ng/mL for monensin steers. Monensin reduced the acetate:propionate ratio (P < 0.001) by 18, 25.1, and 18.4% for the Angus, Brahman, and BrahmanXAngus steers, respectively. Rumen prokaryotic copy number (P < 0.10), methanogen copy number (P < 0.035), and methanogen:prokaryote ratio (P < 0.001) were reduced within each breedtype by monensin. Addition of monensin reduced methanogen copy number and improved utilization of the diet in temperate and tropically-adapted beef cattle. more...

Somatic-cell nuclear transfer (SCNT) experiments have paved the way to the field of cellular reprogramming. The demonstrated ability to clone over 20 different species to date has proven that the technology is robust but very inefficient, and is prone to developmental anomalies. Yet, the offspring from cloned animals exhibit none of the abnormalities of their parents, suggesting the low efficiency and high developmental mortality are epigenetic in origin. The epigenetic barriers to reprogramming somatic cells into a totipotent embryo capable of developing into a viable offspring are significant and varied. Despite their intimate relationship, chromatin structure and transcription are often not uniformly reprogramed after nuclear transfer, and many cloned embryos develop gene expression profiles that are hybrids between the donor cell and an embryonic blastomere. Recent advances in cellular reprogramming suggest that alteration of donor-cell chromatin structure towards that found in an normal embryo is actually the rate-limiting step in successful development of SCNT embryos. Here we review the literature relevant to the transformation of a somatic-cell nucleus into an embryo capable of full-term development. Interestingly, while resetting somatic transcription and associated epigenetic marks are absolutely required for development of SCNT embryos, life does not demand perfection. more...

Sertoli cells are immune privileged cells, important for controlling the immune response to male germ cells as well as maintaining the tolerogenic environment in the testis. Additionally, ectopic Sertoli cells have been shown to survive and protect co-grafted cells when transplanted across immunological barriers. The survival of ectopic Sertoli cells has led to the idea that they could be used in cell based gene therapy. In this review, we provide a brief overview of testis immune privilege and Sertoli cell transplantation, factors contributing to Sertoli cell immune privilege, the challenges faced by viral vector gene therapy, the use of immune privileged cells in cell based gene therapy and describe several recent studies on the use of genetically engineered Sertoli cells to provide continuous delivery of therapeutic proteins. more...

In studies of somatic cell nuclear transfer (SCNT), the ability of factors within the oocyte to epigenetically reprogram transferred nuclei is essential for embryonic development of the clone to proceed. However, irregular patterns of X-chromosome inactivation, abnormal expression of imprinted genes, and genomic DNA hypermethylation are frequently observed in reconstructed embryos, suggesting abnormalities in this process. To better understand the epigenetic events underlying SCNT reprogramming, we sought to determine if the abnormal DNA methylation levels observed in cloned embryos result from a failure of the oocyte to properly reprogram transcription versus differential biochemical regulation of the DNA methyltransferase family of enzymes (DNMTs) between embryonic and somatic nuclei. To address this question, we conducted real-time quantitation of Dnmt transcripts in bovine preimplantation embryos generated though in vitro fertilization (IVF), parthenogentic activation, and SCNT. By the 8-cell stage, transcripts encoding Dnmt1 become significantly down-regulated in cloned embryos, likely in response to the state of genomic hypermethylation, while the de novo methyltransferases maintain an expression pattern indistinguishable from their IVF and parthenote counterparts. Depletion of embryonic/maternal Dnmt1 transcripts within IVF embryos using short-interfering RNAs, while able to lower genomic DNA methylation levels, resulted in developmental arrest at the 8/16-cell stage. In contrast, SCNT embryos derived from a stable, Dnmt1-depleted donor cell line develop to blastocyst stage, but failed to carry to term. Our results indicate an essential role for Dnmt1 during bovine preimplantation development, and suggest proper transcriptional reprogramming of this gene family in SCNT embryos. Mol. Reprod. Dev. 78:306–317, 2011. © 2011 Wiley-Liss, Inc. more...