The influence of maternal nutrition on expression of genes responsible for adipogenesis and myogenesis in the bovine fetus.

The objective of this study was to determine whether altered maternal energy supply during mid-gestation results in differences in muscle histology or genes regulating fetal adipose and muscle development. In total, 22 Angus cross-bred heifers (BW=527.73±8.3 kg) were assigned randomly to the three dietary treatments providing 146% (HIGH; n=7), 87% (INT; n=7) or 72% (LOW; n=8) of the energy requirements for heifers from day 85 to day 180 of gestation. Fetuses were removed via cesarean section at day 180 of gestation and longissimus muscle (LM) and subcutaneous fat were collected and prepared for analysis of gene expression. Samples from the LM and semitendinosus (ST) were evaluated for muscle fiber diameter, area and number. The right hind limb was dissected and analyzed to determine compositional analysis. Fetal growth and muscle histology characteristics of the LM and ST were similar among treatments. Preadipocyte factor-1 expression was up-regulated in fetal LM (P<0.05) of HIGH fetuses as compared with INT, whereas LOW fetuses showed increased CCAAT/enhancer-binding protein-ß (C/EBP-ß) expression in LM as compared with INT (P<0.05). Peroxisome proliferator-activated receptor γand C/EBP-α did not differ as a result of dietary treatment in LM or subcutaneous fat samples. There was a tendency for increased expression of fatty acid synthase in LM of LOW fetuses as compared with INT (P<0.10). Myogenin was more highly expressed (P<0.05) in LM of the LOW fetuses, whereas µ-calpain expression was increased in the HIGH treatment compared with INT. A tendency for increased expression of IGF-II was observed for both LOW and HIGH fetuses compared with INT (P<0.10). Expression of stearoyl-CoA desaturase, myoblast determination protein 1, myogenic factor 5, myogenic regulatory factor-4, m-calpain, calpastatin, IGF-I and myostatin was similar between treatments. Collectively, these results suggest that fetal growth characteristics are not affected by the level of maternal nutritional manipulation imposed in this study during mid-gestation. However, differences in expression of fetal genes regulating adipose and muscle tissue growth and development could lead to differences in postnatal composition and warrants further investigation.

The influence of maternal energy status during mid-gestation on beef offspring tenderness, muscle characteristics, and gene expression.

The objective of this study was to determine if maternal energy status during mid-gestation influences the expression of genes regulating muscle and fat development, and muscle characteristics that may impact meat tenderness. Cows grazed dormant, native range (Positive Energy Status [PES]) or were fed at 80% of maintenance energy requirements (Negative Energy Status [NES]) during mid-gestation. Steer offspring were harvested after 21 d in the feedlot (weaning subsample) or after 208 d in the feedlot (final subsample). Greater 21-d tenderness was observed in NES steers, resulting from reduced collagen content in longissimus lumborum steaks. In the semitendinosus, NES steers had greater soluble collagen, and down-regulated expression of MHC-IIA and TIMP-3 at weaning, while MHC-IIA expression was up-regulated in NES steers in the final harvest. Data show mid-gestational maternal energy status may impact offspring tenderness and collagen, but differences were not detected in expression of genes important in myogenesis and adipogenesis in muscle samples obtained from steers at weaning or slaughter.

Measuring bovine viral diarrhea virus vaccine response: using a commercially available ELISA as a surrogate for serum neutralization assays.

Genetic selection in livestock offers the opportunity to improve bovine viral diarrhea virus (BVDV) vaccine response, but first we must define how vaccine response should be measured. For measuring humoral vaccine response, serum neutralization (SN) measures antibodies that can neutralize BVDV, but relative to enzyme-linked immunosorbent assay (ELISA) is time consuming, technically demanding, and expensive. The ELISA, however, measures total BVDV-specific antibodies, regardless of whether the antibodies can neutralize BVDV. Our objective was to test whether a commercially available BVDV antibody ELISA could be used as a surrogate (or indicator trait) for neutralizing antibodies as measured by SN. Angus and Angus-cross calves (n=193) from two South Dakota research herds were vaccinated for BVDV-1 and BVDV-2. Sera and plasma samples (n=406) were collected from these calves at the time of vaccination and post-vaccination (20-72 days post-vaccination). The BVDV-specific antibody concentration was measured on each serum and plasma sample by (1) a commercially available total antibody ELISA, (2) BVDV-1 SN, and (3) BVDV-2 SN. Correlation between the ELISA and SN tests was estimated with a Spearman correlation coefficient. Higher BVDV ELISA sample-to-positive (S/P) ratios were positively correlated with higher BVDV-1 (ρ=0.809) and BVDV-2 (ρ=0.638) SN titers (P<0.0001), although the relationship was weaker when SN titers were <1:64. Higher BVDV-1 SN titers were also positively correlated with higher BVDV-2 SN titers (ρ=0.708; P<0.0001). The correlation between ELISA S/P ratios and SN titers was lower when calves were ≤2 months of age (ρ=0.344-0.566). Our results suggest that increased ELISA S/P ratios were associated with higher SN titers. We conclude that this BVDV antibody ELISA can be used as a surrogate for BVDV-1 and -2 SN titers when investigating genetic determinants of vaccine response, as long as samples are collected at 2 months of age or older.

Influence of standing estrus before an injection of GnRH during a beef cattle fixed-time AI protocol on LH release, subsequent concentrations of progesterone, and steriodogenic enzyme expression.

Beef cows that exhibit estrus before fixed-time AI have been reported to have increased pregnancy success and increased concentrations of progesterone during the subsequent estrous cycle. Therefore, these experiments were conducted to evaluate if initiation of standing estrus before an injection of GnRH during a fixed-time AI protocol affected LH pulses, subsequent concentrations of progesterone, and luteal steroidogenic enzyme expression. In Experiments 1 and 2, cows were treated with the CO-Synch protocol (100 µg GnRH day -9, 25 mg PGF(2α) day -2, and 100 µg GnRH day 0) and allotted to one of two treatments: 1) cows that initiated estrus before GnRH on day 0 (estrus; n = 5) or 2) cows that did not initiate estrus and were induced to ovulate by the GnRH on day 0 (no estrus; n = 5). In Experiment 1, blood samples were collected at 15-min intervals from 0 to 6 (bleed 1), 12 to 20 (bleed 2), 26 to 34 (bleed 3), and 40 to 48 (bleed 4) h after GnRH. Daily blood samples were collected for 17 d. Initiation of estrus before the GnRH injection had no effect on LH release or the pattern of progesterone increase; however, cows detected in estrus had overall increased (P = 0.002) concentrations of progesterone compared with cows not in estrus. In Experiment 2, estrus was detected with the HeatWatch system. Location and size of the ovulatory follicle was determined on day 0 by transrectal ultrasonography at time of injection with GnRH. Blood samples were collected on days 3, 4, 5, 7, and 9; luteal tissue was collected on day 10 (n = 4 estrus and n = 9 no estrus) from corpus luteum (CL) originating from similar-sized follicles (13.0 to 16.0 mm). Total cellular RNA was extracted, and relative mRNA levels were determined by real-time reverse transcription PCR and corrected for GAPDH. There was no effect of estrus on CL weight or concentrations of progesterone. In addition, there was no effect of estrus, follicle size, or CL weight on luteal expression of LH receptor, StAR, CYP11A1, or 3ßHSD. However, there was a correlation between follicle size and CL weight (P = 0.01; R(2) = 0.43); for every increase of 1 mm in follicle size, CL weight increased by 1.5 g. In summary, estrus did not influence release of LH, CL weight, progesterone concentrations, or expression of steriodogenic enzymes. However, as follicle size increased, CL weight increased; therefore, both follicle size and CL weight were associated with progesterone concentrations.

Gene expression profiling of chondrocytes from a porcine impact injury model.

OBJECTIVE: To identify differentially expressed genes between axially impacted and control articular cartilage taken from porcine patellae maintained in organ culture for 14 days. METHODS: Porcine patellae were impacted perpendicular to the articular surface to create an impact injury. Intact patellae (control and impacted) were maintained in culture for 14 days. Total RNA was then extracted from the articular cartilage beneath the impaction and used to prepare two Serial Analysis of Gene Expression (SAGE) libraries. Approximately 42,500 SAGE long tags were sequenced from the libraries. The expression of select genes was confirmed by quantitative real-time PCR analysis. RESULTS: Thirty-nine SAGE tags were significantly differentially expressed in the impacted and control libraries, representing 30 different annotated pig genes. These genes represented gene products associated with matrix molecules, iron and phosphate transport, protein biosynthesis, skeletal development, cell proliferation, lipid metabolism and the inflammatory response. Twenty-three of the 30 genes were down-regulated in the impacted library and five were up-regulated in the impacted library. Quantitative real-time PCR follow-up of four genes supported the results found with SAGE. CONCLUSION: We have identified 30 putative genes differentially expressed in a porcine impact injury model and validated these findings for four of these genes using real-time PCR. Results using this impact injury model have contributed further evidence that damaged chondrocytes may de-differentiate into fibroblast-like cells and proliferate in an attempt to repair themselves. Additional work is underway to study these genes in further detail at earlier time points to provide a more complete story about the fate of chondrocytes in articular cartilage following an injury.

Identification of a QTL on BTA20 affecting susceptibility to Mycobacterium avium ssp. paratuberculosis infection in US Holsteins.

The objective of this study was to identify QTL affecting susceptibility to Mycobacterium paratuberculosis infection in US Holsteins. Twelve paternal half-sib families were selected for the study based on large numbers of daughters in production and limited relationships among sires. Serum and faecal samples from 4350 daughters of these 12 sires were obtained for disease testing. Case definition for an infected cow was an ELISA sample-to-positive ratio >/=0.25, a positive faecal culture or both. Three families were selected for genotyping based on a high apparent prevalence (6.8-10.4% infected cows), high faecal culture prevalence (46.2-52.9% positive faecal cultures) and large numbers of daughters tested for disease (264-585). DNA pooling was used to genotype cows, with an average of 159 microsatellites within each sire family. Infected cows (the positive pool) were matched with two of their non-infected herdmates in the same lactation (the negative pool) to control for herd and age effects. Eight chromosomal regions putatively linked with susceptibility to M. paratuberculosis infection were identified using a Z-test (P < 0.01). Significant results were more rigorously tested by individually genotyping cows with three to five informative microsatellites within 15 cM of the significant markers identified with the DNA pools. Probability of infection based on both diagnostic tests was estimated for each individual and used as the dependent variable for interval mapping. Based on this analysis, evidence for the presence of a QTL segregating within families on BTA20 was found (chromosome-wide P-value = 0.0319).

Effect of Mycobacterium paratuberculosis infection on production, reproduction, and health traits in US Holsteins.

Our objective was to estimate the effect of Mycobacterium paratuberculosis infection on milk, fat, and protein yield deviations, pregnancy rate, lactation somatic cell score, and projected total months in milk (productive life). A serum ELISA and fecal culture for M. paratuberculosis were performed on 4375 Holsteins in 232 DHIA herds throughout the US. Primarily first through third lactation cows (99% of total) were assayed for infection. Trait information (except productive life) was obtained for the lactation concurrent with disease tests. Productive life was total months in milk through a cow's life, which was projected if a cow was still milking. For most analyses, case definition for M. paratuberculosis infection was defined as either an ELISA S/P ratio>or=0.25 or a positive fecal culture for M. paratuberculosis or both. To determine if diagnostic test affected estimates, case definition was redefined to include only cows with ELISA S/P ratios>or=0.25 or only fecal culture-positive cows. Linear models were used to estimate effect of M. paratuberculosis infection on traits. M. paratuberculosis-infected cows (7.89% of cows) produced 303.9 kg less milk/lactation, 11.46 kg less fat/lactation, and 9.49 kg less protein/lactation (P

Genetic variation of Mycobacterium avium ssp. paratuberculosis infection in US Holsteins.

The objective of this study was to estimate genetic variability of Mycobacterium avium ssp. paratuberculosis infection in US Holsteins. Blood and fecal samples were collected primarily from daughters of 12 bulls in their second or third lactation. Routine disease testing of the sires documented that they were not infected. Herds without a "suspect" or positive ELISA (sample/positive ratio > or = 0.10) or positive fecal culture test were deleted from the data set. The remaining 4,603 cows from 238 herds and 46 sires were used to estimate heritability of M. paratuberculosis infection. Heritability was estimated with 3 Johne's disease diagnostic tests: 1) fecal culture alone, 2) serum antibody ELISA alone, and 3) both tests (combined) with a positive animal defined as all animals with either a positive fecal culture or ELISA test. Four statistical models were used to estimate heritability: 1) linear (ELISA), 2) threshold (fecal culture and combined), 3) ordered threshold (ELISA), and 4) bivariate linear-threshold (ELISA-fecal culture). A sire model and Bayesian approach using Markov chain Monte Carlo methods were used in each case. Heritability of infection based on the fecal culture test was 0.153 [posterior standard deviation (PSD) = 0.115]. Heritability with the ELISA was 0.159 (PSD = 0.090) with a linear model and 0.091 (PSD = 0.053) with an ordered threshold model. Heritability of the combined tests was 0.102 (PSD = 0.066). Heritability estimates of fecal culture and ELISA with the bivariate model varied slightly from estimates obtained with the univariate models (0.125 and 0.183, respectively), with a corresponding increase in precision (PSD = 0.096 and 0.082, respectively). This study demonstrates that exploitable genetic variation exists in dairy cattle for M. paratuberculosis infection susceptibility.

Identification of an ovulation rate QTL in cattle on BTA14 using selective DNA pooling and interval mapping.

Increased twinning incidence in beef cattle has the potential to improve production efficiency. However, phenotypic selection for twinning rate is difficult because of the trait's low heritability and the long time interval necessary to collect phenotypic records. Therefore, this trait and the correlated trait of ovulation rate are ideal candidates for marker-assisted selection. The objective of this study was to complete a genome-wide search for ovulation rate quantitative trait loci (QTL) in two related sire families. The families (paternal halfsib sires 839802 and 839803) were from a population of cattle selected for ovulation rate at the USDA Meat Animal Research Center, Clay Center, Nebraska. Putative ovulation rate QTL have previously been identified in the 839802 family on chromosomes 7 and 19; however, marker coverage in the original scan was not complete. This study fills the gaps in marker coverage of the earlier study by adding approximately 60 informative microsatellites to each sire family. Each family was genotyped using selective DNA pooling. Sons and daughters were included in either the high or low pool based on their estimated breeding value deviations from the mid-parent average (EBVMD) for ovulation rate. Approximately 40% (839802) and 26% (839803) of available progeny comprised the high and low pools combined. Pooled typing revealed possible associations (nominal P < 0.05) between ovulation rate and marker genotype for 11 and 15 microsatellites in the 839802 and 839803 families, respectively. Subsequent interval mapping strengthened support for the presence of an ovulation rate QTL on BTA14 (chromosome-wise P < 0.02).