Quantification of cytosol and membrane proteins in rumen epithelium of sheep with low or high CH4 emission phenotype.

BACKGROUND: Ruminant livestock are a major contributor to Australian agricultural sector carbon emissions. Variation in methane (CH4) produced from enteric microbial fermentation of feed in the reticulo-rumen of sheep differs with different digestive functions. METHOD: We isolated rumen epithelium enzymatically to extract membrane and cytosol proteins from sheep with high (H) and low (L) CH4 emission. Protein abundance was quantified using SWATH-mass spectrometry. RESULTS: The research found differences related to the metabolism of glucose, lactate and processes of cell defence against microbes in sheep from each phenotype. Enzymes in the methylglyoxal pathway, a side path of glycolysis, resulting in D-lactate production, differed in abundance. In the H CH4 rumen epithelium the enzyme hydroxyacylglutathione hydrolase (HAGH) was 2.56 fold higher in abundance, whereas in the L CH4 epithelium lactate dehydrogenase D (LDHD) was 1.93 fold higher. Malic enzyme 1 which converts D-lactate to pyruvate via the tricarboxylic cycle was 1.57 fold higher in the L CH4 phenotype. Other proteins that are known to regulate cell defence against microbes had differential abundance in the epithelium of each phenotype. CONCLUSION: Differences in the abundance of enzymes involved in the metabolism of glucose were associated with H and L CH4 phenotype sheep. Potentially this represents an opportunity to use protein markers in the rumen epithelium to select low CH4 emitting sheep.

Live animal predictions of carcass components and marble score in beef cattle: model development and evaluation.

Until recently, beef carcass payment grids were predominantly based on weight and fatness categories with some adjustment for age, defined as number of adult teeth, to determine the price received by Australian beef producers for slaughter cattle. With the introduction of the Meat Standards Australia (MSA) grading system, the beef industry has moved towards payments that account for intramuscular fat (IMF) content (marble score (MarbSc)) and MSA grades. The possibility of a payment system based on lean meat yield (LMY, %) has also been raised. The BeefSpecs suite of tools has been developed to assist producers to meet current market specifications, specifically P8-rump fat and hot standard carcass weight (HCW). A series of equations have now been developed to partition empty body fat and fat-free weight into carcass fat-free mass (FFM) and fat mass (FM) and then into flesh FFM (FleshFFM) and flesh FM (FleshFM) to predict carcass components from live cattle assessments. These components then predict denuded lean (kg) and finally LMY (%) that contribute to emerging market specifications. The equations, along with the MarbSc equation, are described and then evaluated using two independent datasets. The decomposition of evaluation datasets demonstrates that error in prediction of HCW (kg), bone weight (BoneWt, kg), FleshFFM (kg), FleshFM (kg), MarbSc and chemical IMF percentage (ChemIMF%) is shown to be largely random error (%) in evaluation dataset 1, though error for ChemIMF% was primarily slope bias (%) in evaluation dataset 1, and BoneWt had substantial mean bias (%) in evaluation dataset 2. High modelling efficiencies of 0.97 and 0.95 for predicting HCW for evaluation datasets 1 and 2, respectively, suggest a high level of accuracy and precision in the prediction of HCW. The new outputs of the model are then described as to their role in estimating MSA index scores. The modelling system to partition chemical components of the empty body into carcass components is not dependent on the base modelling system used to derive empty body FFM and FM. This can be considered a general process that could be used with any appropriate model of body composition.

Protein profiles of enzymatically isolated rumen epithelium in sheep fed a fibrous diet.

BACKGROUND: The rumen wall plays a major role in efficient transfer of digested nutrients in the rumen to peripheral tissues through the portal venous system. Some of these substrates are metabolised in the epithelium during this process. To identify the specific proteins involved in these processes, we used proteomic technologies. Protein extracts were prepared from ventral rumen tissue of six sheep fed a fibrous diet at 1.5× maintenance energy requirements. Using a newly developed method, we were able to enzymatically isolate the epithelial cells from underlying tissue layers, thus allowing cytosol and membrane fractions to be independently analysed using liquid chromatography tandem mass spectrometry (LC MS/MS). RESULTS: Using our procedure we identified 570 epithelial proteins in the Ovis aries sequence database. Subcellular locations were largely cytosolic (n = 221) and extracellular (n = 85). However, a quarter of the proteins identified were assigned to the plasma membrane or organelle membranes, some of which transport nutrients and metabolites. Of these 91 were transmembrane proteins (TMHMM), 27 had an N-terminal signal peptide (signalP) and TMHMM motif, 13 had a glycosylphosphatidylinositol (GPI) anchor and signalP sequence, 67 had beta (ß) strands or 17 ß strands and a transit peptide sequence, indicating the identified proteins were integral or peripheral membrane proteins. Subunits of the 5 protein complexes involved in mitochondrial cellular energy production were well represented. Structural proteins (15%), proteins involved in the metabolism of lipids and proteins (26%) and those with steroid or cytokine action were a feature of the proteome. CONCLUSION: Our research has developed a procedure to isolate rumen epithelium proteins from the underlying tissue layers so that they may be profiled using proteomic technologies. The approach improves the number of proteins that can be profiled that are specific to the epithelium of the rumen wall. It provides new insights into the proteins of structural and nutritional importance in the rumen epithelium, that carry out nutrient transport and metabolism, cell growth and signalling.

Across-Experiment Transcriptomics of Sheep Rumen Identifies Expression of Lipid/Oxo-Acid Metabolism and Muscle Cell Junction Genes Associated With Variation in Methane-Related Phenotypes.

Ruminants are significant contributors to the livestock generated component of the greenhouse gas, methane (CH4). The CH4 is primarily produced by the rumen microbes. Although the composition of the diet and animal intake amount have the largest effect on CH4 production and yield (CH4 production/dry matter intake, DMI), the host also influences CH4 yield. Shorter rumen feed mean retention time (MRT) is associated with higher dry matter intake and lower CH4 yield, but the molecular mechanism(s) by which the host affects CH4 production remain unclear. We integrated rumen wall transcriptome data and CH4 phenotypes from two independent experiments conducted with sheep in Australia (AUS, n = 62) and New Zealand (NZ, n = 24). The inclusion of the AUS data validated the previously identified clusters and gene sets representing rumen epithelial, metabolic and muscular functions. In addition, the expression of the cell cycle genes as a group was consistently positively correlated with acetate and butyrate concentrations (p < 0.05, based on AUS and NZ data together). The expression of a group of metabolic genes showed positive correlations in both AUS and NZ datasets with CH4 production (p < 0.05) and yield (p < 0.01). These genes encode key enzymes in the ketone body synthesis pathway and included members of the poorly characterized aldo-keto reductase 1C (AKR1C) family. Several AKR1C family genes appear to have ruminant specific evolution patterns, supporting their specialized roles in the ruminants. Combining differential gene expression in the rumen wall muscle of the shortest and longest MRT AUS animals (no data available for the NZ animals) with correlation and network analysis, we identified a set of rumen muscle genes involved in cell junctions as potential regulators of MRT, presumably by influencing contraction rates of the smooth muscle component of the rumen wall. Higher rumen expression of these genes, including SYNPO (synaptopodin, p < 0.01) and NEXN (nexilin, p < 0.05), was associated with lower CH4 yield in both AUS and NZ datasets. Unlike the metabolic genes, the variations in the expression of which may reflect the availability of rumen metabolites, the muscle genes are currently our best candidates for causal genes that influence CH4 yield.

Live animal assessments of rump fat and muscle score in Angus cows and steers using 3-dimensional imaging.

The objective of this study was to develop a proof of concept for using off-the-shelf Red Green Blue-Depth (RGB-D) Microsoft Kinect cameras to objectively assess P8 rump fat (P8 fat; mm) and muscle score (MS) traits in Angus cows and steers. Data from low and high muscled cattle (156 cows and 79 steers) were collected at multiple locations and time points. The following steps were required for the 3-dimensional (3D) image data and subsequent machine learning techniques to learn the traits: 1) reduce the high dimensionality of the point cloud data by extracting features from the input signals to produce a compact and representative feature vector, 2) perform global optimization of the signatures using machine learning algorithms and a parallel genetic algorithm, and 3) train a sensor model using regression-supervised learning techniques on the ultrasound P8 fat and the classified learning techniques for the assessed MS for each animal in the data set. The correlation of estimating hip height (cm) between visually measured and assessed 3D data from RGB-D cameras on cows and steers was 0.75 and 0.90, respectively. The supervised machine learning and global optimization approach correctly classified MS (mean [SD]) 80 (4.7) and 83% [6.6%] for cows and steers, respectively. Kappa tests of MS were 0.74 and 0.79 in cows and steers, respectively, indicating substantial agreement between visual assessment and the learning approaches of RGB-D camera images. A stratified 10-fold cross-validation for P8 fat did not find any differences in the mean bias ( = 0.62 and = 0.42 for cows and steers, respectively). The root mean square error of P8 fat was 1.54 and 1.00 mm for cows and steers, respectively. Additional data is required to strengthen the capacity of machine learning to estimate measured P8 fat and assessed MS. Data sets for and continental cattle are also required to broaden the use of 3D cameras to assess cattle. The results demonstrate the importance of capturing curvature as a form of representing body shape. A data-driven model from shape to trait has established a proof of concept using optimized machine learning techniques to assess P8 fat and MS in Angus cows and steers.

One-hour portable chamber methane measurements are repeatable and provide useful information on feed intake and efficiency.

Feed intake (FI), live weight (LW), and ADG were recorded over 31 d in ninety-six 12-month-old ewes (progeny of 4 sires) given ad libitum access to chaffed lucerne/cereal hay. Methane (CH) and CO emissions of each ewe were measured for 40 to 60 min in portable accumulation chambers (PAC) and in respiration chambers (RC) over 22 h. Testing in RC increased the variability of FI on the test day and depressed the amount eaten from an average of 1,384 to 1,062 g/d; FI depression increased by 0.63 ± 0.24 percentage points for every kilogram of additional LW. Repeatabilities of PAC measurements were 0.76 (CH) and 0.81 (CO). After adjusting for LW and ADG, repeatabilities were 0.47 (PAC CH) and 0.43 (PAC CO). Daily FI measurements had similar repeatability (0.76 before and 0.42 after adjustment for LW and ADG). The PAC measurements were highly correlated with mean 31-d FI ( = 0.81 for both CH and CO). After adjustment for LW and ADG, PAC measurements were moderately correlated with residual feed intake (RFI; = 0.37 for CH, 0.31 for CO). The CH:CO ratio was also significantly correlated with mean 31-d FI ( = 0.52). After most of the ewes had given birth and raised lambs, repeat PAC measurements were available for 91 of the ewes at 2 years of age (with ad libitum access to the same feed). Correlations with the 2012 PAC measurements were 0.64 (CH) and 0.75 (CO). After adjusting 2014 PAC measurements for LW, correlations with RFI in 2012 were 0.34 (CH) and 0.33 (CO), with a clear relationship between sire means for RFI in 2012 and PAC CH adjusted for LW in 2014. These results suggest that PAC tests under similar feeding conditions are repeatable over an extended time period and can provide useful information on FI and feed efficiency as well as methane emissions. Analyses of RC measurements might need to consider FI depression.

Benefits of including methane measurements in selection strategies.

Estimates of genetic/phenotypic covariances and economic values for slaughter weight, growth, feed intake and efficiency, and three potential methane traits were compiled to explore the effect of incorporating methane measurements in breeding objectives for cattle and meat sheep. The cost of methane emissions was assumed to be zero (scenario A), A$476/t (based on A$14/t CO equivalent and methane's 100-yr global warming potential [GWP] of 34; scenario B), or A$2,580/t (A$30/t CO equivalent combined with methane's 20-yr GWP of 86; scenario C). Methane traits were methane yield (MY; methane production divided by feed intake based on measurements over 1 d in respiration chambers) or short-term measurements of methane production adjusted for live weight (MPadjWt) in grazing animals, e.g., 40-60 min measurements in portable accumulation chambers (PAC) on 1 or 3 occasions, or measurements for 1 wk using a GreenFeed Emissions Monitor (GEM) on 1 or 3 occasions. Feed costs included the cost of maintaining the breeding herd and growth from weaning to slaughter. Sheep were assumed to be grown and finished on pasture (A$50/t DM). Feed costs for cattle included 365 d on pasture for the breeding herd and averages of 200 d postweaning grow-out on pasture and 100 d feedlot finishing. The greatest benefit of including methane in the breeding objective for both sheep and cattle was as a proxy for feed intake. For cattle, 3 GEM measurements were estimated to increase profit from 1 round of selection in scenario A (no payment for methane) by A$6.24/animal (from A$20.69 to A$26.93) because of reduced feed costs relative to gains in slaughter weight and by A$7.16 and A$12.09/animal, respectively, for scenarios B and C, which have payments for reduced methane emissions. For sheep, the improvements were more modest. Returns from 1 round of selection (no methane measurements) were A$5.06 (scenario A), A$4.85 (scenario B), and A$3.89 (scenario C) compared to A$5.26 (scenario A), A$5.12 (scenario B), and A$4.72 (scenario C) for 1 round of selection with 3 PAC measurements. Including MY in the selection index was less profitable because it did not reduce feed costs relative to weight gain. Consequently, for strategies measuring MY but not MPadjWt (and with no estimate of feed intake in the production environment), proportionately greater emphasis was placed on increasing slaughter weight, and as a result, the decreases in methane emissions per animal and per unit of feed intake were smaller than for strategies that measured MPadjWt.

Comparison of repeated measurements of methane production in sheep over 5 years and a range of measurement protocols.

Emissions of 710 ewes at pasture were measured for 1 h (between 09:00-16:30 h) in batches of 15 sheep in portable accumulation chambers (PAC) after an overnight fast continuing until 2 h before measurement, when the sheep had access to baled hay for 1 h. The test was used to identify a group of 104 low emitters (I-Low) and a group of 103 high emitters (I-Hi) for methane emissions adjusted for liveweight (CHawt). The 207 ewes selected at the initial study were remeasured in 5 repeat tests from 2009 through 2014 at another location. The first repeat used the original measurement protocol. Two modified protocols, each used in 2 yr, drafted unfasted sheep on the morning of the test into a yard or holding paddock until measurement. Emissions of the I-Hi sheep were higher (102-112%) than I-Low sheep in all subsequent PAC tests, with statistical significance ( < 0.05) in 3 tests. Tests without overnight fasting were simpler to conduct and had repeatabilities of 51 to 60% compared with 31 and 43% for the initial and first repeat tests, respectively. After habituation to a diet fed at 20 g/kg liveweight, 160 of the 207 sheep were measured in respiration chambers (RC); 10 high (Hi-10) and 10 low (Low-10) sheep were chosen, representing extremes (top and bottom 6.25%) for methane yield (MY; g CH/kg DMI). The Hi-10 group emitted 14% more methane (adjusted for feed intake) in a follow-up RC test, but Low-10 and Hi-10 sheep differed in only 1 of the 5 PAC tests, when Hi-10 sheep emitted less CHawt than Low-10 sheep ( = 0.002) and tended to eat less in the feeding opportunity ( = 0.085). Compared with their weight on good pasture, Low-10 sheep were proportionately lighter than Hi-10 sheep in the relatively poor pasture conditions of the initial test. Sheep identified as low emitters by PAC tests using the initial protocol did not produce less CH (mg/min) when fed a fixed level of intake in RC. Correlations between estimates of an animal's CHawt measured in PAC and CH adjusted for feed intake in RC were quite low ( = 0-19%) and significant ( < 0.05) in only 1 test of unfasted sheep. With moderate repeatability over the 5 yr, PAC tests of CHawt could be a viable way to select for reduced emissions of grazing sheep. As well as exploiting any variation in MY, selecting for reduced CHawt in PAC could result in lower feed intake than expected for the animals' liveweight and might affect the diurnal feeding pattern. Further work is required on these issues.

Animal board invited review: genetic possibilities to reduce enteric methane emissions from ruminants.

Measuring and mitigating methane (CH4) emissions from livestock is of increasing importance for the environment and for policy making. Potentially, the most sustainable way of reducing enteric CH4 emission from ruminants is through the estimation of genomic breeding values to facilitate genetic selection. There is potential for adopting genetic selection and in the future genomic selection, for reduced CH4 emissions from ruminants. From this review it has been observed that both CH4 emissions and production (g/day) are a heritable and repeatable trait. CH4 emissions are strongly related to feed intake both in the short term (minutes to several hours) and over the medium term (days). When measured over the medium term, CH4 yield (MY, g CH4/kg dry matter intake) is a heritable and repeatable trait albeit with less genetic variation than for CH4 emissions. CH4 emissions of individual animals are moderately repeatable across diets, and across feeding levels, when measured in respiration chambers. Repeatability is lower when short term measurements are used, possibly due to variation in time and amount of feed ingested prior to the measurement. However, while repeated measurements add value; it is preferable the measures be separated by at least 3 to 14 days. This temporal separation of measurements needs to be investigated further. Given the above issue can be resolved, short term (over minutes to hours) measurements of CH4 emissions show promise, especially on systems where animals are fed ad libitum and frequency of meals is high. However, we believe that for short-term measurements to be useful for genetic evaluation, a number (between 3 and 20) of measurements will be required over an extended period of time (weeks to months). There are opportunities for using short-term measurements in standardised feeding situations such as breath 'sniffers' attached to milking parlours or total mixed ration feeding bins, to measure CH4. Genomic selection has the potential to reduce both CH4 emissions and MY, but measurements on thousands of individuals will be required. This includes the need for combined resources across countries in an international effort, emphasising the need to acknowledge the impact of animal and production systems on measurement of the CH4 trait during design of experiments.

Sire and liveweight affect feed intake and methane emissions of sheep confined in respiration chambers.

Daily methane production and feed intake were measured on 160 adult ewes, which were the progeny of 20 sires and 3 sire types (Merino, dual-purpose and terminal) from a genetically diverse flock. All animals were housed in individual pens and fed a 50/50 mix of chaffed lucerne and oaten hays at 20 g/kg liveweight (LW), with feed refusals measured for at least 10 days before the first of three 22-h measurements in respiration chambers (RC). Feed was withdrawn at 1600 h on the day before each RC test to encourage the ewes to eat the entire ration provided for them in the RC. After the first 1-day RC test, the sheep were returned to their pens for a day, then given a second 1-day RC test, followed by another day in their pens, then a third RC test. After all animals had been tested, they were ranked according to methane emissions adjusted for feed intake in the RC and on the previous day, enabling 10 low and 10 high methane animals to be chosen for repeat measurement. No variation between sires nor consistent effects of LW on feed eaten (%FE, expressed as per cent of feed offered) was evident in the 10 days before the first RC measurement. However, significant differences between sires (equivalent to an estimated heritability of 41%) were identified for %FE during the 2(nd) and 3(rd) days of RC testing (2 and 4 days after the initial RC test). The analysis of all data showed that methane emissions in the RC were related to feed intake on the day of testing and the two previous days (all P<0.0005). Before correcting for feed intake on previous days, there was some variation between sires in methane yield, equivalent to an estimated heritability of 9%. Correction for feed intake on the 2 previous days halved the residual variation, allowing other effects to be detected, including effects of LW, twins reared as singles, test batch, RC and test-day effects, but estimated sire variation fell to zero. In order to avoid potential biases, statistical models of methane emissions in the RC need to consider potential confounding factors, such as those identified as significant in this study.

Genetic and environmental variation in methane emissions of sheep at pasture.

A total of 2,600 methane (CH4) and 1,847 CO2 measurements of sheep housed for 1 h in portable accumulation chambers (PAC) were recorded at 5 sites from the Australian Sheep CRC Information Nucleus, which was set up to test leading young industry sires for an extensive range of current and novel production traits. The final validated dataset had 2,455 methane records from 2,279 animals, which were the progeny of 187 sires and 1,653 dams with 7,690 animals in the pedigree file. The protocol involved rounding up animals from pasture into a holding paddock before the first measurement on each day and then measuring in groups of up to 16 sheep over the course of the day. Methane emissions declined linearly (with different slopes for each site) with time since the sheep were drafted into the holding area. After log transformation, estimated repeatability (rpt) and heritability (h(2)) of liveweight-adjusted CH4 emissions averaged 25% and 11.7%, respectively, for a single 1-h measurement. Sire × site interactions were small and nonsignificant. Correlations between EBV for methane emissions and Sheep Genetics Australia EBV for production traits were used as approximations to genetic correlations. Apart from small positive correlations with weaning and yearling weights (r = 0.21-0.25, P < 0.05), there were no significant relationships between production trait and methane EBV (calculated from a model adjusting for liveweight by fitting separate slopes for each site). To improve accuracy, future protocols should use the mean of 2 (rpt = 39%, h(2) = 18.6%) or 3 (rpt = 48%, h(2) = 23.2%) PAC measurements. Repeat tests under different pasture conditions and time of year should also be considered, as well as protocols measuring animals directly off pasture instead of rounding them up in the morning. Reducing the time in the PAC from 1 h to 40 min would have a relatively small effect on overall accuracy and partly offset the additional time needed for more tests per animal. Field testing in PAC has the potential to provide accurate comparisons of animal and site methane emissions, with potentially lower cost/increased accuracy compared to alternatives such as SF6 tracers or open path lasers. If similar results are obtained from tests with different protocols/seasonal conditions, use of PAC measurements in a multitrait selection index with production traits could potentially reduce methane emissions from Australian sheep for the same production level.

Lack of association between allelic status and myostatin content in lambs with the myostatin g+6723G>A allele.

Lambs with the myostatin (MSTN) g+6723G>A mutation have a greater muscle mass, which is believed to be associated with reduced myostatin protein abundance. This experiment was designed to determine if differences in allelic frequency of the MSTN g+6723G>A mutation affected abundance of myostatin protein from birth to 24 wk of age. A Poll Dorset cross White Suffolk ram (MSTN A/G) was mated to 35 White Suffolk cross Border Leicester cross Merino ewes (MSTN A/G, n=21, and MSTN G/G, n=14). The progeny of these matings delivered 44 lambs with MSTN A/A (n=9), MSTN A/G (n=21), and MSTN G/G (n=14) genotypes. At approximately 1, 4, and 12 wk of age, a biopsy sample was collected and a blood sample was taken to measure the abundance of myostatin protein in muscle and plasma. At approximately 24 wk of age, the wether lambs were slaughtered to determine carcass characteristics and muscle samples were taken from the bicep femoris. The abundance of mature myostatin protein in muscle from 1 wk old lambs was less (P=0.05) in MSTN A/A and MSTN A/G compared with MSTN G/G lambs. However, at 4 and 24 wk the MSTN A/A lambs had a greater (P=0.04) abundance of myostatin protein compared with the MSTN A/G and MSTN G/G lambs. The abundance of mature myostatin did not differ between genotypes in plasma but the myostatin protein did increase as the lambs aged. At slaughter the MSTN A/A wether lambs had greater dressing percentages (P=0.04), shortloin (P=0.01), topside (P<0.001), and round (P=0.01) weights but did not differ in final BW or HCW (P>0.05). The MSTN A/A lambs had more muscle fibers (P=0.02) in the cross-section of LM between the 12th and 13th rib. The MSTN A/A lambs also had greater lean (P=0.002), less fat (P=0.009), and reduced organ (heart, liver, spleen, and kidneys) mass as determined by computed tomography scanning than MSTN G/G lambs. The results of this study demonstrated that lambs homozygous for the MSTN g+6723G>A mutation have changes in carcass characteristics (dressing and total lean), organ weights, and muscle fiber number. This may be due to reduced myostatin protein early in utero, but after 4 wk of age there was no difference in the abundance of mature myostatin protein in muscle or plasma among MSTN A/A, MSTN A/G, and MSTN G/G genotypes.

Myostatin allelic status interacts with level of nutrition to affect growth, composition, and myofiber characteristics of lambs.

The objective of this experiment was to determine if growth, carcass composition, and myofiber characteristics of lambs were affected by heterozygosity for a myostatin mutation (g+6723G>A) when offered differing allowances of feed administered with or without ractopamine. Heterozygote [MSTN A/G (n = 40)] and homozygote wildtype [MSTN G/G (n = 39)] castrate male lambs were individually fed ad libitum (HI; 1.8 × estimated ME(m)) or a restricted allowance (LO; 1.1 × estimated ME(m)) of a diet (191 g of CP/kg of DM and 12 MJ of ME/kg of DM), supplemented with (0.4 mg/kg of BW) or without the ß-adrenergic agonist ractopamine (RAC or NO RAC) for 47 d. The lambs were scanned by computed tomography at the beginning and completion of the feeding experiment to calculate composition of lean, fat, and bone in the carcass component of the body. The MSTN A/G HI intake lambs had significantly greater total daily carcass growth (P = 0.045) and loin eye depth (P = 0.022) and tended to have a greater daily growth of lean (P = 0.09) in the carcass, compared with MSTN G/G HI intake lambs. Conversely, MSTN A/G LO intake lambs tended to have less daily lean deposition (P = 0.09), significantly less total daily carcass growth (P = 0.045), and had a greater percentage of type IIX myofibers (P < 0.01) and total myofiber area (relative area) of type IIX myofibers (P = 0.013). The inclusion of RAC increased final BW (P = 0.03) and ADG (P = 0.02), percentage of type IIC (P < 0.001) and IIA (P = 0.012) myofibers, cross-sectional area of types I (P = 0.04) and IIAX (P = 0.04) fibers, and the relative area of type IIC (P = 0.003) and IIA (P = 0.01) myofibers in the LM. The experiment demonstrated that including RAC in the diet of lambs increased final BW and ADG, but not HCW, and increased proportion of type IIC and IIA myofibers and cross-sectional area of type I and IIAX myofibers. Our data suggest that RAC and the heterozygous myostatin mutation act together to increase growth of muscle on a high plane of nutrition. The experiment also demonstrated that poor nutritional background of lambs heterozygous for the myostatin mutation may negatively influence their growth rates and myofiber characteristics.

Effects of various adjuvants on efficacy of a vaccine against Streptococcus bovis and Lactobacillus spp in cattle.

OBJECTIVE: To determine efficacy of vaccines incorporating QuilA, alum, dextran combined with mineral oil, or Freund adjuvant for immunization of feedlot cattle against Streptococcus bovis and Lactobacillus spp. ANIMALS: 24 steers housed under feedlot conditions. PROCEDURE: Steers were randomly assigned to 4 experimental groups and a control group. Animals in experimental groups were inoculated on days 0 and 26 with vaccines containing Freund adjuvant (FCA), QuilA, dextran combined with mineral oil (Dex), or alum as adjuvant. Serum anti-S bovis and anti-Lactobacillus IgG concentrations were measured, along with fecal pH, ruminal fluid pH, and number of S bovis and Lactobacillus spp in ruminal fluid. RESULTS: Throughout the study, serum anti-S bovis and anti-Lactobacillus IgG concentrations for animals in the Dex, QuilA, and alum groups were similar to or significantly higher than concentrations for animals in the FCA group. Serum anti-S bovis and anti-Lactobacillus IgG concentrations were significantly increased on days 26 through 75 in all 4 experimental groups, and there was a linear relationship between anti-S bovis and anti-Lactobacillus IgG concentrations. For animals in the QuilA and Dex groups, mean pH of feces throughout the period of experiment were significantly higher and numbers of S bovis and Lactobacillus spp in ruminal fluid on day 47 were significantly lower than values for control cattle. CONCLUSIONS AND CLINICAL RELEVANCE: Results suggest that immunization of feedlot steers against S bovis and Lactobacillus spp with vaccines incorporating Freund adjuvant, QuilA, dextran, or alum as an adjuvant effectively induced high, long-lasting serum anti-S bovis and anti-Lactobacillus IgG concentrations. Of the adjuvants tested, dextran may be the most effective.

Hind-limb protein metabolism and calpain system activity influence post-mortem change in meat quality in lamb.

This study is concerned with the rate of protein turnover in the hind limb muscle bed of intact lambs, the activity of calpain proteolytic system in the M. biceps femoris, and subsequent rates of myofibre breakdown and tenderisation in the M. longissimus dorsi. Feed restriction increased protein degradation in hind-limb muscle of lambs (p<0.1), with a concominant decrease in the extractable activity of calpastatin (p<0.01), the endogenous inhibitor of calpain. IGF-1 analog treatment decreased both protein degradation and assayed µ-calpain activity (p<0.05) with no effect on the activity of calpastatin. ß-Agonist treatment increased hind-limb protein synthesis (p<0.01), calpastatin activity (p<0.1) and decreased (p<0.01) µ-calpain activity, but did not effect protein degradation. Significant correlations were observed between Myofibril Fragmentation Index (MFI) values during post-mortem storage and initial post-slaughter calpastatin activity at days 3 (r=-0.34, p<0.1), 5 (r=-0.58, p<0.01) and 9 (r=-0.58, p<0.1), and µ-calpain activity at days 5 (r=0.35, p<0.1) and 9 (r=0.41, p<0.05). However, stronger correlations were observed between the ratio of µ-calpain to calpastatin, an estimate of potential µ-calpain proteolytic activity, and the rate of myofibril fragmentation (r=0.75, p<0.001) and tenderisation (r=-0.64, p<0.01) during aging. These results are consistent with the calpain system being the major proteolytic system involved in myofibril fragmentation and hence aging-related tenderisation of meat.

Prediction of the chemical composition of lamb carcasses from multi-frequency impedance data.

Multi-frequency bioimpedance analysis (MFBIA) was used to determine the impedance, reactance and resistance of 103 lamb carcasses (17.1-34.2 kg) immediately after slaughter and evisceration. Carcasses were halved, frozen and one half subsequently homogenized and analysed for water, crude protein and fat content. Three measures of carcass length were obtained. Diagonal length between the electrodes (right side biceps femoris to left side of neck) explained a greater proportion of the variance in water mass than did estimates of spinal length and was selected for use in the index L2/Z to predict the mass of chemical components in the carcass. Use of impedance (Z) measured at the characteristic frequency (Zc) instead of 50 kHz (Z50) did not improve the power of the model to predict the mass of water, protein or fat in the carcass. While L2/Z50 explained a significant proportion of variation in the masses of body water (r(2) 0.64), protein (r(2) 0.34) and fat (r(2) 0.35), its inclusion in multi-variate indices offered small or no increases in predictive capacity when hot carcass weight (HCW) and a measure of rib fat-depth (GR) were present in the model. Optimized equations were able to account for 65-90% of the variance observed in the weight of chemical components in the carcass. It is concluded that single frequency impedance data do not provide better prediction of carcass composition than can be obtained from measures of HCW and GR. Indices of intracellular water mass derived from impedance at zero frequency and the characteristic frequency explained a similar proportion of the variance in carcass protein mass as did the index L2/Z50.

Insulin-like growth factor I inhibits degradation and improves retention of protein in hindlimb muscle of lambs.

We infused recombinant human insulin-like growth factor I (IGF-I) for 4 h at 12.3 micrograms.h-1.kg live weight-1 directly into the left femoral artery and measured the rates of synthesis, degradation, and gain of protein by the treated and contralateral limbs of well-fed (n = 8), feed-restricted (n = 10), and fasted (n = 9) castrated male lambs. Reducing feed intake decreased net protein gain of hindlimb muscle, reduced hindlimb glucose uptake, and lowered arterial plasma concentrations of IGF-I, insulin, glucose, phenylalanine, tyrosine, and isoleucine. The effect of nutrition on IGF-binding proteins (IGFBP) was generally small; IGFBP-2 was more abundant in fasted lambs. Infusion of IGF-I into the left femoral artery increased plasma levels of IGF-I 2- to 4-fold in the left femoral vein and by 1.5- to 3-fold in the artery and right femoral vein. In the treated limb, IGF-I reduced protein degradation, increased protein gain, and increased glucose uptake without altering blood flow or oxygen uptake, regardless of feed intake. Systemically, IGF-I reduced plasma insulin, phenylalanine, tyrosine, isoleucine, and leucine in all nutrition groups. Plasma IGFBP-3 was increased by 4 h of IGF-I treatment in fasted but not in fed lambs. In fed but not fasted lambs, IGF-I increased blood glucose concentration. Effects of IGF-I on protein metabolism in the contralateral limb were affected by nutrition, generally more so in fasted than in unrestricted fed lambs.

Humoral responses to a multivalent vaccine in age-matched lambs of different bodyweight and nutrition.

K-agglutination, pilus-enzyme-linked immunosorbent assay (ELISA) and outer membrane protein-ELISAs were used to assess humoral responses after vaccination with a commercial, multivalent, ovine foot rot vaccine (Dichelobacter nodosus whole cells) in three groups of nine-month-old lambs of markedly different bodyweight, nutritional history and dietary protein supply. Mean bodyweights of lambs in low (L), medium (M) and high (H) bodyweight/nutrition groups were 22, 32 and 48 kg, respectively, at the time of vaccination. Few significant differences in humoral responses to vaccine antigens were found between groups. However, lambs in group H tended to have lower levels of antibody to a greater number of component antigens than did lambs in the other groups. These results suggest that low bodyweight due to poor nutrition is unlikely to affect the response of sheep to multivalent foot rot vaccines.

Protein synthesis and degradation in the mammary gland of lactating goats.

Lactating goats were given a close arterial infusion of [1-14C]leucine and [4,5-3H]4-methyl-2-oxopentanoic acid into one half of the mammary gland at 2-3 weeks and 34-39 weeks after kidding. Rates of protein synthesis, degradation and net output were determined from measurements of arteriovenous difference and blood flow using a model of leucine metabolism previously developed for muscle (Oddy & Lindsay, 1986). Protein leucine output in milk (Y mumol/min) correlated well with the difference between synthesis and degradation (X mumol/min) derived from the model: Y = 1.30 + 1.24X (r2 = 0.9; n = 9, P less than 0.01). There was substantial synthesis and degradation of protein within the mammary gland. Although only an approximate value could be obtained for the partitioning of protein synthesis and degradation between tissue and milk proteins, there was evidence of appreciable turnover of both. There was no significant difference between mammary leucine and protein metabolism in early and late lactation other than that imparted by a greater mass of mammary tissue in early lactation, although there was a tendency for greater oxidation of leucine in late lactation.