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.

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.

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.

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.

Bone cement implantation syndrome.

Bone cement implantation syndrome (BCIS) is poorly understood. It is an important cause of intraoperative mortality and morbidity in patients undergoing cemented hip arthroplasty and may also be seen in the postoperative period in a milder form causing hypoxia and confusion. Hip arthroplasty is becoming more common in an ageing population. The older patient may have co-existing pathologies which can increase the likelihood of developing BCIS. This article reviews the definition, incidence, clinical features, risk factors, aetiology, pathophysiology, risk reduction, and management of BCIS. It is possible to identify high risk groups of patients in which avoidable morbidity and mortality may be minimized by surgical selection for uncemented arthroplasty. Invasive anaesthetic monitoring should be considered during cemented arthroplasty in high risk patients.