Effect of residual methane emission on physiological characteristics and carcass performance in Japanese Black cattle.

This study investigated the physiological characteristics and carcass performance associated with residual methane emissions (RME), and the effects of bull differences on CH4-related traits in Japanese Black cattle. Enteric methane (CH4) emissions from 156 Japanese Black cattle (111 heifers and 45 steers) were measured during early fattening using the sniffer method. Various physiological parameters were investigated to clarify the physiological traits between the high, middle, and low RME groups. CH4-related traits were examined to determine whether bull differences affected progeny CH4 emissions. Ruminal butyrate and NH3 concentrations were significantly higher in the high-RME group than in the low-RME group, whereas the propionate content was significantly higher in the low-RME group. Blood urea nitrogen, ß-hydroxybutyric acid, and insulin concentrations were significantly higher, and blood amino acids were lower in the high-RME group than in the other groups. No significant differences were observed in the carcass traits and beef fat composition between RME groups. CH4-related traits were significantly different among bull herds. Our results show that CH4-related traits are heritable, wherein bull differences affect progeny CH4 production capability, and that the above-mentioned rumen fermentations and blood metabolites could be used to evaluate enteric methanogenesis in Japanese Black cattle.

Assessing the impact of three feeding stages on rumen bacterial community and physiological characteristics of Japanese Black cattle.

In Japan, Japanese Black cattle, known for their exceptional meat quality owing to their abundant intramuscular fat, undergo a unique three-stage feeding system with varying concentrate ratios. There is limited research on physiological and rumen microbial changes in Japanese Black cattle during these stages. Therefore, this study aimed to examine Japanese Black steers in these three stages: early (T1, 12-14 months), middle (T2, 15-22 months), and late (T3, 23-30 months). The rumen bacteria of 21 cattle per phase was analyzed using 16S rRNA gene sequencing. Rumen bacterial diversity was significantly higher in T1, with a distinct distribution, than in T2 and T3. Specific phyla and genera were exclusive to each stage, reflecting the shifts in feed composition. Certain genera dominated each stage: T1 had Flexilinea, Streptococcus, Butyrivibrio, Selenomonas, and Kandleria; T2 had Bifidobacterium, Shuttleworthia, and Sharpea; and T3 had Acetitomaculum, Mycoplasma, Atopobium, and Howardella. Correlation analysis revealed significant associations between certain microbial populations and physiological parameters. These findings indicate that changes in energy content and feed composition are associated with physiological and ruminal alterations. This study may guide strategies to improve rumen health and productivity in Japanese Black cattle by modifying diets to specific fattening stages.

Gas chromatography-mass spectrometry-based quantitative method using tert-butyldimethylsilyl derivatization for plasma levels of free amino acids and related metabolites in Japanese Black cattle.

The quantification of amino acid and related metabolite levels is important for evaluating amino acid metabolism and function in animals. However, a useful quantitative method is not enough. In this study, we developed and validated tert-butyldimethylsilyl derivatization method using gas chromatography-mass spectrometry to quantify plasma levels of free amino acids and related metabolites in Japanese Black cattle. Of the 51 metabolites examined, 24, including 20 amino acids, one amine, and three keto acids, could be quantified. Compared with the trimethylsilyl derivatization method using gas chromatography-mass spectrometry, which has been used for untargeted metabolomic analysis, the present method had higher analytical reliability. This method is advantageous for assessing branched-chain amino acid (BCAA) metabolism because it enables the quantification of not only BCAA levels (valine, leucine, and isoleucine) but also their bioactive metabolite keto acid levels (2-ketoisovaleric acid, 2-ketoisocaproic acid, and 2-keto-3-methylvaleric acid) in the plasma. In addition, this method can quantify the plasma levels of not only tryptophan but also its bioactive metabolites kynurenine and serotonin. These results suggest that this quantitative method has the potential to further our understanding of amino acid metabolic processes and their functions in Japanese Black cattle.

Variation among individual beef cattle in methane-to-carbon dioxide ratio measured under on-farm conditions using the sniffer method.

This study proposed a method for measuring the methane (CH4 )/carbon dioxide (CO2 ) ratio from individual beef cattle under on-farm conditions and estimated the variance components of the CH4 /CO2 ratio. Gas measurements were conducted using 166 Japanese Black cattle group-housed in pens equipped with individual feed bins. The gas containing the animal's breath was measured individually after concentrate feeding by covering the feed bin with a sheet with sampling inlets. Measurements were performed six times (three consecutive days, twice daily) per individual. Most of the sampled gas contained more than 1000 ppm of the mean background-corrected CO2 , suggesting that the method proposed in this study successfully collected sufficient breath concentration to accurately measure the CH4 /CO2 ratio. The between-animal variance accounted for 31.7% of the total variance in the CH4 /CO2 ratio. The results showed that the gas collection method proposed in this study could be a useful tool for measuring the CH4 /CO2 ratio under on-farm conditions. The variance component obtained from this study will help to establish protocols for generating data for genetic evaluation and performing dietary experiments with sufficient statistical power.

Effects of crude protein and neutral detergent fiber percentages in the diet of Japanese Black steers on rumen fluid properties, blood biochemical properties, and carcass characteristics.

The effects of crude protein (CP) and neutral detergent fiber (NDF) percentages in the diet of Japanese Black steers on rumen fluid properties, blood biochemical properties, and carcass characteristics were examined. Twelve 13-month-old Japanese Black steers were used for this study and slaughtered at 30 months of age. Steers were assigned to a control group (n = 6) and test group (n = 6) and were fed a concentrate containing 12.9%-13.9% CP and 26.5%-29.8% NDF or 9.1%-9.6% CP and 29.9%-31.2% NDF, respectively. Lipopolysaccharide activity levels in rumen fluid were lower in the test group than in the control group. Plasma urea nitrogen concentration and activities of aspartate aminotransferase and γ-glutamyltransferase remained lower in the test group than in the control group. In contrast, plasma vitamin A concentrations remained higher in the test group than in the control group. Carcass characteristics did not significantly differ between the two groups. These results suggest that dietary CP and NDF percentages in feed for Japanese Black steers older than 13 months of age affected rumen fluid properties and blood biochemical properties, indicating a reduced load on the liver with a small effect on carcass characteristics.

Physiological roles and regulation of hepatic angiopoietin-like protein 3 in Japanese Black cattle (Bos taurus) during the fattening period.

Angiopoietin-like protein 3 (ANGPTL3) is expressed predominantly in the liver and plays a major role in regulating the circulating triglyceride and lipoprotein fraction concentrations by inhibiting lipoprotein lipase (LPL) activity. Given these physiological roles, ANGPTL3 may play an important role in metabolic changes related to fat accumulation during the fattening period in Japanese Black. This study aimed to reveal the physiological roles of hepatic ANGPTL3 in Japanese Black steers (Bos taurus) during the fattening period and investigate the regulatory effects of hepatic ANGPTL3. To investigate the gene expression and protein localization of ANGPTL3, 18 tissue samples were collected from tree male Holstein bull calves aged 7 wk. Biopsied liver tissues and blood samples were collected from 21 Japanese Black steers during the early (T1; 13 mo of age), middle (T2; 20 mo), and late fattening phases (T3; 28 mo). Relative mRNA expression, blood metabolite concentrations, hormone concentrations, growth, and carcass traits were analyzed. To identify the regulatory factors of hepatic ANGPTL3, primary bovine hepatocytes collected by two Holstein calves aged 7 wk were incubated with insulin, palmitate, oleate, propionate, acetate, or beta-hydroxybutyric acid (BHBA). The ANGPTL3 gene was most highly expressed in the liver, with minor expression in the renal cortex, lungs, reticulum, and jejunum in Holstein bull calves. In Japanese Black steers, relative ANGPTL3 mRNA expressions were less as fattening progressed, and blood triglyceride, total cholesterol, and nonesterified fatty acid (NEFA) concentrations increased. Relative ANGPTL8 and Liver X receptor alpha (LXRα) mRNA expressions decreased in late and middle fattening phases, respectively. Furthermore, relative ANGTPL3 mRNA expression was positively correlated with ANGPTL8 (r = 0.650; P < 0.01) and ANGPTL4 (r = 0.540; P < 0.05) in T3 and T1, respectively, and LXRα showed no correlation with ANGPTL3. Relative ANGTPL3 mRNA expression was negatively correlated with total cholesterol (r = -0.434; P < 0.05) and triglyceride (r = -0.645; P < 0.01) concentrations in T3 and T1, respectively; There was no significant correlation between ANGTPL3 and carcass traits. Relative ANGTPL3 mRNA expression in cultured bovine hepatocytes was downregulated in oleate treatment. Together, these findings suggest that ANGPTL3 downregulation in late fattening phases is associated with the changes in lipid metabolism.

The role of angiopoietin-like protein 3 (ANGPTL3) in various animal species under different physiological conditions remains largely unknown. We evaluated the physiological roles of hepatic ANGPTL3 in Japanese Black steers (Bos taurus) during the fattening period and investigated the expressional regulation of ANGPTL3 in bovine hepatocytes. Relative ANGPTL3 mRNA expression decreased late in the fattening phases. Relative ANGPTL3 mRNA expression was positively correlated with ANGPTL4 and ANGPTL8 and was negatively correlated with blood triglyceride concentrations in early fattening phases. Relative ANGPTL3 mRNA expression in cultured bovine hepatocytes was downregulated in oleate treatment. Fatty acids may influence ANGPTL3 expression in cultured bovine hepatocytes through possible regulatory factors. Our findings suggest that the physiological roles of ANGPTL3 are associated with the changes of lipid metabolism during the fattening period, and the ANGPTL family seem to be associated with blood lipid metabolites.

Physiological responses and adaptations to high methane production in Japanese Black cattle.

In this study, using enteric methane emissions, we investigated the metabolic characteristics of Japanese Black cattle. Their methane emissions were measured at early (age 13 months), middle (20 months), and late fattening phases (28 months). Cattle with the highest and lowest methane emissions were selected based on the residual methane emission values, and their liver transcriptome, blood metabolites, hormones, and rumen fermentation characteristics were analyzed. Blood ß-hydroxybutyric acid and insulin levels were high, whereas blood amino acid levels were low in cattle with high methane emissions. Further, propionate and butyrate levels differed depending on the enteric methane emissions. Hepatic genes, such as SERPINI2, SLC7A5, ATP6, and RRAD, which were related to amino acid transport and glucose metabolism, were upregulated or downregulated during the late fattening phase. The above mentioned metabolites and liver transcriptomes could be used to evaluate enteric methanogenesis in Japanese Black cattle.

Changes in the liver transcriptome and physiological parameters of Japanese Black steers during the fattening period.

We investigated the physiological changes during the fattening period and production characteristics in Japanese Black steers bred and raised using the typical feeding system in Japan. Here, 21 Japanese Black steers aged 12 months were used, with experimental period divided into early (12-14 months of age), middle (15-22 months), and late fattening phases (23-30 months). The liver transcriptome, blood metabolites, hormones, and rumen fermentation characteristics were analyzed. Blood triglyceride and non-esterified fatty acid concentrations increased, whereas blood ketone levels decreased, with fattening phases. Blood insulin increased with fattening phases and was positively correlated with carcass weight and marbling in late fattening phases. Rumen fermentation characteristics showed high propionate levels and low butyrate levels in late fattening phases, likely due to increased energy intake. Genes related to glucose metabolism, such as SESN3, INSR, LEPR, and FOXO3, were down-regulated in late fattening phases. Genes related to lipid metabolism, such as FABP4, were up-regulated, whereas FADS1 and FADS2 were down-regulated. These findings suggest that the physiological changes resulted from changes in the energy content and composition of diets. Liver metabolism changed with changes in fat metabolism. Insulin was strongly associated with physiological changes and productivity in Japanese Black cattle.

Estimation of genetic parameters for carcass grading traits, image analysis traits, and monounsaturated fatty acids in Japanese Black cattle from Hyogo Prefecture.

Genetic parameters for carcass grading traits, image analysis traits, and monounsaturated fatty acid (MUFA) percentages were estimated in 29,942 Japanese Black cattle from Hyogo Prefecture. The analyzed traits included five carcass grading traits, two image analysis traits, fat area ratio and fineness index, and two MUFA traits, one measured in intermuscular fat using near-infrared spectroscopy (NIRS) and the other in intramuscular fat using gas chromatography (GC). The heritability estimates of image analysis traits and MUFA were moderate to high, ranging from 0.395 to 0.740, and it was considered that they could be improved simultaneously with carcass grading traits because no severe genetic antagonism was observed. Although the heritability of the NIRS-based intermuscular MUFA was slightly lower than that of the GC-based intramuscular MUFA, the genetic correlation between the two methods was as high as 0.804. These results indicate that the NIRS method can be used as an alternative evaluation procedure to predict MUFA in intramuscular fat in the longissimus muscle.

Changes in the predicted function of the rumen bacterial community of Japanese Black beef cattle during the fattening stages according to Kyoto Encyclopedia of Genes and Genomes (KEGG) analyses.

We investigated changes in the predicted functions of the rumen bacterial community in Japanese Black beef cattle during fattening. Nine cattle were fed a high-concentrate diet during the early, middle, and late fattening stages consecutively (10-14, 15-22, and 23-30 months of age, respectively). The rumen fluid and solid samples collected at each stage were subjected to sequencing analyses. The sequencing results were clustered and classified into operational taxonomic units (OTUs). Representative sequences and a raw counting table for each OTU were submitted to the Piphillin website. The predicted functions were revealed by the Kyoto Encyclopedia of Genes and Genomes database as the ratio of the total sequence. In the early stage, "Biosynthesis of secondary metabolites" was significantly higher in the fluid fraction than in the solid fraction. "Two-component system" in the middle stage was significantly lower and "Purine metabolism" in the late stage was significantly higher in the fluid fraction than those in the solid fraction. The fluid fraction was significantly correlated with acetic acid, propionic acid, and bacterial metabolism, such as "Biosynthesis of secondary metabolites" and "Sugar metabolism." Moreover, the solid fraction was correlated with "Purine metabolism" and "Biosynthesis of secondary metabolism". These results suggest that the rumen bacterial community in Japanese Black beef cattle adapts to changes in rumen conditions by altering their functions in response to a long-term high-grain diet.

Comparison of pH and bacterial communities in the rumen and reticulum during fattening of Japanese Black beef cattle.

We used castrated and fistulated Japanese Black beef cattle (n = 9) to measure the pH and bacterial communities in the rumen liquid, rumen solid, and reticulum liquid during early, middle, and late fattening stages (10-14, 15-22, and 23-30 months of age, respectively). The pH was measured in the rumen and reticulum during the last 13 days of each fattening stage and was significantly lower in the rumen at the early and middle fattening stage and in the reticulum during the late stage. Sequencing analysis indicated similar bacterial compositions in the rumen and reticulum liquid fractions and stability of bacterial diversity in the rumen and reticulum liquid fractions and rumen solid fraction. By contrast, major operational taxonomic units (OTUs), such as Ruminococcus bromii strain ATCC 27255 (OTU1, OTU10, and OTU15), were differently correlated to the fermentation parameters among the rumen and reticulum liquid fractions. Therefore, the long-term feeding of Japanese Black beef cattle with a high-concentrate diet might reverse the trend of pH in the rumen and reticulum during the late fattening stage, and the bacterial communities adapted to changes in fermentation by preserving their diversity throughout fattening.

Long-term high-grain diet alters ruminal pH, fermentation, and epithelial transcriptomes, leading to restored mitochondrial oxidative phosphorylation in Japanese Black cattle.

To increase intramuscular fat accumulation, Japanese Black beef cattle are commonly fed a high-grain diet from 10 to 30 months of age. Castrated and fistulated cattle (n = 9) were fed a high-concentrate diets during the early, middle, and late stages consecutively (10-14, 15-22, 23-30 months of age, respectively). Ruminal pH was measured continuously, and rumen epithelium and fluid samples were collected on each stage. The 24-h mean ruminal pH during the late stage was significantly lower than that during the early stage. Total volatile fatty acid (VFA) and lactic acid levels during the late stage were significantly lower and higher, respectively, than those during the early and middle stages. In silico analysis of differentially expressed genes showed that "Oxidative Phosphorylation" was the pathway inhibited most between the middle and early stages in tandem with an inhibited upstream regulator (PPARGC1A, also called PGC-1α) but the most activated pathway between the late and middle stages. These results suggest that mitochondrial dysfunction and thereby impaired cell viability due to acidic irritation under the higher VFA concentration restored stable mitochondrial oxidative phosphorylation and cell viability by higher lactic acid levels used as cellular oxidative fuel under a different underlying mechanism in subacute ruminal acidosis.

Long-term high-grain diet altered the ruminal pH, fermentation, and composition and functions of the rumen bacterial community, leading to enhanced lactic acid production in Japanese Black beef cattle during fattening.

To increase intramuscular fat accumulation, Japanese Black cattle are commonly fed a high-grain diet from 10 to 30 months of age although it can result in the abnormal accumulation of organic acids in the rumen. We explored the effect of long-term high-concentrate diet feeding on ruminal pH and fermentation, and its effect on the rumen bacterial community in Japanese Black beef cattle during a 20-month fattening period. Nine castrated and fistulated Japanese Black beef cattle were housed with free access to food and water throughout the study period (10-30 months of age). The fattening stages included Early, Middle, and Late stages (10-14, 15-22, and 23-30 months of age, respectively). Cattle were fed high-concentrate diets for the experimental cattle during fattening. The body weight of the cattle was 439 ± 7.6, 561 ± 11.6, and 712 ± 18.5 kg (mean ± SE) during the Early, Middle, and Late stages, respectively. Ruminal pH was measured continuously during the final 7 days of each stage, and rumen fluid and blood samples were collected on day 4 (fourth day during the final 7 days of the pH measurements). The 24-h mean ruminal pH during the Late stage was significantly lower than that during the Early stage. Total volatile fatty acid (VFA) during the Late stage was significantly lower than during the Early and Middle stages, but no changes were noted in individual VFA components. The lactic acid concentration during the Late stage was significantly higher than that during the Early and Middle stages. The bacterial richness indices decreased significantly during the Late stage in accordance with the 24-h mean ruminal pH. Among the 35 bacterial operational taxonomic units (OTUs) shared by all samples, the relative abundances of OTU8 (Family Ruminococcaceae) and OTU26 (Genus Butyrivibrio) were positively correlated with the 24-h mean ruminal pH. Total VFA concentration was negatively correlated with OTU167 (Genus Intestinimonas), and lactic acid concentration was correlated positively with OTU167 and OTU238 (Family Lachnospiraceae). These results suggested that long-term high-grain diet feeding gradually lowers ruminal pH and total VFA production during the Late fattening stage. However, the ruminal bacterial community adapted to feeding management and the lower pH during the Late stage by preserving their diversity or altering their richness, composition, and function, to enhance lactic acid production in Japanese Black beef cattle.

Effects of an increased concentrate diet on rumen pH and the bacterial community in Japanese Black beef cattle at different fattening stages.

This study evaluated the effects of increasing the proportion of concentrate in the diet on the rumen pH and bacterial community in Japanese Black beef cattle at different fattening stages. Six rumen-cannulated beef cattle were studied in the middle (Mid group, n=3, age 21-22 months) and late (Late group, n=3, age 31 months) fattening stages. The cattle were fed rice straw with control (CON period) or high-concentrate (HC period) diets for 14 consecutive days in each period. Rumen pH was measured continuously and the rumen fluids were collected on the last day of each period. The 24-hr mean and minimum rumen pH in the Mid group were significantly (P<0.05) lower during the HC period compared with the CON period, whereas those in the Late group were continuously low during both periods. In the Late group, the ruminal volatile fatty acid and lactic acid concentrations were significantly (P<0.05) higher during the HC period. During the HC period, the proportions of Prevotella and Caloramator were significantly (P<0.05) higher and lower, respectively, in the Mid group. From these findings, significant changes in the rumen pH and bacterial community induced by dietary changes were mainly observed in the Mid group. Therefore, the ruminal fermentative function in response to a higher concentrate diet might adapt differently in Japanese Black beef cattle at the two different fattening stages.