A rumen virosphere with implications of contribution to fermentation and methane production, and endemism in cattle breeds and individuals.

Viruses have a potential to modify the ruminal digestion via infection and cell lysis of prokaryotes, suggesting that viruses are related to animal performance and methane production. This study aimed to elucidate the genome-based diversity of rumen viral communities and the differences in virus structure between individuals and cattle breeds and to understand how viruses influence on the rumen. To these ends, a metagenomic sequencing of virus-like particles in the rumen of 22 Japanese cattle, including Japanese Black (JB, n = 8), Japanese Shorthorn (n = 2), and Japanese Black sires × Holstein dams crossbred steers (F1, n = 12) was conducted. Additionally, the rumen viromes of six JB and six F1 that were fed identical diets and kept in a single barn were compared. A total of 8,232 non-redundant viral genomes (≥5-kb length and ≥50% completeness), including 982 complete genomes, were constructed, and rumen virome exhibited lysogenic signatures. Furthermore, putative hosts of 1,223 viral genomes were predicted using tRNA and clustered regularly interspaced short palindromic repeat (CRISPR)-spacer matching. The genomes included 1 and 10 putative novel complete genomes associated with Fibrobacter and Ruminococcus, respectively, which are the main rumen cellulose-degrading bacteria. Additionally, the hosts of 22 viral genomes, including 2 complete genomes, were predicted as methanogens, such as Methanobrevibacter and Methanomethylophilus. Most rumen viruses were highly rumen and individual specific and related to rumen-specific prokaryotes. Furthermore, the rumen viral community structure was significantly different between JB and F1 steers, indicating that cattle breed is one of the factors influencing the rumen virome composition.IMPORTANCEHere, we investigated the individual and breed differences of the rumen viral community in Japanese cattle. In the process, we reconstructed putative novel complete viral genomes related to rumen fiber-degrading bacteria and methanogen. The finding strongly suggests that rumen viruses contribute to cellulose and hemicellulose digestion and methanogenesis. Notably, this study also found that rumen viruses are highly rumen and individual specific, suggesting that rumen viruses may not be transmitted through environmental exposure. More importantly, we revealed differences of viral communities between JB and F1 cattle, indicating that cattle breed is a factor that influences the establishment of rumen virome. These results suggest the possibility of rumen virus transmission from mother to offspring and its potential to influence beef production traits. These rumen viral genomes and findings provide new insights into the characterizations of the rumen viruses.

Identification of 146 Metagenome-assembled Genomes from the Rumen Microbiome of Cattle in Japan.

The rumen contains a complex microbial ecosystem that degrades plant materials, such as cellulose and hemicellulose. We herein reconstructed 146 nonredundant, rumen-specific metagenome-assembled genomes (MAGs), with ≥50% completeness and <10% contamination, from cattle in Japan. The majority of MAGs were potentially novel strains, encoding various enzymes related to plant biomass degradation and volatile fatty acid production. The MAGs identified in the present study may be valuable resources to enhance the resolution of future taxonomical and functional studies based on metagenomes and metatranscriptomes.

Effects of calcium salt of linseed oil fatty acid with different oil adsorbents on in vitro gas production and ruminal fermentation characteristics.

We investigated the effects of supplementary calcium salt of fatty acid (CSFA) from linseed oil with different oil adsorbents on in vitro gas production and rumen fermentation characteristics in barley substrate condition. A non-supplementation treatment (CONT) and treatments of six products, CSFA without oil adsorbent (2.1 fatty acid/Ca molar ratio) and CSFAs with silica gel, zeolite, bentonite, diatomite, and vermiculite (2.8 fatty acid/Ca molar ratio), were prepared. The supplementary 2% and 4% CSFA with silica gel (+S) in the substrate reduced CH4 production 56% and 79%, respectively, compared with that in CONT (p < 0.01). The products, except for +S, did not decrease CH4 production. The dry matter (DM) disappearance in CSFAs with oil adsorbents was lower than that in the CSFA without oil adsorbent (74.8%-77.3% vs. 79.3%, p < 0.01), and crude protein (CP) disappearance in +S supplementation was lower than that of the other products (53.5% vs. 57.2%-59.1%, p < 0.01). The +S supplementation decreased acetate proportion and increased propionate proportion (p < 0.01). Our study indicated that although the disappearance of DM and CP might decrease, using silica gel as an oil adsorbent of linseed oil calcium salt with high fatty acid/Ca molar ratio has the potential to mitigate CH4 emissions from ruminants.

Taxonomic and functional characterization of the rumen microbiome of Japanese Black cattle revealed by 16S rRNA gene amplicon and metagenome shotgun sequencing.

This study aimed to determine the taxonomic and functional characteristics of the Japanese Black (JB) steer rumen microbiome. The rumen microbiomes of six JB steers (age 14.7 ± 1.44 months) and six JB sires × Holstein dams crossbred (F1) steers (age 11.1 ± 0.39 months), fed the same diet, were evaluated. Based on 16S rRNA gene sequencing, the beta diversity revealed differences in microbial community structures between the JB and F1 rumen. Shotgun sequencing showed that Fibrobacter succinogenes and two Ruminococcus spp., which are related to cellulose degradation were relatively more abundant in the JB steer rumen than in the F1 rumen. Furthermore, the 16S rRNA gene copy number of F. succinogenes was significantly higher in the JB steer rumen than in the F1 rumen according to quantitative real-time polymerase chain reaction analysis. Genes encoding the enzymes that accelerate cellulose degradation and those associated with hemicellulose degradation were enriched in the JB steer rumen. Although Prevotella spp. were predominant both in the JB and F1 rumen, the genes encoding carbohydrate-active enzymes of Prevotella spp. may differ between JB and F1.

Effects of Heat Stress on Heart Rate Variability in Free-Moving Sheep and Goats Assessed With Correction for Physical Activity.

Heart rate variability (HRV) is the heart beat-to-beat variation under control of the cardiovascular function of animals. Under stressed conditions, cardiac activity is generally regulated with an upregulated sympathetic tone and withdrawal of vagal tone; thus, HRV monitoring can be a non-invasive technique to assess stress level in animals especially related to animal welfare. Among several stress-induced factors, heat stress is one of the most serious causes of physiological damage to animals. The aim of this study was to assess the effects of heat stress on HRV in small ruminants under free-moving conditions. In three experimental periods (June, August, and October), inter-beat intervals in sheep and goats (three for each) in two consecutive days were measured. HRV parameters were calculated from the inter-beat interval data by three types of analyses: time domain, frequency domain, and non-linear analyses. The temperature-humidity index (THI) was used as an indicator of heat stress, and vectorial dynamic body acceleration (VeDBA) was calculated to quantify the physical activity of the animals tested. First, we investigated correlations of THI and VeDBA with HRV parameters; subsequently, THI was divided into five categories according to the values obtained (≤ 65, 65-70, 70-75, 75-80, and >80), and the effects of the THI categories on HRV parameters were investigated with and without correcting for the effects of physical activity based on the VeDBA. The results indicated that HRV significantly decreased with increasing THI and VeDBA. For non-linear HRV parameters that were corrected for the effects of physical activity, it was suggested that there would be a threshold of THI around 80 that strongly affected HRV; high heat stress can affect the autonomic balance of animals non-linearly by inducing the sympathetic nervous system. In conclusion, to assess psychophysiological conditions of unrestrained animals by HRV analysis, the confounding effect of physical activity on HRV should be minimized for a more precise interpretation of the results.

Estimation of the marbling development pattern in Japanese Black cattle by using serial ultrasound measurement data.

The objective of this study was to develop mathematical equations for describing the change in marbling in Japanese Black steers using longitudinal measurements. Serial ultrasound measurements were taken at 14, 16, 20, and 26 months of age and analyzed using an image analysis software. The longitudinal marbling measurements from the ultrasound images and carcasses were fitted into a nonlinear logistic curve. Data used for the analysis consisted of 749 steers that converged in nonlinear curve fitting and showed reasonable estimated parameters of the logistic curves. The average predicted mature beef marbling score (BMS) and maturation rate were 6.26 and 0.353, respectively, and the average maturity levels at 24 months of age were 83.9%. The heritability estimates for the predicted maturity traits were moderate, indicating that these traits may have potential for genetic improvement. There was a negative relationship between the expected progeny differences between carcass BMS and maturity traits, suggesting that genetic improvement by carcass BMS may lead to the selection of bulls with late maturity for marbling. The results indicate that ultrasound and model building for marbling can be useful tools to correctly select candidate bulls with high marbling in the early fattening period.

A deterministic simulation model for the evaluation of reproductive performance in Thoroughbred mares.

The objective of the present study was to develop a deterministic simulation model for evaluating the reproductive performance of Thoroughbred mares. As an application, the model was used to estimate the herd level asymptotic foaling percentage (AFP) for evaluating the performance of mares in stabilized mare herds using the convergent method for estimating a steady-state distribution of mares. In this model, it was assumed that the mares were mated only during the breeding season. The effects of early pregnancy loss and fetal loss on reproductive performance were investigated. The sensitivities of AFP to changes in conception rate (CR), early pregnancy loss rate (EPLR), fetal loss rate (FLR), and postpartum mating interval (PMI) were examined. Furthermore, the AFPs were compared for two types of postpartum first mating schedules: mating during the first postpartum estrus cycle (foal heat) and first mating during the subsequent cycle. The results indicated that AFP was sensitive to changes in EPLR, FLR, and CR. The comparison of the two types of postpartum first mating schedules showed that AFP after foal heat mating was higher than after the subsequent estrus mating even with the decrease in reproductive parameters caused by foal heat mating in the model. In addition, even if mares did not mate during foal heat, AFP could be improved by shortening the estrus cycle with the administration of PGF2α.

Effects of parent-of-origin models with different pedigree information on beef carcass traits and fatty acid composition in Japanese Black cattle.

Genomic imprinting should be considered in animal breeding systems to avoid lead in bias in genetic parameter estimation. The objective of this study was to clarify the effects of pedigree information on imprinting variances for carcass traits and fatty acid composition in Japanese Black cattle. Carcass records [carcass weight, rib eye area, rib thickness (RT), subcutaneous fat thickness and beef marbling score (BMS)] and fatty acid composition were obtained for 11,855 Japanese Black feedlot cattle. To estimate and compare the imprinting variances for the traits, two imprinting models with different pedigree information [the sire-dam gametic relationship matrix (Model 1) and the sire-maternal grandsire (MGS) numerator relationship matrix (Model 2)] were fitted. The ratio of the imprinting variance to the total additive genetic variance for RT (6.33%) and BMS (19.00%) was significant in Model 1, but only that for BMS (21.09%) was significant in Model 2. This study revealed that fitting the sire-MGS model could be useful in estimating imprinting variance under certain conditions, such as when restricted pedigree information is available. Furthermore, the present result suggested that the maternal gametic effects on BMS should be included in breeding programmes for Japanese Black cattle to avoid selection bias caused by imprinting effects.

Calcium salts of long-chain fatty acids from linseed oil decrease methane production by altering the rumen microbiome in vitro.

Calcium salts of long-chain fatty acids (CSFA) from linseed oil have the potential to reduce methane (CH4) production from ruminants; however, there is little information on the effect of supplementary CSFA on rumen microbiome as well as CH4 production. The aim of the present study was to evaluate the effects of supplementary CSFA on ruminal fermentation, digestibility, CH4 production, and rumen microbiome in vitro. We compared five treatments: three CSFA concentrations-0% (CON), 2.25% (FAL) and 4.50% (FAH) on a dry matter (DM) basis-15 mM of fumarate (FUM), and 20 mg/kg DM of monensin (MON). The results showed that the proportions of propionate in FAL, FAH, FUM, and MON were increased, compared with CON (P < 0.05). Although DM and neutral detergent fiber expressed exclusive of residual ash (NDFom) digestibility decreased in FAL and FAH compared to those in CON (P < 0.05), DM digestibility-adjusted CH4 production in FAL and FAH was reduced by 38.2% and 63.0%, respectively, compared with that in CON (P < 0.05). The genera Ruminobacter, Succinivibrio, Succiniclasticum, Streptococcus, Selenomonas.1, and Megasphaera, which are related to propionate production, were increased (P < 0.05), while Methanobrevibacter and protozoa counts, which are associated with CH4 production, were decreased in FAH, compared with CON (P < 0.05). The results suggested that the inclusion of CSFA significantly changed the rumen microbiome, leading to the acceleration of propionate production and the reduction of CH4 production. In conclusion, although further in vivo study is needed to evaluate the reduction effect on rumen CH4 production, CSFA may be a promising candidate for reduction of CH4 emission from ruminants.

Genetic associations between reproductive traits for first calving and growth curve characteristics of Japanese Black cattle.

The objective of this study was to estimate genetic parameters for first calving reproductive traits and growth curve characteristics in Japanese Black cattle. The Gompertz growth function was fitted to body weight-age data to obtain the mature weight (MWT) and rate of maturing (ROM) of cows. Data of reproductive traits including the first service conception rate (CR) for heifers, age at the first calving (AFC), and gestation length for the first calving were collected. Records of 3,204 animals were used for analysis. Genetic parameters were estimated using a linear uni- and bivariate animal model. The heritability estimates were moderate (0.29 for ROM) and high (0.57 for MWT) for growth curve parameters and low (0.03-0.11) for reproductive traits. There was a negative genetic correlation between MWT and ROM (-0.26), suggesting that an animal with a faster ROM would show a lower MWT. CR was negatively correlated with MWT (-0.42) but significantly and positively correlated with ROM (0.91). There was a negative genetic correlation between AFC and MWT (-0.49). These results suggest that a heifer with a faster ROM and lower MWT would show a higher CR. Meanwhile, a heifer with a lower MWT would show a higher AFC.

Effects of feeding mother liquor, by-product of monosodium glutamate, on digestibility, energy and nitrogen balances, and rumen condition in Thai native bulls.

Four Thai native bulls were used to evaluate the availability of mother liquor (ML), by-product of monosodium glutamate, as a replacement of soybean meal (SBM) consisting of 10% in concentrate. The SBM was replaced by the ML at 0% (C), 20% (T1), 40% (T2), and 60% (T3), and the experiment was a randomized block design experiment. The animals were fed concentrate and roughage (60:40, on a dry matter [DM] basis). There were no significant differences in the digestibility of DM, crude protein, ether extract, acid detergent fiber expressed exclusive of residual ash and non-fibrous carbohydrate, and energy and nitrogen balances among the treatments. However, the digestibility of the neutral detergent fiber in T2 was lower than the other treatments (p < .05). The valeric acid of T2 was lower than those of C and T1 and the iso-valeric acid of T3 was the lowest (p < .05), followed by those of T2, T1, and C at 4 hr post-feeding. No significant differences were observed in the ruminal total VFA concentrations, pH, and NH3 -N among the treatments. These results suggested that SBM could be replaced by the ML up to 60% without adverse effects on nitrogen and energy balance, rumen conditions, and blood metabolites in Thai native bulls.

Correction: Cows painted with zebra-like striping can avoid biting fly attack.

[This corrects the article DOI: 10.1371/journal.pone.0223447.].

Cows painted with zebra-like striping can avoid biting fly attack.

Experimental and comparative studies suggest that the striped coats of zebras can prevent biting fly attacks. Biting flies are serious pests of livestock that cause economic losses in animal production. We hypothesized that cows painted with black and white stripes on their body could avoid biting fly attacks and show fewer fly-repelling behaviors. Six Japanese Black cows were assigned to treatments using a 3 × 3 Latin-square design. The treatments were black-and-white painted stripes, black painted stripes, and no stripes (all-black body surface). Recorded fly-repelling behaviors were head throw, ear beat, leg stamp, skin twitch, and tail flick. Photo images of the right side of each cow were taken using a commercial digital camera after every observation and biting flies on the body and each leg were counted from the photo images. Here we show that the numbers of biting flies on Japanese Black cows painted with black-and-white stripes were significantly lower than those on non-painted cows and cows painted only with black stripes. The frequencies of fly-repelling behaviors in cows painted with black-and-white stripes were also lower than those in the non-painted and black-striped cows. These results thus suggest that painting black-and-white stripes on livestock such as cattle can prevent biting fly attacks and provide an alternative method of defending livestock against biting flies without using pesticides in animal production, thereby proposing a solution for the problem of pesticide resistance in the environment.

Parent-of-origin effects on carcass traits in Japanese Black cattle.

Variances caused by the differential expression of paternally and maternally imprinted genes controlling carcass traits in Japanese Black cattle were estimated in this study. Data on marbling score (BMS), carcass weight, rib thickness, rib-eye area (REA) and subcutaneous fat thickness (SFT) were collected from a total of 13,115 feedlot steers and heifers in a commercial population. A sire-maternal grandsire model was used to analyse the data, and then, imprinting parameters were derived by replacing the genetic effect of the dam with the effect of the maternal grandsire in the imprinting model to calculate the genetic parameter estimates. The proportions of the total genetic variance attributable to imprinted genes ranged from 8.7% (SFT) to 35.2% (BMS). The remarkably large imprinting variance of BMS was mainly contributed by maternally expressed inheritance because the maternal contribution of the trait was much larger than that of the paternal trait. The parent-of-origin effect originating from maternal gene expression was also observed for REA. The results suggested the existence of genomic imprinting effects on the traits of the Japanese Black cattle. Hence, the parent-of-origin effect should be considered for the genetic evaluation of Japanese Black cattle in breeding programmes.

Effects of supplementary mother liquor, by-product of monosodium glutamate, on in vitro ruminal fermentation characteristics.

Mother liquor (ML) is monosodium glutamate by-product and contains much crude protein (CP). The objective of this study was to investigate the effect of supplementation of two types of MLs having different chemical compositions on in vitro ruminal fermentation characteristics. ML1 had 74% organic matter (OM) and 70% CP and ML2 had 85% OM and 83% CP on a dry matter (DM) basis. The experiments were conducted using 0.5 gDM rolled barley or ryegrass straw as substrates. Urea and freeze-dried MLs were added at three levels of nitrogen to be isonitrogenous. The ML1 treatment increased gas production compared with the ML2 treatment in barley substrate condition (p < 0.05). The supplementary ML2 increased gas production compared with the supplementary urea, and the digestibilities of DM and neutral detergent fiber expressed exclusive of residual ash (NDFom) were higher for the supplementary ML2 than for the supplementary ML1 in straw substrate condition (p < 0.05). In both the conditions, ammonia nitrogen concentrations of the ML1 and ML2 treatments were lower than that of the urea treatment (p < 0.05). The results indicated that the supplementary ML1 and ML2 activated in vitro ruminal fermentation particularly in concentrate and roughage substrate conditions respectively.

How do human values influence the beef preferences of consumer segments regarding animal welfare and environmentally friendly production?

Animal welfare and environmental impacts have been emphasized in the sustainable production of livestock. Labels are useful tools for clearly providing such attribute information to consumers. The aim of this study was to evaluate how human values influence consumer segments for beef with information on animal welfare and environmentally friendly production. Using a choice experiment, we examined whether animal welfare and environmentally friendly labels, country of origin and price impact consumer choice. As results, five heterogeneous consumer classes were identified using a latent class model: label conscious, domestic beef preferring, price conscious, animal welfare preferring and not interested in production method. Almost 90% of consumers were interested in and willing to pay for beef with animal welfare or environmentally friendly label. The classes with significant preferences for such labeled beef were affected by "openness to change", "self-enhancement" and "security". Improving consumer attitudes and strengthening consumer perception towards labeled beef by marketers and policy makers will be required.

Correcting the Activity-Specific Component of Heart Rate Variability Using Dynamic Body Acceleration Under Free-Moving Conditions.

Heart rate variability (HRV) analysis is a widely used technique to assess sympatho-vagal regulation in response to various internal or external stressors. However, HRV measurements under free-moving conditions are highly susceptible to subjects' physical activity levels because physical activity alters energy metabolism, which inevitably modulates the cardiorespiratory system and thereby changes the sympatho-vagal balance, regardless of stressors. Thus, researchers must simultaneously quantify the effect of physical activity on HRV to reliably assess sympatho-vagal balance under free-moving conditions. In the present study, dynamic body acceleration (DBA), which was developed in the field of animal ecology as a quantitative proxy for activity-specific energy expenditure, was used as a factor to correct for physical activity when evaluating HRV in freely moving subjects. Body acceleration and heart inter-beat intervals were simultaneously measured in cattle and sheep, and the vectorial DBA and HRV parameters were evaluated at 5-min intervals. Next, the effects of DBA on the HRV parameters were statistically analyzed. The heart rate (HR) and most of the HRV parameters were affected by DBA in both animal species, and the inclusion of the effect of DBA in the HRV analysis greatly influenced the frequency domain and nonlinear HRV parameters. By removing the effect of physical activity quantified using DBA, we could fairly compare the stress levels of animals with different physical activity levels under different management conditions. Moreover, we analyzed and compared the HRV parameters before and after correcting for the mean HR, with and without inclusion of DBA. The results were somewhat unexpected, as the effect of DBA was a highly significant source of HRV also in parameters corrected for mean HR. In conclusion, the inclusion of DBA as a physical activity index is a simple and useful method for correcting the activity-specific component of HRV under free-moving conditions.

Effects of supplementary desalted mother liquor as replacement of commercial salt in diet for Thai native cattle on digestibility, energy and nitrogen balance, and rumen conditions.

Four Thai native cattle were used in a 4 × 4 Latin square design experiment to evaluate the availability of desalted mother liquor (DML) as replacement of salt in concentrate. Each cattle was assigned to one of the following concentrate feeding treatments: C1, 1% NaCl was added as salt; C2, 2% NaCl was added as salt; D1, 1% NaCl was replaced by DML; D2, 2% NaCl was replaced by DML, on a dry matter (DM) basis. The animals were fed rice straw and experimental concentrates (40:60) at 1.9% of body weight on a DM basis, daily. Acid detergent fiber expressed exclusive of residual ash (ADFom) digestibility in DML treatment was higher than salt treatment (p < .05) and D2 feeding showed the highest value (60.8%). There were no significant differences in blood metabolites, nitrogen retention, ruminal ammonia nitrogen, methane emission or energy efficiency among treatments. Molar percent of acetate on volatile fatty acids in rumen fluid 4 hr post-feeding tended to be higher in DML treatment than salt treatment (p = .08). The results indicated that adding DML could improve ADFom digestibility and salt could be replaced by DML up to 2% as NaCl in concentrate without adverse effects on nitrogen balance, rumen conditions, blood metabolites and methane emission.

Interspecific comparison of allometry between body weight and chest girth in domestic bovids.

The sizes of body parts often co-vary through exponential scaling, known as allometry. The evolution of allometry is central to the generation of morphological diversity. To make inferences regarding the evolved responses in allometry to natural and artificial selection, we compared allometric parameters (slope and intercept) among seven species and breeds of domestic bovids using cross-sectional ontogenetic data and attempted to interpret the differences in these parameters. The allometric slopes were not different among some species, whereas those between breeds within species were, indicating that the slopes were typically invariant but could be changed under strong, specific selection. With the exception of yak, the differences in the intercept independent of the slopes (the alternative intercept) among species might better correspond to their divergence times than the differences in allometric slope, and the remarkably higher alternative intercept found in yaks can be explained by their unique morphological evolution. These findings provide evidence that differences in the alternative intercept can retain traces of the phylogenetic changes derived from differentiation and evolution.