Estimates of daily oxygen consumption, carbon dioxide and methane emissions, and heat production for beef and dairy cattle using spot gas sampling.

A simulation study was conducted to examine accuracy of estimating daily O2 consumption, CO2 and CH4 emissions, and heat production (HP) using a spot sampling technique and to determine optimal spot sampling frequency (FQ). Data were obtained from 3 experiments where daily O2 consumption, emissions of CO2 and CH4, and HP were measured using indirect calorimetry (respiration chamber or headbox system). Experiment 1 used 8 beef heifers (ad libitum feeding; gaseous exchanges measured every 30 min over 3 d in respiration chambers); Experiment 2 used 56 lactating Holstein-Friesian cows (restricted feeding; gaseous exchanges measured every 12 min over 3 d in respiration chambers); Experiment 3 used 12 lactating Jersey cows (ad libitum feeding; gaseous exchanges measured every hour for 1 d using headbox style chambers). Within experiment, averages of all measurements (FQALL) and averages of measurements selected at time points with 12, 8, 6, or 4 spot sampling FQ (i.e., sampling every 2, 3, 4, and 6 h in a 24-h cycle, respectively; FQ12, FQ8, FQ6, and FQ4, respectively) were compared. Within study a mixed model was used to compare gaseous exchanges and HP among FQALL, FQ12, FQ8, FQ6, and FQ4, and an interaction of dietary treatment by FQ was examined. A regression model was used to evaluate accuracy of spot sampling within study [i.e., FQALL (observed) vs. FQ12, FQ8, FQ6, or FQ4 (estimated)]. No interaction of diet by FQ was observed for any variables except for CH4 production in experiment 1. No FQ effect was observed for gaseous exchanges and HP except in experiment 2 where CO2 production was less (5,411 vs. 5,563 L/d) for FQ4 compared with FQALL, FQ12, and FQ8. A regression analysis between FQALL and each FQ within study showed that slopes and intercepts became farther from 1 and 0, respectively, for almost all variables as FQ decreased. Most variables for FQ12 and FQ8 had root mean square prediction error (RMSPE) less than 10% of the mean and concordance correlation coefficient (CCC) greater than 0.80, and RMSPE increased and CCC decreased as FQ decreased. When a regression analysis was conducted with combined data from the 3 experiments (mixed model with study as a random effect), results agreed with those from the analysis for the individual studies. Prediction errors increased and CCC decreased as FQ decreased. Generally, all the estimates from FQ12, FQ8, FQ6, and FQ4 had RMSPE less than 10% of the means and CCC greater than 0.90 except for FQ6 and FQ4 for O2 consumption and CH4 production. In conclusion, the spot sampling simulation with 3 indirect calorimetry experiments indicated that FQ of at least 8 samples (every 3 h in a 24-h cycle) was required to estimate daily O2 consumption, CO2 and CH4 production, and HP and to detect changes in those in response to dietary treatments. This sampling FQ may be considered when using techniques that measure spot gas exchanges such as the GreenFeed and face mask systems.

China's low-emission pathways toward climate-neutral livestock production for animal-derived foods.

Animal-derived food production accounts for one-third of global anthropogenic greenhouse gas (GHG) emissions. Diet followed in China is ranked as low-carbon emitting (i.e., 0.21 t CO2-eq per capita in 2018, ranking at 145th of 168 countries) due to the low average animal-derived food consumption rate, and preferential consumption of animal-derived foods with lower GHG emissions (i.e., pork and eggs versus beef and milk). However, the projected increase in GHG emissions from livestock production poses great challenges for achieving China's "carbon neutrality" pledge. We propose that the livestock sector in China may achieve "climate neutrality" with net-zero warming around 2050 by implementing healthy diet and mitigation strategies to control enteric methane emissions.

A Novel Approach for Monitoring the Volatile Metabolome in Biological Samples from Ruminants through Miniaturized Liquid-Liquid Extraction and Multiclass Gas Chromatography Analysis.

A straightforward and versatile methodology for the extraction of volatile metabolites in biological samples from ruminants for gas chromatography analysis is proposed. The methodology was applied in the determination of multiclass metabolites (short-chain fatty acids, aldehydes, alcohols, ketones, esters, phenols, and sulfides) in different analytical matrices (rumen fluid, urine, and feces) collected from Holstein cows. The 24 multiclass volatile metabolites reported in the different biological samples and their respective concentrations were critically discussed in the context of digestive physiology. Most detected compounds are derived from the rumen and lower gut fermentation of carbohydrates, proteins, and lipids or their metabolism, being consistent with the prior state of the art. The proposed method also takes advantage of the already existing tools in animal nutrition laboratories, providing a novel methodological ground that can generate relevant bioanalytical information with a significant impact on ruminant's nutritional studies.

The effect of leucaena hay as a source of effective fibre and nutrients in diets with forage palm for finishing sheep in semi-arid regions.

The present study was conducted to evaluate the intake, digestibility and nitrogen balance of diets for finishing sheep, containing leucaena hay as a source of effective fibre and forage palm. Twenty-one male sheep were used in a randomized block design with three treatments, represented by total diets, where the forage was leucaena hay and forage palm in natura in proportions 350:650, 450:550 and 550:450 g/kg respectively. For the pH and NH3 -N in the ruminal fluid, and urea in the blood serum, plots split in time were adopted. Dry matter and nutrient digestibility, and the nitrogen balance were not influenced (p > .05) by the diets. The neutral detergent fibre digestibility decreased (p < .05) as the proportions of leucaena hay in the diets increased. The pH and NH3 -N were suitable for ruminal fermentation, and blood urea was maintained under normal physiological conditions for sheep. Combining 350 to 550 g/kg of leucaena hay with 450 to 650 g/kg of forage palm in total diets with a forage:concentrate ratio of 60:40 in late-maturing sheep meets the nutritional requirements for daily gain 200 g, characterizing leucaena hay as important source of effective fibre and nutrients in diets.

Development of a CRISPR/Cas9 system against ruminant animal brucellosis.

BACKGROUND: Brucellosis, caused by several Brucella species, such as the bacterium Brucella melitensis, is considered one of the most severe zoonotic diseases worldwide. Not only does it affect ruminant animal populations, leading to a substantial financial burden for stockbreeders, but also poses severe public health issues. For almost four decades in southern Europe and elsewhere, eradication of the disease has been based on ambiguously effective programs, rendering massive sanitation of livestock urgent and indispensable. Gene therapy, which has been proved effective in the clinic, could possibly constitute an alternative option towards a permanent cure for brucellosis, by aiding in the deletion or inactivation of genes associated with the replication of Brucella within the host cells. RESULTS: We infected ovine macrophages with B.melitensis, to simulate the host cell/microorganism interaction in vitro, and transduced the infected cells with CRISPR/Cas9 lentiviral vectors that target Brucella's RNA polymerase subunit A (RpolA) or virulence-associated gene virB10 at a multiplicity of infection of 60. We demonstrate a significant decrease in the bacterial load per cell when infected cells are transduced with the RpolA vector and that the number of internalized brucellae per cell remains unaffected when macrophages are transduced with a conventional lentiviral vector expressing the green fluorescence protein, thus underlining the bactericidal effect of our CRISPR/Cas9 system. CONCLUSIONS: Pending in vivo verification of our findings, overall, these results may prove critical not only for the treatment of human brucellosis, but for other infectious diseases in general.

A Cellular Model of Infection with Brucella melitensis in Ovine Macrophages: Novel Insights for Intracellular Bacterial Detection.

Intracellular bacteria provoking zoonoses, such as those of the genus Brucella, present a host cell tropism mostly limited to the monocyte/macrophage lineage, leading to chronic inflammatory reactions, difficult-to-eradicate-infections, and widespread prevalence among ruminants. Eradication of brucellosis has been based on programs that translate into a substantial financial burden for both the authorities and stockbreeders, if not strictly followed. To this end, we sought to create an in vitro cell model that could be utilized as future reference for adequately measuring the number of engulfed brucellae/cell, using peripheral blood-derived sheep macrophages infected with B. melitensis at decimal multiplicities of infection (MOI = 5000-5), to simulate the host cell/microorganism interaction and monitor bacterial loads up to 6 days post-infection. We show that the MOI = 5000 leads to high numbers of engulfed bacteria without affecting macrophages' viability and that the minimum detection limit of our Real-Time PCR assay was 3.97 ± 5.58 brucellae/cell. Moreover, we observed a time-associated, significant gradual reduction in bacterial loads from Day 2 to Day 6 post-infection (p = 0.0013), as part of the natural bactericidal properties of macrophages. Overall, the work presented here constitutes a reliable in vitro cell model of Brucella melitensis for research purposes that can be utilized to adequately measure the number of engulfed brucellae/cell and provides insights towards future utilization of molecular biology-based methods for detection of Brucella.

A metagenomics investigation of carbohydrate-active enzymes along the goat and camel intestinal tract.

Studies of the digestive microbiota of ruminant animals most often focus on the bacterial diversity in the rumen or the feces of the animals, but little is known about the diversity and functions of their distal intestine. Here, the bacterial microbiota of the distal intestinal tract of two goats and two camels was investigated by metagenomics techniques. The bacterial taxonomic diversity and carbohydrate-active enzyme profile were estimated for samples taken from the small intestine, the large intestine, and the rectum of each animal. The bacterial diversity and abundance in the small intestine were lower than in the rectal and large intestinal samples. Analysis of the carbohydrate-active enzyme profiles at each site revealed a comparatively low abundance of enzymes targeting xylan and cellulose in all animals examined, similar to what has been reported earlier for sheep and therefore suggesting that plant cell wall digestion probably takes place elsewhere, such as in the rumen.

Efficient Transduction and Expansion of Ovine Macrophages for Gene Therapy Implementations.

A number of bacteria provoking zoonotic diseases present intracellular survival and a host cell tropism limited to the monocyte/macrophage lineage. Thus, infection is rendered difficult to eradicate, causing chronic inflammatory reactions to the host and widespread prevalence. Although self-inactivating lentiviral vectors have been successfully tested in the clinic against virally-induced human infectious diseases, little is known about the transduction susceptibility of ruminant animal phagocytes that play a critical role in the outbreak of zoonotic diseases such as brucellosis. In view of the development of a lentiviral vector-based platform targeting and inactivating specific genetic features of intracellular bacteria, we have tested the transducibility of ovine macrophages in terms of transgene expression and vector copy number (VCN). We show that ovine macrophages are relatively resistant to transduction even at a high multiplicity of infection with a conventional lentiviral vector expressing the green fluorescence protein and that addition of transduction enhancers, such as polybrene, increases transgene expression even after a one-week culture of the transduced cells in vitro. Overall, we demonstrate that ovine macrophages may be efficiently expanded and transduced in culture, thus providing the benchmark for gene therapy applications for zoonotic diseases.

Vitamin B12 sources and microbial interaction.

Vitamin B12 is synthesized only by certain bacteria and archaeon, but not by plants. The synthesized vitamin B12 is transferred and accumulates in animal tissues, which can occur in certain plant and mushroom species through microbial interaction. In particular, the meat and milk of herbivorous ruminant animals (e.g. cattle and sheep) are good sources of vitamin B12 for humans. Ruminants acquire vitamin B12, which is considered an essential nutrient, through a symbiotic relationship with the bacteria present in their stomachs. In aquatic environments, most phytoplankton acquire vitamin B12 through a symbiotic relationship with bacteria, and they become food for larval fish and bivalves. Edible plants and mushrooms rarely contain a considerable amount of vitamin B12, mainly due to concomitant bacteria in soil and/or their aerial surfaces. Thus, humans acquire vitamin B12 formed by microbial interaction via mainly ruminants and fish (or shellfish) as food sources. In this review, up-to-date information on vitamin B12 sources and bioavailability are also discussed. Impact statement To prevent vitamin B12 (B12) deficiency in high-risk populations such as vegetarians and elderly subjects, it is necessary to identify foods that contain high levels of B12. B12 is synthesized by only certain bacteria and archaeon, but not by plants or animals. The synthesized B12 is transferred and accumulated in animal tissues, even in certain plant tissues via microbial interaction. Meats and milks of herbivorous ruminant animals are good sources of B12 for humans. Ruminants acquire the essential B12 through a symbiotic relationship with bacteria inside the body. Thus, we also depend on B12-producing bacteria located in ruminant stomachs. While edible plants and mushrooms rarely contain a considerable amount of B12, mainly due to concomitant bacteria in soil and/or their aerial surfaces. In this mini-review, we described up-to-date information on B12 sources and bioavailability with reference to the interaction of microbes as B12-producers.

Comportamento ingestivo e respostas fisiológicas de novilhos submetidos a dietas com torta de amendoim / Intake behavior and physiological responses of steer fed with peanut cake diets

Objetivou-se avaliar a substituição do farelo de soja por torta de amendoim no concentrado da dieta de novilhos, por meio do comportamento ingestivo e respostas fisiológicas. Foram utilizados cinco novilhos, dispostos em um quadrado latino 5 x 5. As dietas foram compostas de feno de tifton, milho moído, farelo de soja e torta de amendoim, constituídas de zero (100 por centode farelo de soja), 25, 50, 75 e 100 por cento de substituição por torta de amendoim no concentrado. Os níveis de torta de amendoim não influenciaram (P>0,05) as eficiências de ingestão e ruminação da matéria seca e da fibra em detergente neutro. Em relação às atividades comportamentais, o único parâmetro em que se observou efeito da substituição foi o número de períodos de ócio no turno da manhã (P<0,05), no qual se constatou efeito linear crescente. A taxa de secreção salivar e a quebra de partículas foram semelhantes, pois não houve efeito (P>0,05) das dietas sobre as características de mastigação. Como não houve efeito (P>0,05) das dietas sobre as frequências respiratória, cardíaca e a temperatura retal, o calor endógeno produzido pela fermentação ruminal foi equivalente. A torta de amendoim, por conter aflatoxinas, quando fornecida em alta relação volumoso x concentrado, pode substituir o farelo de soja na dieta de novilhos, pois ocasiona comportamento ingestivo e respostas fisiológicas semelhantes...

This study aimed to evaluate the replacement of soybean meal with peanut cake in the concentrate of steer diets through intake behavior and physiological responses. Five steers, arranged in a 5 x 5 latin square design were used. The diets were composed of Tifton hay, ground corn, soybean meal and peanut cake, made from zero (100 percent soybean meal), 25, 50, 75 and 100 percent replacement for peanut cake in the concentrate. The levels of peanut cake did not influence (P>0.05) the intake and rumination efficiencies of dry matter and fiber in neutral detergent. Regarding the behavioral activities, the only parameter that observed a substitution effect was the number of idle periods in the morning shift (P<0.05) which had an increasing linear effect. The rate of salivary secretion and the particle breakdown were similar, because there was no effect (P>0.05) of the diets on the chewing characteristics. As there was no effect (P>0.05) of the diets on the respiratory rate, heart rate and rectal temperature, the endogenous heat produced by rumen fermentation was equivalent. The peanut cake, by containing aflatoxin, when provided in high roughage x concentrate rate, can replace soybean meal in the steers' diet because it causes similar intake behavior and physiological responses...