M.D. Carro

New aspects and strategies for methane mitigation from ruminants

The growing demand for sustainable animal production is compelling researchers to explore the potential approaches to reduce emissions of greenhouse gases from livestock that are mainly produced by enteric fermentation. Some potential solutions, for instance, the use of chemical inhibitors to reduce methanogenesis, are not feasible in routine use due to their toxicity to ruminants, inhibition of efficient rumen function or other transitory effects. Strategies, such as use of plant secondary metabolites and dietary manipulations have emerged to reduce the methane emission, but these still require extensive research before these can be recommended and deployed in the livestock industry sector. Furthermore, immunization vaccines for methanogens and phages are also under investigation for mitigation of enteric methanogenesis. The increasing knowledge of methanogenic diversity in rumen, DNA sequencing technologies and bioinformatics have paved the way for chemogenomic strategies by targeting methane producers. Chemogenomics will help in finding target enzymes and proteins, which will further assist in the screening of natural as well chemical inhibitors. The construction of a methanogenic gene catalogue through these approaches is an attainable objective. This will lead to understand the microbiome function, its relation with the host and feeds, and therefore, will form the basis of practically viable and eco-friendly methane mitigation approaches, while improving the ruminant productivity.

Technical note: Comparison of automated ribosomal intergenic spacer analysis and denaturing gradient gel electrophoresis to assess bacterial diversity in the rumen of sheep.

The aim of this study was to compare automated ribosomal intergenic spacer analysis (ARISA) and denaturing gradient gel electrophoresis (DGGE) techniques to assess bacterial diversity in the rumen of sheep. Sheep were fed 2 diets with 70% of either alfalfa hay or grass hay, and the solid (SOL) and liquid (LIQ) phases of the rumen were sampled immediately before feeding (0 h) and at 4 and 8 h postfeeding. Both techniques detected similar differences between forages, with alfalfa hay promoting greater (P < 0.05) bacterial diversity than grass hay. In contrast, whereas ARISA analysis showed a decrease (P < 0.05) of bacterial diversity in SOL at 4 h postfeeding compared with 0 and 8 h samplings, no variations (P > 0.05) over the postfeeding period were detected by DGGE. The ARISA technique showed lower (P < 0.05) bacterial diversity in SOL than in LIQ samples at 4 h postfeeding, but no differences (P > 0.05) in bacterial diversity between both rumen phases were detected by DGGE. Under the conditions of this study, the DGGE was not sensitive enough to detect some changes in ruminal bacterial communities, and therefore ARISA was considered more accurate for assessing bacterial diversity of ruminal samples. The results highlight the influence of the fingerprinting technique used to draw conclusions on factors affecting ruminal bacterial diversity.

Influence of protein fermentation and carbohydrate source on in vitro methane production

Incubations were carried out with batch cultures of ruminal micro-organisms from sheep to analyse the influence of the N source on inxa0vitro CH4 production. The two substrates were mixtures of maize starch and cellulose in proportions of 75:25 and 25:75 (STAR and CEL substrates, respectively), and the three nitrogen (N) sources were ammonia (NH4 Cl), casein (CA) and isolated soya bean protein (SP). Five isonitrogenous treatments were made by replacing non-protein-N (NPN) with CA or SP at levels of 0 (NPN), 50 (CA50 and SP50, respectively) and 100% (CA100 and SP100) of total N. All N treatments were applied at a rate of 35xa0mg of N/g of substrate organic matter and incubations lasted 16.5xa0h. With both proteins, N sourcexa0×xa0substrate interactions (pxa0=xa00.065 to 0.002) were detected for CH4 production and CH4 /total VFA ratio. The increases in CH4 production observed by replacing the NPN with protein-N were higher (pxa0<xa00.05) for STAR than for CEL substrate, but the opposite was observed for the increases in volatile fatty acid (VFA) production. As a consequence, replacing the NPN by increased levels of CA or SP led to linear increases (pxa0<xa00.05) in CH4 /total VFA ratio with STAR, whereas CH4 /total VFA ratio tended (pxa0<xa00.10) to be decreased with CEL substrate. Increasing the amount of both proteins decreased linearly (pxa0<xa00.05) ammonia-N concentrations, which may indicate an incorporation of amino acids and peptides into microbial protein without being first deaminated into ammonia-N. In incubations with the tested N sources as the only substrate, the fermentation of 1xa0mg of CA or SP produced 1.24 and 0.60xa0μmol of CH4 respectively. The results indicate the generation of CH4 from protein fermentation, and that the response of CH4 production to protein-N supply may differ with the basal substrate.

Treatment of tropical forages with exogenous fibrolytic enzymes: effects on chemical composition and in vitro rumen fermentation

The effects of three treatments of fibrolytic enzymes (cellulase from Trichoderma longibrachiatum (CEL), xylanase from rumen micro-organisms (XYL) and a 1:1 mixture of CEL and XYL (MIX) on the in vitro fermentation of two samples of Pennisetum clandestinum (P1 and P2), two samples of Dichanthium aristatum (D1 and D2) and one sample of each Acacia decurrens and Acacia mangium (A1 and A2) were investigated. The first experiment compared the effects of two methods of applying the enzymes to forages, either at the time of incubation or 24xa0h before, on the in vitro gas production. In general, the 24xa0h pre-treatment resulted in higher values of gas production rate, and this application method was chosen for a second study investigating the effects of enzymes on chemical composition and in vitro fermentation of forages. The pre-treatment with CEL for 24xa0h reduced (pxa0<xa00.05) the content of neutral detergent fibre (NDF) of P1, P2, D1 and D2, and that of MIX reduced the NDF content of P1 and D1, but XYL had no effect on any forage. The CEL treatment increased (pxa0<xa00.05) total volatile fatty acid (VFA) production for all forages (ranging from 8.6% to 22.7%), but in general, no effects of MIX and XYL were observed. For both P.xa0clandestinum samples, CEL treatment reduced (pxa0<xa00.05) the molar proportion of acetate and increased (pxa0<xa00.05) that of butyrate, but only subtle changes in VFA profile were observed for the rest of forages. Under the conditions of the present experiment, the treatment of tropical forages with CEL stimulated their in vitro ruminal fermentation, but XYL did not produce any positive effect. These results showed clearly that effectiveness of enzymes varied with the incubated forage and further study is warranted to investigate specific, optimal enzyme-substrate combinations.

Protection of sunflower seed and sunflower meal protein with malic acid and heat: effects on in vitro ruminal fermentation and methane production

BACKGROUNDnCombined malic acid-heat treatments of protein supplements have been shown to reduce ruminal protein degradation, but there is no information on their possible influence on ruminal fermentation and methane emissions. This study aimed to investigate the effects of the treatment of sunflower meal (SM) and sunflower seed (SS) with malic acid and subsequent drying at 150°C for 1 (MAL1) or 3 h (MAL3) on in vitro rumen fermentation and methane emission using ruminal fluid from sheep as inoculum.nnnRESULTSnCompared with untreated samples, the MAL3 treatment reduced (P < 0.05) the dry matter effective degradability (DMED) by 78% and 46% for SS and SM, respectively, indicating heat damage. The MAL1 treatment reduced the DMED of SS by 22%, but did not affect (P > 0.05) total volatile fatty acid production for any feed. This treatment also increased (P < 0.05) the propionate proportion (by 17.7% and 15.6% for SS and SM, respectively) and decreased (P < 0.05) methane production (by 15.5% and 11.3%, respectively) and ammonia-N concentrations (by 26.5% and 14.5%, respectively).nnnCONCLUSIONnThe MAL1 treatment was effective in reducing both ammonia-N concentrations and methane emissions without depressing SS and SM fermentation, but more research is needed to formulate environmentally cleaner diets for ruminants.

Shifts in microbial populations in Rusitec fermenters as affected by the type of diet and impact of the method for estimating microbial growth (15N v. microbial DNA)

Rusitec fermenters are in vitro systems widely used to study ruminal fermentation, but little is known about the microbial populations establishing in them. This study was designed to assess the time evolution of microbial populations in fermenters fed medium- (MC; 50% alfalfa hay : concentrate) and high-concentrate diets (HC; 15 : 85 barley straw : concentrate). Samples from solid (SOL) and liquid (LIQ) content of fermenters were taken immediately before feeding on days 3, 8 and 14 of incubation for quantitative polymerase chain reaction and automated ribosomal intergenic spacer analysis analyses. In SOL, total bacterial DNA concentration and relative abundance of Ruminococcus flavefaciens remained unchanged over the incubation period, but protozoal DNA concentration and abundance of Fibrobacter succinogenes, Ruminococcus albus and fungi decreased and abundance of methanogenic archaea increased. In LIQ, total bacterial DNA concentration increased with time, whereas concentration of protozoal DNA and abundance of methanogens and fungi decreased. Diet×time interactions were observed for bacterial and protozoal DNA and relative abundance of F. succinogenes and R. albus in SOL, as well as for protozoal DNA in LIQ. Bacterial diversity in SOL increased with time, but no changes were observed in LIQ. The incubated diet influenced all microbial populations, with the exception of total bacteria and fungi abundance in LIQ. Bacterial diversity was higher in MC-fed than in HC-fed fermenters in SOL, but no differences were detected in LIQ. Values of pH, daily production of volatile fatty acids and CH4 and isobutyrate proportions remained stable over the incubation period, but other fermentation parameters varied with time. The relationships among microbial populations and fermentation parameters were in well agreement with those previously reported in in vivo studies. Using 15N as a microbial marker or quantifying total microbial DNA for estimating microbial protein synthesis offered similar results for diets comparison, but both methods presented contrasting results for microbial growth in SOL and LIQ phases. The study showed that fermentation parameters remained fairly stable over the commonly used sampling period (days 8 to 14), but shifts in microbial populations were detected. Moreover, microbial populations differed markedly from those in the inocula, which indicates the difficulty of directly transposing results on microbial populations developed in Rusitec fermenters to in vivo conditions.

The effect of cellobiose on the health status of growing rabbits depends on the dietary level of soluble fiber

The aim of this study was to examine whether the combination of dietary soluble fiber and cellobiose exerts a synergistic effect on growth performance, health status, fermentation traits, and immune response in rabbits. Six treatments in a 3 × 2 factorial arrangement were used: 3 cellobiose concentrations in drinking water (0.0, 7.5, and 15.0 g/L) × 2 dietary levels of soluble fiber (84.0 and 130 g/kg DM, for the low soluble fiber [LSF] and high soluble fiber [HSF] diets, respectively). A total of 318 young rabbits (53/treatment) were weaned at 34 d of age and had ad libitum access to feed and water. At 46 d of age, 9 rabbits/treatment were slaughtered and ileal and cecal digesta were collected to analyze VFA profile and the immune response in the cecal appendix mucosa. At 48 d of age, the cellobiose supplementation was withdrawn and the experimental diets were replaced by a standard commercial diet until 61 d of age. From 34 to 48 d of age, there was a linear increase of mortality with the level of cellobiose in the HSF group (0% vs. 17.1%; P = 0.017). In contrast, a quadratic effect of cellobiose level on mortality was observed in the LSF group, the rabbits offered 7.5-cellobiose showing the lowest mortality (5.7% vs. 21.4%; P = 0.030). Cellobiose level had a quadratic effect on ADFI, ADG, and G:F in this period (P ≤ 0.047), with the 7.5-cellobiose groups having the best growth performance. In contrast, only minor changes on these traits were observed from 48 d of age onwards. Cellobiose level influenced quadratically the ileal VFA concentrations (P = 0.014), showing the maximal value in the 7.5-cellobiose groups. In rabbits fed 7.5-cellobiose-LSF, a change of acetate to propionate, butyrate, and valerate was observed in the ileum. Increasing cellobiose levels reduced linearly cecal VFA concentrations in HSF fed rabbits, but no effect was detected in LSF groups (P = 0.046). The level of soluble fiber increased VFA concentrations in both the ileum (by 22%; P < 0.001), and the cecum (by 11%; P = 0.005). The relative gene expression of IL-6, IL-10, TNF-α, iNOS, MUC-1, and toll-like receptors (TLR-2 and TLR-4) in the cecal appendix increased linear and quadratically with increasing levels of cellobiose (P ≤ 0.063). In conclusion, in rabbits fed LSF diets, a dose of 7.5 g cellobiose/L drinking water would be recommended, whereas these levels of cellobiose supplementation should be avoided in rabbits fed HSF diets.

Effects of dietary fish oil supplementation on performance, meat quality, and cecal fermentation of growing rabbits

The objective of this study was to investigate the effect of dietary supplementation with fish oil on growth performance (during all fatening period), carcass characteristics and fatty acid (FA) profile of muscle and fat tissues (at slaughter), as well as cecal fermentation and ileal mucosa morphology of growing rabbits (at 30, 45, and 60 d of age). Two isonitrogenous and isoenergetic diets, only differing in their fat source, were formulated and provided each to 24 does (12 per diet) and their offspring during pregnancy and lactation. The control diet contained 4.59 g of n-3 per 100 g of total FA, and the enriched diet contained 14.9 g of n-3 per 100 g of total FA. From weaning (30 d of age) to slaughter (60 d), the litters (12 per diet; 8 kits each) continued fed the corresponding experimental diet. There were no differences ( > 0.05) between groups in ADFI, ADG and G:F ratio during the growing period. At slaughter, BW, full gastrointestinal tract weight, carcass yield, meat color and pH, drip loss percentage, content of scapular fat and tissue composition of the left hind leg were similar between groups ( > 0.05), but perirenal fat was lower ( = 0.020) and skin weight and abdominal fat tended to be lower ( = 0.055 and = 0.063, respectively) in enriched rabbits than in control ones. Total PUFA content in both LM and perirenal fat was greater ( = 0.021 and < 0.001, respectively) in enriched rabbits, that also showed lower n-6/n-3 ratios in LM (1.61 vs. 5.80; < 0.001) and perirenal fat (4.71 vs. 12.0; < 0.001) than those fed the control diet. Cecal concentrations of total VFA were greater ( < 0.001) in enriched than in control group at 30, 45 and 60 d of age, but diet did not affect ( ≥ 0.332) VFA profile, with the exception of a lower ( = 0.013) proportion of minor VFA (sum of isobutyrate, isovalerate, and valerate) in control group. Diet did not affect ( > 0.255) either pH and NH-N concentrations in the cecum or ileal morphology (crypt depth and villi length). The results showed that dietary fish oil supplementation enhanced beneficial long-chain n-3 FA and decreased n-6/n-3 ratio in rabbit meat and fat, being healthier for human consumption, without having negative effects on growth performance, cecal fermentation, and ileal morphology or carcass characteristics.

Effect of disodium/calcium malate or supplementation on growth performance, carcass quality, ruminal fermentation products, and blood metabolites of heifers

The aim of this study was to assess the effects of malate salts and culture on growth performance, carcass quality, ruminal fermentation products, and blood metabolites in heifers raised under southern Europe practical farm conditions. A total of 108 Charolaise cross heifers (214 ± 27.3 kg BW and 6.4 ± 1.1 mo of age) were housed in 18 pens of 6 animals each and used in a 114-d feedlot study. There was a totally randomized experimental design, and 6 pens were assigned to each of the following experimental diets: a control (no supplementation), the control plus 4 g of disodium/calcium malate mixture per kilogram of concentrate (2.12 g malate/kg), and the control plus 0.15 g of CBS 493.94 per kilogram of concentrate (1.5 × 10 cfu/kg). The control diet consisted of wheat-barley-based pelleted concentrate (32% starch, DM basis) and full-length barley straw. Concentrate and straw were fed separately ad libitum (5% orts) in an 88:12 ratio. On Days 0, 56, and 114, ruminal fluid and blood samples were obtained from each heifer between 2 and 2.5 h after the morning feeding by ruminocentesis and tail venipuncture, respectively. Body weight, concentrate ADFI, and G:F were recorded at 28, 56, 84, and 114 d. At slaughter, hot carcass weight and yield and carcass classification were determined in 2 representative heifers per pen (12 animals per dietary treatment). Supplementation with malate salts or did not affect concentrate ADFI ( = 0.98), ADG ( = 0.74), or G:F ( = 0.50) at any time during the experiment. At slaughter, there were no differences in carcass weight ( = 0.86), classification ( = 0.18), or carcass yield ( = 0.84) among experimental groups. Also, there were no differences treatments on ruminal pH ( = 0.24), ruminal fermentation products ( = 0.69, = 0.88, and = 0.93 for total VFA, NH-N, and lactate, respectively), and blood metabolites ( = 0.96, = 0.82, and = 0.15 for glucose, urea N, and lactate, respectively). In conclusion, under the feeding and management conditions of this study, diet supplementation with malate salts or did not have any significant effects on growth performance, carcass quality, ruminal fermentation products, and blood metabolites.

Protecting protein against ruminal degradation could contribute to reduced methane production

Ruminants have a low efficiency of nitrogen (N) utilization that has negative implications for animal production and the environment, but reducing the ruminal degradation of protein can help to reduce N losses. The objective of this study was to evaluate the inclusion of sunflower meal (SM) and sunflower seed (SS) protected against ruminal degradation in high-cereal diets on in vitro ruminal fermentation and CH4 production. Samples of SS and SM were sprayed with a solution of malic acid 1xa0M (400xa0ml/kg sample) and dried at 150°C for 1xa0hr as a protective treatment. Four diets were formulated to contain either 13 (low) or 17 (high) g of crude protein (CP)/100xa0g dry matter (DM), and included SM and SS either untreated (13CON and 17CON diets) or treated as before described (13TR and 17TR diets). Diets were incubated in vitro with rumen fluid from sheep for 8 and 24xa0hr. The treatment did not affect (pxa0≥xa00.57) total volatile fatty acid (VFA) production at any incubation time, but it reduced (pxa0<xa00.05) NH3 -N concentrations by 19.2 and 12.5% at 8 and 24xa0hr respectively. Both CH4 production and CH4 /VFA ratio were lower (pxa0<xa00.02) in TR than in CON diets at 8xa0hr, but differences disappeared (pxa0>xa00.05) at 24xa0hr. The treatment increased the molar proportion of propionate (pxa0=xa00.001) and reduced that of isovalerate (pxa0=xa00.03) at 8xa0hr compared with CON diets, but only a reduction of isovalerate proportion (pxa0=xa00.03) was detected at 24xa0hr. There were no treatment x crude protein level interactions (pxa0>xa00.05) in any parameter, but high-protein diets had greater NH3 -N concentrations (pxa0<xa00.001) and lower VFA production (pxa0<xa00.001) than low-protein diets at 24xa0hr. The treatment reduced protein degradation, and CH4 production was decreased by 4.6 and 10.8% for low- and high-protein diets, respectively, at short incubation times without affecting VFA production, thus improving fermentation efficiency and decreasing polluting emissions.