Comparison of automated ribosomal intergenic spacer analysis (ARISA) and denaturing gradient gel electrophoresis (DGGE) techniques for analysing the influence of diet on ruminal bacterial diversity.

The objective of this study was to compare the automated ribosomal intergenic spacer analysis (ARISA) and the denaturing gradient gel electrophoresis (DGGE) techniques for analysing the effects of diet on diversity in bacterial pellets isolated from the liquid (liquid-associated bacteria (LAB)) and solid (solid-associated bacteria (SAB)) phase of the rumen. The four experimental diets contained forage to concentrate ratios of 70:30 or 30:70 and had either alfalfa hay or grass hay as forage. Four rumen-fistulated animals (two sheep and two goats) received the diets in a Latin square design. Bacterial pellets (LAB and SAB) were isolated at 2 h post-feeding for DNA extraction and analysed by ARISA and DGGE. The number of peaks in individual samples ranged from 48 to 99 for LAB and from 41 to 95 for SAB with ARISA, and values of DGGE-bands ranged from 27 to 50 for LAB and from 18 to 45 for SAB. The LAB samples from high concentrate-fed animals tended (p < 0.10) to show greater peak numbers and Shannon index values than those isolated from high forage-fed animals with ARISA, but no differences were identified with DGGE. The SAB samples from high concentrate-fed animals had lower (p < 0.05) peak numbers and Shannon index values than those from animals fed high-forage diets with ARISA, but only a trend was noticed for these parameters with DGGE (p < 0.10). The ARISA detected that animals fed alfalfa hay diets showed lower (p < 0.05) SAB diversity than those fed grass hay diets, but no differences were observed with DGGE. No effect of forage type on LAB diversity was detected by any technique. In this study, ARISA detected some changes in ruminal bacterial communities that were not detected by DGGE, and therefore ARISA was considered more appropriate for assessing bacterial diversity of ruminal bacterial pellets. The results highlight the impact of the fingerprinting technique used to draw conclusions on dietary factors affecting bacterial diversity in ruminal bacterial pellets.

Effects of Garlic Oil and Cinnamaldehyde on Sheep Rumen Fermentation and Microbial Populations in Rusitec Fermenters in Two Different Sampling Periods.

Garlic oil (GO) and cinnamaldehyde (CIN) have shown potential to modify rumen fermentation. The aim of this study was to assess the effects of GO and CIN on rumen fermentation, microbial protein synthesis (MPS), and microbial populations in Rusitec fermenters fed a mixed diet (50:50 forage/concentrate), as well as whether these effects were maintained over time. Six fermenters were used in two 15-day incubation runs. Within each run, two fermenters received no additive, 180 mg/L of GO, or 180 mg/L of CIN. Rumen fermentation parameters were assessed in two periods (P1 and P2), and microbial populations were studied after each of these periods. Garlic oil reduced the acetate/propionate ratio and methane production (p < 0.001) in P1 and P2 and decreased protozoal DNA concentration and the relative abundance of fungi and archaea after P1 (p < 0.05). Cinnamaldehyde increased bacterial diversity (p < 0.01) and modified the structure of bacterial communities after P1, decreased bacterial DNA concentration after P2 (p < 0.05), and increased MPS (p < 0.001). The results of this study indicate that 180 mg/L of GO and CIN promoted a more efficient rumen fermentation and increased the protein supply to the animal, respectively, although an apparent adaptive response of microbial populations to GO was observed.

The Effect of Supplementation with Betaine and Zinc on In Vitro Large Intestinal Fermentation in Iberian Pigs under Heat Stress.

We investigated the effects of betaine and zinc on the in vitro fermentation of pigs under heat stress (HS). Twenty-four Iberian pigs (43.4 ± 1.2 kg) under HS (30 °C) were assigned to treatments for 4 weeks: control (unsupplemented), betaine (5 g/kg), and zinc (0.120 g/kg) supplemented diet. Rectal content was used as the inoculum in 24-hincubations with pure substrates (starch, pectin, inulin, cellulose). Total gas, short-chain fatty acid (SCFA), and methane production and ammonia concentration were measured. The abundance of total bacteria and several bacterial groups was assessed. Betaine increased the acetate production with pectin and inulin, butyrate production with starch and inulin, and ammonia concentration, and decreased propionate production with pectin and inulin. The abundance of Bifidobacterium and two groups of Clostridium decreased with betaine supplementation. Zinc decreased the production of SCFA and gas with starch and inulin, associated with diminished bacterial activity. Propionate production decreased with starch, pectin, and inulin while butyrate production increased with inulin, and isoacid production increased with cellulose and inulin in pigs supplemented with zinc. The ammonia concentration increased for all substrates. The Clostridium cluster XIV abundance decreased in pigs fed zinc supplemented diets. The results reported were dependent on the substrate fermented, but the augmented butyrate production with both betaine and zinc could be of benefit for the host.

Effects of Thymbra capitata essential oil on in vitro fermentation end-products and ruminal bacterial communities.

An in vitro trial was carried out to investigate the effects of natural Thymbra capitata essential oil (NEO) and its main compounds [including carvacrol, p-cymene, γ-terpinene given alone or in a synthetic combination (SEO)] on ruminal fermentation and the bacterial community using batch cultures inoculated with ruminal digesta and incubating two different basal diets [high-forage (F) and high-concentrate (C) diet]. After 24 h of incubation, primary fermentation end-products [gas, methane, volatile fatty acids (VFAs) and ammonia] and rumen microbial diversity were determined. NEO reduced the total VFA concentration (P < 0.05) only in the C diet. In contrast, SEO and carvacrol decreased the total VFA concentration (P < 0.05) only in the F diet. Methane production was not affected (P > 0.05) by any of the experimental treatments or diets evaluated. Microbial diversity analysis showed only a moderate effect of carvacrol and SEO on 13 genera, including, mainly, Atopobium and Blautia (involved in subacute ruminal acidosis) or Candidatus Saccharimonas (related to laminitis). In conclusion, T. capitata EO has a limited potential to attain nutritional or environmental benefits, but further research should be carried out to clarify its effects on animal health and microbial food safety.

Rumen protozoa and methanogenesis: not a simple cause-effect relationship.

Understanding the interactions between hydrogen producers and consumers in the rumen ecosystem is important for ruminant production and methane mitigation. The present study explored the relationships between rumen protozoa, methanogens and fermentation characteristics. A total of six donor sheep harbouring (F, faunated) or not (D, defaunated) protozoa in their rumens (D animals were kept without protozoa for a period of a few months (D - ) or for more than 2 years (D+)) were used in in vitro and in vivo experiments. In vitro the absence of protozoa decreased NH3 and butyrate production and had no effect on methane. In contrast, the liquid-associated bacterial and methanogens fraction of D+ inocula produced more methane than D -  and F inoculum (P < 0·05). In vivo fermentation parameters of donor animals showed the same trend on NH3 and butyrate and showed that D+ animals were high methane emitters, while D -  were the lowest ( - 35 %). The concentration of dissolved dihydrogen measured after feeding followed the opposite trend. Methane emissions did not correlate with the relative abundance of methanogens in the rumen measured by quantitative PCR, but there was a trend for higher methanogens concentration in the solid-associated population of D+ animals compared with D -  animals. In contrast, PCR-denaturing gradient gel electrophoresis profiles of methanogens' methyl coenzyme-M reductase A gene showed a clear clustering in liquid-associated fractions for all three groups of donors but fewer differences in solid-associated fractions. These results show that the absence of protozoa may affect differently the methanogen community and methane emissions in wethers.

Potential of Recycling Cauliflower and Romanesco Wastes in Ruminant Feeding: In Vitro Studies.

The nutritive values for ruminants of cauliflower (CAU) and Romanesco (ROM) wastes (leaves, stems and sprouts) were assessed by analyzing their chemical composition, in vitro ruminal fermentation, and in vitro intestinal digestibility. In addition, the in vitro ruminal fermentation of diets containing increasing amounts of CAU was studied. The dry matter (DM) content of leaves, stems and sprouts of both vegetables was lower than 10%, but they contained high crude protein (CP; 19.9 to 33.0%) and sugar (16.3 to 28.7%) levels, and low neutral detergent fiber (21.6 to 32.3%). Stems and sprouts were more rapidly and extensively fermented in the rumen than leaves, but there were only minor differences the fermentation profiles of both vegetables. The estimated metabolizable energy content ranged from 9.3 (leaves) to 10.8 (sprouts) MJ/kg DM. The CP rumen degradability (12-h in situ incubations) was greater than 80.0% for all fractions, and the in vitro intestinal digestibility of CP ranged from 85.7 to 93.2%. The inclusion of up to 24% of dried CAU in the concentrate of a mixed diet (40:60 alfalfa hay:concentrate) increased the in vitro rumen fermentation of the CAU diet, but did not affect methane (CH4) production, indicating the lack of antimethanogenic compounds in CAU.

In Vitro and In Situ Evaluation of Broccoli Wastes as Potential Feed for Ruminants.

The potential of broccoli wastes (florets and stems) as ruminant feed was analyzed using in vitro and in situ techniques. Both stems and florets had high moisture content (90.6 and 86.1%, respectively), but the stems contained (% dry matter) lower levels (p < 0.05) of crude protein (CP; 23.2 vs. 30.8%) and ether extract (2.91 vs. 6.15%) and tended to have greater sugars content (p = 0.071; 33.4 vs. 19.6%) than florets. Stems had greater in vitro dry matter rumen degradability (45.3%; 24 h incubation) and lower in vitro CP intestinal digestibility (82.7%) compared with florets (42.2 and 90.1%, respectively). Rumen degradability of protein was high (<85%) for both fractions. In a second experiment, diets including different proportions of broccoli were formulated and fermented in vitro. The replacement of 24% of conventional feeds (wheat, soybean meal and wheat bran) in a concentrate by dried broccoli increased the amount of organic matter fermented in vitro and the NH3-N concentrations of a mixed diet including 40% of the concentrate. Including dried broccoli in the diet produced only small modifications in the volatile fatty acid profile and did not affect CH4 emission.

Effects of Replacing Extruded Maize by Dried Citrus Pulp in a Mixed Diet on Ruminal Fermentation, Methane Production, and Microbial Populations in Rusitec Fermenters.

Citrus pulp is a highly abundant by-product of the citrus industry. The aim of this study was to assess the effects of replacing extruded maize (EM; 20% of total diet) by dried citrus pulp (DCP; 20%) in a mixed diet on rumen fermentation and microbial populations in Rusitec fermenters. The two diets contained 50% alfalfa hay and 50% concentrate, and the same protein level. Four Rusitec fermenters were used in a cross-over design with two 13-d incubation runs. After 7-d of diet adaptation, diet disappearance, fermentation parameters, microbial growth, and microbial populations were assessed. Fermenters receiving the DCP showed greater pH values and fiber disappearance (p < 0.001) and lower methane production (p = 0.03) than those fed EM. Replacing EM by DCP caused an increase in the proportions of propionate and butyrate (p < 0.001) and a decrease in acetate (p = 0.04). Microbial growth, bacterial diversity, and the quantity of bacteria and protozoa DNA were not affected by the diet, but the relative abundances of fungi and archaea were greater (p < 0.03) in solid and liquid phases of DCP fermenters, respectively. Results indicate that DCP can substitute EM, promoting a more efficient ruminal fermentation.

Heat Stress Increases In Vitro Hindgut Fermentation of Distinct Substrates in Iberian Pigs.

Heat stress reduces the feed intake and growth of pigs. We hypothesized that heat stress affects the intestinal fermentation capacity of pigs. Sixteen Iberian pigs (44 ± 1.0 kg) were randomly assigned to one of two treatments (eight pigs/treatment) for 4 weeks-heat stress (HS; 30 °C) ad libitum or thermoneutral (TN; 20 °C) pair feeding. Frozen rectum contents were used as inocula for 24 h in vitro incubations in which a mixture of starches, citrus pectin, inulin from chicory, and cellulose were the substrates. Cellulose was poorly degraded, whereas pectin and the mixture of starches were the most fermentable substrates according to total short-chain fatty acid (SCFA) production. The mixture of starches and inulin produced the greatest amount of gas. For all substrates, heat stress enhanced gas production (8%, p = 0.001), total SCFA production (16%, p = 0.001), and the production of acetate and propionate (12% and 42%, respectively; p = 0.001). The increased isoacid production (33%, p = 0.001) and ammonia concentration (12%, p = 0.001) may indicate protein fermentation under heat stress. In conclusion, the in vitro intestinal fermentation capacity of pigs under heat stress was increased compared to thermoneutral conditions, which may indicate an adaptive response to heat stress.

Replacing Forage by Crude Olive Cake in a Dairy Sheep Diet: Effects on Ruminal Fermentation and Microbial Populations in Rusitec Fermenters.

Olive oil extraction generates large amounts of a highly pollutant by-product called olive cake (OC), and its use in ruminant feeding could be an alternative. This study was designed to evaluate the effects of partially replacing forage by crude OC (COC) in a mixed dairy diet on rumen fermentation and microbial populations in Rusitec fermenters. The COC replaced 33% of the forage (66% maize silage and 33% barley straw) and was included at 16.6% of the total diet. Four fermenters were used in a cross-over design with two 13-day incubation periods. Experimental diets had a 50:50 forage-to-concentrate ratio and were formulated to contain the same protein (16.0%) and neutral detergent fiber (32.5%) levels. Compared with control fermenters, those fed the COC diet showed greater (p ≤ 0.02) pH (6.07 vs. 6.22), diet disappearance (0.709 vs. 0.748), and butyrate proportions (18.0 vs. 19.4), but there were no differences in volatile fatty acids and ammonia production. Microbial growth, bacterial diversity, protozoal abundance, and relative abundance of fungi and archaea were unaffected by diet, although the solid phase of COC-fed fermenters showed greater (p = 0.01) bacterial abundance than control ones. Results indicate that COC could replace 33% of the forage in a mixed dairy diet.

Evaluation of Brassica Vegetables as Potential Feed for Ruminants.

The objective of this study was to analyze the chemical composition, in vitro ruminal fermentation, and intestinal digestibility of discarded samples of four Brassica vegetables: Brussels sprouts (BS), white cabbage, Savoy cabbage, and red cabbage, and to assess the effects of including increasing amounts of BS in the concentrate of a dairy sheep diet on in vitro fermentation, CH4 production, and in situ degradation of the diets. All cabbages had low dry matter content (DM; <16.5%), but their DM had high crude protein (19.5-24.8%) and sugars (27.2-41.4%) content and low neutral detergent fiber (17.5-28%) and was rapidly and extensively fermented in the rumen. Rumen degradability of protein at 12 h of in situ incubation was greater than 91.5% for all cabbages, and in vitro intestinal digestibility of protein ranged from 61.4 to 90.2%. Replacing barley, corn, and soybean meal by 24% of dried BS in the concentrate of a diet for dairy sheep (40:60 alfalfa hay:concentrate) increased in vitro diet fermentation and in situ degradability of DM and protein, and reduced in vitro CH4/total volatile fatty acid ratio. In vivo trials are necessary to confirm these results.

Replacing Soybean Meal with Urea in Diets for Heavy Fattening Lambs: Effects on Growth, Metabolic Profile and Meat Quality.

Thirty-six Assaf male lambs (29.4 ± 3.10 kg body weight (BW)) were used to study the feasibility of including urea (at 0, 0.6 or 0.95% of dry matter for Control, Urea1, and Urea2 diets, respectively) in substitution of soybean meal in fattening diets. Animals were individually penned and feed intake was recorded daily. Blood samples were taken at days 35 and 63 of the experimental period to determine the acid-base status and the biochemical profile. At the end of the experiment (nine weeks), lambs were slaughtered, ruminal contents were collected and carcass and meat quality were evaluated. There were not differences (p > 0.05) among treatments in dry matter intake, animal performance, ruminal fermentation pattern, and carcass and meat parameters. Serum albumin concentration was higher and concentration of HCO3 and total CO2 in blood were lower in Urea2 compared to Urea1 and Control lambs. These results, together with the tendency to lower (p = 0.065) blood pH in this group might suggest a moderate metabolic acidosis. Partial replacement of soybean meal with urea did not impair growth rate in heavy fattening Assaf lambs (from 29 to 50 kg body weight), reduced feeding costs and had no adverse effects on feed efficiency, rumen fermentation and carcass and meat quality.

Chemical Composition, In Vitro Digestibility and Rumen Fermentation Kinetics of Agro-Industrial By-Products.

The nutritive value of 26 agro-industrial by-products was assessed from their chemical composition, in vitro digestibility and rumen fermentation kinetics. By-products from sugar beet, grape, olive tree, almond, broccoli, lettuce, asparagus, green bean, artichoke, peas, broad beans, tomato, pepper, apple pomace and citrus were evaluated. Chemical composition, in vitro digestibility and fermentation kinetics varied largely across the by-products. Data were subjected to multivariate and principal component analyses (PCA). According to a multivariate cluster analysis chart, samples formed four distinctive groups (A-D). Less degradable by-products were olive tree leaves, pepper skins and grape seeds (group A); whereas the more degradable ones were sugar beet, orange, lemon and clementine pulps (group D). In the PCA plot, component 1 segregated samples of groups A and B from those of groups C and D. Considering the large variability among by-products, most of them can be regarded as potential ingredients in ruminant rations. Depending on the characteristic nutritive value of each by-product, these feedstuffs can provide alternative sources of energy (e.g., citrus pulps), protein (e.g., asparagus rinds), soluble fibre (e.g., sugar beet pulp) or less digestible roughage (e.g., grape seeds or pepper skin).

Selection of internal reference genes for normalization of reverse transcription quantitative polymerase chain reaction (RT-qPCR) analysis in the rumen epithelium.

The rumen is lined on the luminal side by a stratified squamous epithelium that is responsible for not only absorption, but also transport, extensive short-chain fatty acid (SCFA) metabolism and protection. Butyrate has been demonstrated to initiate the differentiation of the tissue following introduction of solid feed to the weaning neonate as well as affecting the metabolism of other nutrients and absorption of nutrients in in vitro experiments. The objective of the present study was to validate expression stability of eight putative reference genes bovine rumen, considering the intrinsic heterogeneity of bovine rumen with regard to different luminal characteristics due to direct infusion of butyrate to double the intra-ruminal content of the rumen liquor. Our focus was on identifying stable reference genes which are suitable to normalize real-time RT-qPCR experiments from rumen samples collected from clinical assays, irrespective of localization within the organ and the across physiological state. The most stably expressed genes included: ACTB, UXT, DBNDD2, RPS9, DDX54 and HMBS. Their high stability values suggest these reference genes will facilitate better evaluation of variation of across an array of conditions including: localization within the rumen, differences among cattle fed an array of rations, as well as response to development in the weaning animal. Moreover, we anticipate these reference genes may be useful for expression studies in other ruminants.