Ruminal microbiome changes across lactation in primiparous Holstein cows with varying methane intensity: Heritability assessment.

Methane is a potent greenhouse gas produced during the ruminal fermentation and is associated with a loss of feed energy. Therefore, efforts to reduce methane emissions have been ongoing in the last decades. Methane production is highly influenced by factors such as the ruminal microbiome and host genetics. Previous studies have proposed to use the ruminal microbiome to reduce long-term methane emissions, as ruminal microbiome composition is a moderately heritable trait and genetic improvement accumulates over time. Lactation stage is another important factor that might influence methane production but potential associations with the ruminal microbiome have not been evaluated previously. This study sought to examine the changes in ruminal microbiome over the lactation period of primiparous Holstein cows differing in methane intensity and estimate the heritability of the abundance of relevant microorganisms. Ruminal content samples from 349 primiparous Holstein cows with 14 - 378 d in milk were collected from May 2018 to June 2019. Methane intensity (MI) of each cow was calculated as methane concentration/milk yield. Up to 64 taxonomic features (TF) from 20 phyla had a significant differential abundance between cows with low and high MI early in lactation, 16 TF during mid lactation, and none late in lactation. Taxonomical features within the Firmicutes, Proteobacteria, Melainabacteria, Cyanobacteria, Bacteroidetes and Actinobacteria phyla were associated to low MI, whereas eukaryotic TF and those within the Euryarchaeota, Verrucomicrobia, Kiritimatiellaeota, Lentisphaerae phyla were associated to high MI. Out of the 60 TF that were found to be differentially abundant between early and late lactation in cows with low MI, 56 TF were also significant when cows with low and high MI were compared in the first third of the lactation. In general, microbes associated with low MI were more abundant early in lactation (e.g., Acidaminococcus, Aeromonas and Weimeria genera) and showed low to moderate heritabilities (0.03 to 0.33). These results suggest some potential to modulate the rumen microbiome composition through selective breeding for lower MI. Differences in the ruminal microbiome of cows with extreme MI levels likely result from variations in the ruminal physiology of these cows and were more noticeable early in lactation probably due to important interactions between the host phenotype and environmental factors associated to that period. Our results suggest that the ruminal microbiome evaluated early in lactation may be more precise for MI difference, and hence, this should be considered to optimize sampling periods to establish a reference population in genomic selection scenarios.

Effects of agroindustrial by-product supplementation on dairy goat milk characteristics, nutrient utilization, ruminal fermentation, and methane production.

The use of agroindustrial by-products, such as dried distillers grains with solubles (DDGS) and dried citrus pulp (DCP), has been widely investigated in dairy cows, but information on their effects in dairy goats is limited. The influence of feeding olive cake (a by-product of olive oil production) to dairy goats has been assessed in some studies, but exhausted olive cake (EOC) has been much less investigated. Twelve Murciano-Granadina goats were used in a crossover design trial with 2 periods to assess the effects of including agroindustrial by-products on nutrient digestibility, ruminal fermentation, methane production, urinary excretion of purine derivatives, and milk yield and composition. In each period, 6 goats received daily a control diet comprising 1 kg of alfalfa hay and 1 kg of high-cereal concentrate, and another 6 goats received a diet (BYP) comprising 1 kg of alfalfa hay and 1 kg of a concentrate including corn DDGS, DCP, and EOC in proportions of 180, 180, and 80 g/kg of concentrate (as-fed basis), respectively. Diet had no effect on total dry matter intake, but intake of alfalfa hay, CP, and fat was greater for the BYP group than for the control group. There were no differences between diets in nutrient apparent digestibility, with the exception of fat, which was greater for the BYP diet compared with the control diet. Although fecal N tended to be greater for the BYP diet, there were no differences in N utilization. Compared with the control diet, milk yield tended to be greater and daily production of milk CP, fat, whey protein, and TS as well as milk gross energy were greater for the BYP diet. The concentration of C12:0, C14:0, and C16:0 fatty acids (FA) was or tended to be lower and the concentration of polyunsaturated FA was greater in the milk of BYP-fed goats compared with goats fed the control diet. Diet had no effect on ruminal parameters (pH, volatile FA, and NH3-N concentrations) and methane emissions, but urinary excretion of total purine derivatives tended to be lower in BYP-fed goats than in those fed the control diet. A mixture of corn DDGS (180 g), DCP (180 g), and EOC (80 g) could replace 44% of cereal grains and protein feeds in the concentrate for dairy goats without compromising nutrient utilization, ruminal fermentation, or milk yield and led to a more unsaturated FA profile in milk.

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 in vitro 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 35 mg of N/g of substrate organic matter and incubations lasted 16.5 h. With both proteins, N source × substrate interactions (p = 0.065 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 (p < 0.05) 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 (p < 0.05) in CH4 /total VFA ratio with STAR, whereas CH4 /total VFA ratio tended (p < 0.10) to be decreased with CEL substrate. Increasing the amount of both proteins decreased linearly (p < 0.05) 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 1 mg of CA or SP produced 1.24 and 0.60 µmol 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 24 h before, on the in vitro gas production. In general, the 24 h 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 24 h reduced (p < 0.05) 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 (p < 0.05) 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. clandestinum samples, CEL treatment reduced (p < 0.05) the molar proportion of acetate and increased (p < 0.05) 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.

Effect of dietary supplementation of two fiber sources differing on fermentability and hydration capacity on performance, nutrient digestibility and cecal fermentation in broilers from 1 to 42 d of age.

A total of 378 Cobb-500 male broilers were used to evaluate the effects of 2 fiber sources, differing in hydration capacity and fermentability, on gastrointestinal tract development, apparent ileal digestibility and performance from 1 to 42d of age. There were 9 replicates per each of the 3 dietary treatments, all in mash form: a wheat-soybean control (CON) diet, CON diet diluted with 1.5% of wood lignocellulose (LC diet) as a non-fermentable insoluble fiber with high hydration capacity; and CON diluted with 1.5% of a mixture of fibers (ISFC diet) containing both lignified insoluble fiber and a prebiotic soluble fiber fraction from fructooligosaccharides. Additionally, the fermentability of both fiber sources (LC and ISFC) was determined by in vitro using cecal inoculum from broilers fed the experimental diets. Both LC and ISFC treatments impaired by 4% feed conversion ratio only during the first 7d (P = 0.003) compared with CON group. In the grower period (21-42d), the ISFC group showed the best growth (P = 0.039), and at 42d tended to show the highest body weight (P = 0.095). This agrees well with the highest ileal dry matter (P = 0.033) and organic matter (P = 0.043) digestibility observed in ISFC group and the similar trend observed for ileal protein digestibility (P = 0.099) at 42d. Also, at 42 d, absolute and relative (% body weight) digestive tract weights (P ≤ 0.041) and empty gizzard weights (P ≤ 0.034) were greater for LC and ISFC groups compared to CON. The cecal molar proportion of valeratewas greatest in ISFC group (P = 0.039). In vitro gas production was higher for ISFC than for LC substrate when using either a diet-adapted or non-adapted cecal inoculum (P < 0.05). These results show the interest in combining IF with prebiotic highly fermentable fiber, such as fructooligosaccharides, in broilers to improve nutrient digestibility and finishing performance.

The effects of allopurinol, uric acid, and inosine administration on xanthine oxidoreductase activity and uric acid concentrations in broilers.

The purpose of these studies was to determine the effects of uric acid (UA) and inosine administration on xanthine oxidoreductase activity in broilers. In experiment one, 25 broilers were assigned to 5 treatment groups: control, AL (25 mg of allopurinol/kg of body mass), AR (AL for 2 wk followed by allopurinol withdrawal over wk 3), UAF (AL plus 6.25 g of UA sodium salt/kg of feed), and UAI (AL plus 120 mg of UA sodium salt injected daily). The UA administration had no effect on plasma concentration of UA (P > 0.05), and all allopurinol-treated birds had lower (P < 0.05) UA levels than controls. The UA concentrations were restored in both plasma and kidney of AR birds at wk 3, but liver UA concentrations remained lower. Whereas xanthine oxidoreductase (XOR) activity in the liver (LXOR) was reduced (P < 0.05) by allopurinol treatment, XOR activity in the kidney (KXOR) was not affected (P = 0.05). In experiment two, 3 groups of 5 birds each were fed 0 (control), 0.6 M inosine/kg of feed (INO), or INO plus 50 mg of allopurinol/kg of body mass (INOAL). The INOAL birds showed lower total LXOR activity, but KXOR activity was not affected. Both INO and INOAL birds had higher plasma and kidney UA concentrations than controls. The results suggest that regulation of UA production is tissue dependent.

Comparison of fermentation of diets of variable composition and microbial populations in the rumen of sheep and Rusitec fermenters. I. Digestibility, fermentation parameters, and microbial growth.

Four ruminally and duodenally cannulated sheep and 8 Rusitec fermenters were used to determine the effects of forage to concentrate (F:C) ratio and type of forage in the diet on ruminal fermentation and microbial protein synthesis. The purpose of the study was to assess how closely fermenters can mimic the dietary differences found in vivo. The 4 experimental diets contained F:C ratios of 70:30 or 30:70 with either alfalfa hay or grass hay as the forage. Microbial growth was determined in both systems using (15)N as a microbial marker. Rusitec fermenters detected differences between diets similar to those observed in sheep by changing F:C ratio on pH; neutral detergent fiber digestibility; total volatile fatty acid concentrations; molar proportions of acetate, propionate, butyrate, isovalerate, and caproate; and amylase activity. In contrast, Rusitec fermenters did not reproduce the dietary differences found in sheep for NH(3)-N and lactate concentrations, dry matter (DM) digestibility, proportions of isobutyrate and valerate, carboxymethylcellulase and xylanase activities, and microbial growth and its efficiency. Regarding the effect of the type of forage in the diet, Rusitec fermenters detected differences between diets similar to those found in sheep for most determined parameters, with the exception of pH, DM digestibility, butyrate proportion, and carboxymethylcellulase activity. Minimum pH and maximal volatile fatty acid concentrations were reached at 2h and at 6 to 8h postfeeding in sheep and fermenters, respectively, indicating that feed fermentation was slower in fermenters compared with that in sheep. There were differences between systems in the magnitude of most determined parameters. In general, fermenters showed lower lactate concentrations, neutral detergent fiber digestibility, acetate:propionate ratios, and enzymatic activities. On the contrary, fermenters showed greater NH(3)-N concentrations, DM digestibility, and proportions of propionate, butyrate, isovalerate, valerate, and caproate. Values of efficiency of microbial growth were greater in fermenters compared with sheep for 70:30 diets, but they were lower for 30:70 diets. Differences between fermentation in sheep and fermenters can be mainly attributed to the lack of absorption in fermenters, differences in solid retention time, and compartmentalization in the Rusitec system. In general, the Rusitec system simulated more closely the in vivo fermentation of high-forage diets compared with high-concentrate diets.

Comparison of fermentation of diets of variable composition and microbial populations in the rumen of sheep and Rusitec fermenters. II. Protozoa population and diversity of bacterial communities.

Four ruminally and duodenally cannulated sheep and 8 Rusitec fermenters were used to determine the effects of dietary characteristics on microbial populations and bacterial diversity. The purpose of the study was to assess how closely fermenters can mimic the differences between diets found in vivo. The 4 experimental diets contained forage to concentrate (F:C) ratios of 70:30 (high forage; HF) or 30:70 (high concentrate; HC) with either alfalfa hay (A) or grass hay (G) as the forage. Total bacterial numbers were greater in the rumen of sheep fed HF diets compared with those fed HC diets, whereas the opposite was found in fermenters. The numbers of cellulolytic bacteria were not affected by F:C ratio in any fermentation system, but cellulolytic numbers were 2.7 and 1.8 times greater in sheep than in fermenters for HF and HC diets, respectively. Neither total bacterial nor cellulolytic numbers were affected by the type of forage in sheep or fermenters. Decreasing F:C ratio increased total protozoa and Entodiniae numbers in sheep by about 29 and 25%, respectively, but it had no effect in fermenters. Isotrichidae and Ophryoscolecinae numbers in sheep were not affected by changing F:C ratio, but both disappeared completely from fermenters fed HC diets. Total protozoa and Entodiniae numbers were greater in sheep fed A diets than in those fed G diets, whereas the opposite was found in fermenters. Results indicate that under the conditions of the present study, protozoa population in Rusitec fermenters was not representative of that in the rumen of sheep fed the same diets. In addition, protozoa numbers in fermenters were 121 and 226 times lower than those in the sheep rumen for HF and HC diets, respectively. The automated ribosomal intergenic spacer analysis of the 16S ribosomal DNA was used to analyze the diversity of liquid- and solid-associated bacteria in both systems. A total of 170 peaks were detected in the automated ribosomal intergenic spacer analysis electropherograms of bacterial pellets across the full set of 64 samples, from which 160 were detected in at least 1 individual from each system (sheep or fermenter). Diversity of liquid-associated bacterial pellets was greater with G diets in fermenters but seemed to be unaffected by diet in sheep. Bacterial diversity in solid-associated bacteria pellets was greater for G diets compared with A diets in sheep and fermenters. Different conditions in the fermenters compared with sheep rumen might have caused a selection of some bacterial strains.

Ruminal fermentation, microbial growth and amino acid flow in single-flow continuous culture fermenters fed a diet containing olive leaves.

Six single-flow continuous culture fermenters were used to determine fermentation profile, microbial growth and amino acid (AA) flow promoted by olive leaves supplemented with barley grains and faba beans (OLSUP), and alfalfa hay (AH). Two incubation runs were carried out with three fermenters inoculated with ruminal fluid from wethers and three from goats. The inoculum source did not affect (p = 0.059 to 0.980) any of the parameters. Daily volatile fatty acid (VFA) production and carbohydrate digestibility were greater (p = 0.009 and 0.024, respectively) for AH, therefore the pH values were lower (p = 0.015) than for OLSUP. Acetate was greater (p < 0.001) and isobutyrate, isovalerate and caproate lower (p < 0.001 to 0.006) for AH with greater acetate/propionate (p = 0.014) and 'VFA/digested carbohydrate' (p = 0.026) ratios. Daily microbial N flow and efficiency were greater (p = 0.016 and p = 0.041) for diet AH. Individual AA flows were greater (p < 0.001 to 0.016) for AH, but microbial essential AA proportion was greater for OLSUP (p = 0.015). The results indicate that OLSUP promoted lower bacterial growth and AA flow than AH, which could have been partially due to a limitation of N availability to ruminal microbes.

Effects of dilution rate and retention time of concentrate on efficiency of microbial growth, methane production, and ruminal fermentation in Rusitec fermenters.

The objective of this study was to investigate the effects of 2 dilution rates (DL) and 2 concentrate retention times (RT) on microbial growth, methane production, and fermentation of a 30:70 alfalfa hay:concentrate diet in Rusitec fermenters maintained at similar pH. The DL were 3.78 (low DL, LDL) and 5.42%/h (high DL, HDL), and concentrate RT was either 24 h (T24) or 48 h (T48). Forage RT was 48 h in all fermenters. Apparent disappearance of diet DM and NDF was greater in HDL fermenters compared with LDL fermenters, but there was a significant DL x concentrate RT interaction, showing that the effect of DL was more pronounced in T48 compared with T24 fermenters. Methane production was not affected by DL, but was greater in T48 compared with T24 fermenters, which was consistent with the increased fiber degradation in T48 fermenters. Increasing DL augmented volatile fatty acid production and molar proportions of propionate, isovalerate, and valerate, and reduced those of caproate, but no effects were observed on acetate, butyrate, and isobutyrate proportions. Increasing concentrate RT resulted in greater volatile fatty acid production and proportions of acetate, butyrate, and caproate, but reduced those of propionate, valerate, and isovalerate. Ammonia-N production was not affected by concentrate RT, but was greater at HDL compared with LDL. Microbial growth was not affected by DL, but microbial growth efficiency was lower in HDL compared with LDL fermenters. Concentrate RT affected microbial growth and its efficiency, with both being greater in T48 compared with T24 fermenters. Carboxymetylcellulase and xylanase activities in ruminal fluid were greater in HDL compared with LDL fermenters, but were not affected by concentrate RT. There were DL x concentrate RT interactions for diet apparent disappearance, molar proportions of propionate, butyrate, isovalerate, and caproate, and acetate:propionate ratio, indicating that effects of DL on these variables were influenced by concentrate RT. The results would indicate that using higher DL and shorter concentrate RT than those typically used in Rusitec fermenters would contribute to improving the simulation of in vivo fermentation of high-concentrate diets.

Influence of detachment procedure and diet on recovery of solid-associated bacteria from sheep ruminal digesta and representativeness of bacterial isolates as assessed by automated ribosomal intergenic spacer analysis-polymerase chain reaction.

Six ruminally and duodenally cannulated sheep were used in a partially replicated 4 x 4 Latin square experiment designed to evaluate the efficiency of 3 detachment procedures (DP) to recover solid-associated bacteria (SAB) from ruminal digesta. The 4 experimental diets contained forage to concentrate (F:C) ratios of 70:30 or 30:70 with either alfalfa hay or grass hay as the forage. Bacterial biomass was labeled with 15NH4Cl. The DP were 1) MET: digesta was incubated at 38 degrees C for 15 min with saline solution (0.9% NaCl) containing 0.1% methylcellulose under continuous shaking; 2) STO: digesta was mixed with cold saline solution and homogenized with a stomacher for 5 min at 230 rpm; 3) FRE: digesta was immediately frozen at -20 degrees C for 72 h, thawed at 4 degrees C, mixed with saline solution and subjected to STO procedure. Common to all treatments was storing at 4 degrees C for 24 h after the treatment, homogenization, filtration, and resuspension of digesta 2 times in the treatment solutions. The automated ribosomal intergenic spacer analysis of the 16S ribosomal DNA was used to analyze the similarity between bacterial communities attached to the digesta and those in the pellet obtained after each DP. There were no significant F:C x DP or forage x DP interactions for any variable. On average, STO treatment detached 65.8% of SAB from ruminal digesta, about 1.2 and 1.5 times more than FRE and MET treatments, respectively. Total recovery of SAB in STO pellets (48.9%) was greater compared with FRE (31.7%) and MET (33.1%), values being greater for high-forage compared with high-concentrate diets. Similarity index between the bacteria attached to digesta and those in the pellets were lower for FRE (48.2%) compared with MET (54.1%) and STO (54.1%), which suggests that FRE could have destroyed cell integrity of some bacterial species, thus reducing the bacterial diversity present in the pellets. The STO method was the most effective removing SAB from digesta, but only a moderate similarity between the bacterial communities attached to digesta and those recovered in the bacterial pellets was obtained. Values of duodenal microbial flow estimated using SAB as reference bacteria were greater with FRE compared with STO and MET, but all DP detected similar differences between diets, and therefore did not influence the interpretation of results.

Determination of xanthine oxidoreductase activity in broilers: effect of pH and temperature of the assay and distribution in tissues.

Xanthine oxidoreductase (XOR) is the enzyme responsible for the synthesis of uric acid, which exists primarily in the dehydrogenase form in birds. Uric acid is the major end product of the metabolism of nitrogen-containing compounds in birds and it functions as an antioxidant to reduce oxidative stress. Despite the importance of this enzyme, the tissue distribution of XOR in physiologically normal chickens is not well known. In this study, we analyzed XOR activity in extracts of 8 tissues from broilers at 7 and 10 wk of age. No differences in XOR activity due to the age were found in any tissue. Liver and kidney showed the greatest activity, that in the kidney being about 89% of the activity in the liver. Enzyme activity in intestine and pancreas was about 60 and 37% of that in the liver. All breast muscle, heart, and lung samples showed enzyme activity, but values were only 3.0, 1.2, and 0.6% of those found in the liver. Traces of enzyme activity were also detected in 3 out of 10 brain samples, and no activity was found in the plasma. Our results show that XOR distribution in chickens differs from that in mammals, in which the highest levels have been found in liver and intestine. An additional objective was the evaluation of the effect of pH (7.2, 7.7, 8.2, and 8.7) and temperature (25 and 41 degrees C) on the enzyme activity in liver and kidney samples. Temperature had a similar effect on both tissues, with the activity at 25 degrees C being about 30% of that measured at 41 degrees C. At 41 degrees C, the enzyme activity in liver and kidney decreased quadratically as pH decreased from 8.7 to 7.2. The highest activity in kidney was measured at pH 8.2, although there were no differences between enzyme activities at pH 8.7 or 8.2 in the liver. Our results indicate that the optimum pH of the enzyme in chicken liver and kidney is around 8.2.

Comparison of bacterial pellets and microbial markers for the estimation of the microbial nitrogen and amino acids flows from single flow continuous culture fermenters fed diets containing two-stage olive cake.

The effects of using effluent bacteria (EB) and solid- (SAB) and liquid- (LAB) associated bacteria and diaminopimelic acid (DAPA) or purine bases (PB) and partially substituting alfalfa hay (AH) by a concentrate including olive cake on the microbial N flow (MNF) and amino acids (AA) flow were investigated with continuous culture fermenters fed AH and a mixture of AH and a concentrate containing barley grains and two-stage olive cake (2:1 ratio) without (AHCO) or with polyethylene glycol (PEG) (AHCOP). The MNF was not different among diets with SAB or LAB (p = 0.302 and 0.203, respectively) and DAPA, but differed with PB (p = 0.021 and 0.014, respectively). With EB both markers detected similar differences, AHCOP showing a higher value (p < 0.05) than AH and AHCO. The MNF was higher (p < 0.001) with PB than DAPA. Daily flow of non-essential AA was not different (p = 0.356) among diets but essential AA flow was higher (p < 0.05) for AH and AHCOP than for AHCO. The SAB presented lower (p < 0.05) total AA than LAB and higher total AA (p < 0.05) for diet AH than AHCO. The AA profile of EB was similar to that of LAB, but alanine and leucine were higher (p < 0.05) in EB than in LAB. Microbial contribution to AA flow was 45.4%, 55.6% and 58.1% for diets AH, AHCO and AHCOP respectively. With both markers, microbial AA flow was higher (p < 0.05) for diet AHCOP compared with AH (451 and 355 mg/day, respectively), but not different (p > 0.05) for AHCOP and AHCO (389 mg/day). The results would indicate that olive cake could be used in the practical feeding of small ruminants without negatively affecting microbial AA N supply.

Nutritive Value of Tomato Pomace for Ruminants and Its Influence on In Vitro Methane Production.

The objective of this study was to determine the variability in nutritive value for ruminants of tomato pomace (TP) samples and analyze its effect on in vitro fermentation when it was included in a high-concentrate diet. Twelve TP samples were obtained from two processing plants at weekly intervals and analyzed for chemical composition, in vitro rumen fermentation, and intestinal digestibility. The chemical composition of TP did not differ between processing plants and only slight variations were observed among sampling times. Tomato pomace had a low dry matter content (<300 g/kg), a high content of neutral detergent fiber, crude protein, and ether extract (572, 160, and 82.7 g/kg dry matter on average, respectively), and was rapidly fermented in the rumen. Protein degradability at 16 h in situ incubation was 510 g/kg and in vitro intestinal digestibility of protein was low (430-475 g/kg). Replacing soybean meal and barley straw by dried TP increased the in vitro fermentation rate and the production of volatile fatty acids and reduced NH3-N concentrations without affecting CH4. In summary, TP samples showed little variability in nutritive value over sampling time and TP of up to 180 g/kg could be included in high-concentrate diets without negatively affecting rumen fermentation.

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.

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.

Effect of garlic oil and four of its compounds on rumen microbial fermentation.

Different concentrations (3, 30, 300, and 3000 mg/L of culture fluid) of garlic oil (GAR), diallyl sulfide (DAS), diallyl disulfide (DAD), allicin (ALL), and allyl mercaptan (ALM) were incubated for 24 h in diluted ruminal fluid with a 50:50 forage:concentrate diet (17.7% crude protein; 30.7% neutral detergent fiber) to evaluate their effects on rumen microbial fermentation. Garlic oil (30 and 300 mg/L), DAD (30 and 300 mg/L), and ALM (300 mg/L) resulted in lower molar proportion of acetate and higher proportions of propionate and butyrate. In contrast, at 300 mg/L, DAS only increased the proportion of butyrate, and ALL had no effects on volatile fatty acid proportions. In a dual-flow continuous culture of rumen fluid fed the same 50:50 forage:concentrate diet, addition of GAR (312 mg/L), DAD (31.2 and 312 mg/L), and ALM (31.2 and 312 mg/L) resulted in similar changes to those observed in batch culture, with the exception of the lack of effect of DAD on the proportion of propionate. In a third in vitro study, the potential of GAR (300 mg/L), DAD (300 mg/L), and ALM (300 mg/L) to decrease methane production was evaluated. Treatments GAR, DAD, and ALM resulted in a decrease in methane production of 73.6, 68.5, and 19.5%, respectively, compared with the control. These results confirm the ability of GAR, DAD, and ALM to decrease methane production, which may help to improve the efficiency of energy use in the rumen.

Plasmatic profiles of pregnancy-associated glycoprotein and progesterone levels during gestation in Churra and Merino sheep.

This study was carried out to determine ovine pregnancy-associated glycoprotein (oPAG) and progesterone (P4) levels in the serum of Churra and Merino ewes throughout gestation and the first month post partum. The oPAG levels were determined with an heterologus RIA using bovine PAG as standard and tracer and rabbit antiserum against oPAG, sensitivity was 4.0 ng/ml. The P4 levels were measured with a radioimmunological procedure, including a specific extraction step with petroleum ether (bp 60-80 degrees C) with a sensitivity of less than 0.1 ng/ml. There were no differences (P<0.10) in the oPAG profile between breeds from Weeks 1 to 18. From Week 18 to lambing, oPAG concentrations increased rapidly in Churra ewes (on average, from 250 to 650 ng/ml) while remaining relatively constant in the Merino ewes (around 250 ng/ml). No significant differences (P>0.05) were observed for mean weekly P4 levels between the 2 breeds. In both breeds, P4 increased throughout the whole length of gestation, with the highest level measured at Weeks 19-20, then declined 2 wk before parturition. No correlation was found between P4 and oPAG concentrations during gestation in either of the breeds. After lambing, oPAG and P4 levels decreased rapidly in 4 wk to basal values. In both breeds the oPAG concentrations at Weeks 19, 20 and 21 of gestation in ewes carrying male fetuses were higher than in those carrying female fetuses. From the results, we conclude that the breed and sex of the fetus could influence the production of oPAG.

Diet and procedures used to detach particle-associated microbes from ruminal digesta influence chemical composition of microbes and estimation of microbial growth in Rusitec fermenters.

Four different detachment methods were evaluated for their ability to remove particle-associated microorganisms (PAM) from ruminal digesta in semicontinuous fermenters fed two diets differing in their forage:concentrate ratio (80:20 [C20] and 20:80 [C80]). In the methylcellulose method, ruminal digesta was incubated at 38 degrees C for 15 min with saline solution containing 0.1% methylcellulose before being stored at 4 degrees C for 24 h. In the other procedures, samples were incubated with 0.1% methylcellulose before storage for 24 h at 4 degrees C in different solutions (pH = 2): 1) saline solution with 0.1% Tween 80; 2) saline solution with 0.1% Tween 80 and 1% tertiary butanol; and 3) saline solution with 0.1% Tween 80, 1% methanol, and 1% tertiary butanol. Common to all treatments was subsequent homogenization, followed by filtration and resuspension of the residue five times in the treatment solutions. Microbial removal was estimated indirectly by measuring removal of 15N. There were no differences (P > 0.05) among detachment procedures, neither in the detaching efficiency (mean values of 79.7 and 88.1% for C20 and C80 diets, respectively) nor in the total recovery of PAM (54.9 and 34.9% for C20 and C80, respectively). There were no differences (P > 0.05) among PAM pellets obtained by the different detachment procedures in their N content, purine bases (PB) concentration, or PB:N ratio. For the C80 diet, 15N enrichment was greater (P < 0.05) in PAM pellets obtained with methylcellulose than in those obtained by the other methods. However, there were no differences (P > 0.05) due to the detachment procedure in the values of daily microbial growth estimated using as reference the different PAM pellets. The PAM pellets for diet C20 presented greater (P < 0.01) N content and lower (P < 0.01) PB concentration than those for diet C80 (mean values of 74.3 vs 49.1 mg of N/g of dry matter, and 22.8 vs 26.0 micromol PB/mg of dry matter, respectively). Daily microbial growth was greater (P < 0.05) for the C80 diet than for the C20 diet (121 vs 114 mg of microbial N, respectively). Results suggest that the treatment of ruminal digesta with a saline solution with 0.1% methylcellulose at 38 degrees C for 15 min combined with homogenizing and chilling at 4 degrees C for 24 h removed a major proportion of PAM, although further research is needed to decrease microbial losses during the isolation process.