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.     

GC–MS analysis of the ruminal metabolome response to thiamine supplementation during high grain feeding in dairy cows

Introduction

Thiamine is known to attenuate high-concentrate diet induced subacute ruminal acidosis (SARA) in dairy cows, however, the underlying mechanisms remain unclear.

Objectives

The major objective of this study was to investigate the metabolic mechanisms of thiamine supplementation on high-concentrate diet induced SARA.

Methods

Six multiparous, rumen-fistulated Holstein cows were used in a replicated 3?×?3 Latin square design. The treatments included a control diet (CON; 20% starch, dry matter basis), a SARA-inducing diet (SAID; 33.2% starch, dry matter basis) and SARA-inducing diet supplemented with 180 mg of thiamine/kg of dry matter intake (SAID?+?T). On d21 of each period, ruminal fluid samples were collected at 3 h post feeding, and GC/MS was used to analyze rumen fluid samples.

Results

PCA and OPLS-DA analysis demonstrated that the ruminal metabolite profile were different in three treatments. Compared with CON treatment, SAID feeding significantly decreased rumen pH, acetate, succinic acid, increased propionate, pyruvate, lactate, glycine and biogenic amines including spermidine and putrescine. Thiamine supplementation significantly decreased rumen content of propionate, pyruvate, lactate, glycine and spermidine; increase rumen pH, acetate and some medium-chain fatty acids. The enrichment analysis of different metabolites indicated that thiamine supplementation mainly affected carbohydrates, amino acids, pyruvate and thiamine metabolism compared with SAID treatment.

Conclusions

These findings revealed that thiamine supplementation could attenuate high-concentrate diet induced SARA by increasing pyruvate formate-lyase activity to promote pyruvate to generate acetyl-CoA and inhibit lactate generation. Besides, thiamine reduced biogenic amines to alleviate ruminal epithelial inflammatory response.

     

Transient reductions in milk fat synthesis and their association with the ruminal and metabolic profile in dairy cows fed high-starch,low-fat diets

Sub-acute ruminal acidosis (SARA) is sometimes observed along with reduced milk fat synthesis. Inconsistent responses may be explained by dietary fat levels. Twelve ruminally cannulated cows were used in a Latin square design investigating the timing of metabolic and milk fat changes during Induction and Recovery from SARA by altering starch levels in low-fat diets. Treatments were (1) SARA Induction, (2) Recovery and (3) Control. Sub-acute ruminal acidosis was induced by feeding a diet containing 29.4% starch, 24.0% NDF and 2.8% fatty acids (FAs), whereas the Recovery and Control diets contained 19.9% starch, 31.0% NDF and 2.6% FA. Relative to Control, DM intake (DMI) and milk yield were higher in SARA from days 14 to 21 and from days 10 to 21, respectively (P < 0.05). Milk fat content was reduced from days 3 to 14 in SARA (P < 0.05) compared with Control, while greater protein and lactose contents were observed from days 14 to 21 and 3 to 21, respectively (P < 0.05). Milk fat yield was reduced by SARA on day 3 (P < 0.05), whereas both protein and lactose yields were higher on days 14 and 21 (P < 0.05). The ruminal acetate-to-propionate ratio was lower, and the concentrations of propionate and lactate were higher in the SARA treatment compared with Control on day 21 (P < 0.05). Plasma insulin increased during SARA, whereas plasma non-esterified fatty acids and milk β-hydroxybutyrate decreased (P < 0.05). Similarly to fat yield, the yield of milk preformed FA (>16C) was lower on day 3 (P < 0.05) and tended to be lower on day 7 in SARA cows (P < 0.10), whereas yield of de novo FA (<16C) was higher on day 21 (P < 0.01) in the SARA group relative to Control. The t10- to t11-18:1 ratio increased during the SARA Induction period (P < 0.05), but the concentration of t10-18:1 remained below 0.5% of milk fat, and t10,c12 conjugated linoleic acid remained below detection levels. Odd-chain FA increased, whereas branched-chain FA was reduced during SARA Induction from days 3 to 21 (P < 0.05). Sub-acute ruminal acidosis reduced milk fat synthesis transiently. Such reduction was not associated with ruminal biohydrogenation intermediates but rather with a transient reduction in supply of preformed FA. Subsequent rescue of milk fat synthesis may be associated with higher availability of substrates due to increased DMI during SARA.     

A rapid shift to high-grain diet results in dynamic changes in rumen epimural microbiome in sheep

The rapid shift to high-grain (HG) diets in ruminants can affect the function of the rumen epithelium, but the dynamic changes in the composition of the epithelium-associated (epimural) bacterial community in sheep still needs further investigation. Twenty male lambs were randomly allocated to four groups (n = 5). Animals of the first group received hay diet and represented a control group (CON). Simultaneously, animals in the other three groups (HG groups) were rapidly shifted to an HG diet (60% concentrate)which continued for 7 (HG7), 14 (HG14) and 28 (HG28) days, correspondingly. Results showed that ruminal pH dramatically decreased due to the rapid shift to the HG diet (P <0.001), while, the concentrations of butyrate (P <0.001), lactate (P = 0.001), valerate (P = 0.008) and total volatile fatty acids (P = 0.001) increased. Diversity estimators showed a dramatic decrease after the shift without recovering as the HG feeding continued. The principal coordinates analysis showed that CON group clustered separately from all HG groups with the presence of significant difference only between HG7 and HG28 (P = 0.034). The non-parametric multivariate analysis (npmv R-package) deduced that the primary significant differences in phyla and phylogenetic investigation of communities by reconstruction of unobserved states (PICRUSt)-predicted Kyoto Encyclopedia of Genes and Genomes (KEGGs) was attributed mainly to the diet composition (P <0.001, P = 0.001) compared to its application period (P = 0.140, 0.545) which showed a significant effect only on the genus (P = 0.001) and the operational taxonomic units (OTUs) level (P = 0.011). The Kruskal–Wallis test deduced that six phyla showed a significant effect due to the shift in diet composition. At the genus level, HG feeding altered the abundance of 12 taxa, four of which showed a significant variation due to the duration of the HG diet application. Similarly, we found that 21 OTUs showed significant variations due to the duration of the HG diet application. Furthermore, the genes abundance predicted by PICRUSt revealed that the HG feeding significantly affected seven metabolic pathways identified in the KEGG. Particularly, the abundance of gene families associated with carbohydrates metabolism were significantly higher in HG feeding groups (P = 0.027). Collectively, these results revealed that the rapid transition to an HG diet causes dramatic alterations in ruminal fermentation and the composition and function of ruminal epithelium-associated microbiome in sheep, while, the duration of the HG diet application causes drastic alterations to the abundance of some species.     

Supplementation of a clay mineral-based product modulates plasma metabolomic profile and liver enzymes in cattle fed grain-rich diets

Grain-rich diets often lead to subacute ruminal acidosis (SARA) impairing rumen and systemic cattle health. Recent data suggest beneficial effects of a clay mineral (CM)- based product on the rumen microbiome of cattle during SARA. This study sought to investigate whether the CM supplementation can counteract SARA-induced perturbations of the bovine systemic health. The study used an intermittent diet-induced SARA-model with eight dry Holstein cows receiving either no additive as control or CM via concentrates (n=8 per treatment). Cows received first a forage diet (Baseline) for 1 week, followed by a 1-week SARA-challenge (SARA 1), a 1-week recovery phase (Recovery) and finally a second SARA-challenge for 2 weeks (SARA 2). Cows were monitored for feed intake, reticular pH and chewing behavior. Blood samples were taken and analyzed for metabolites related to glucose and lipid metabolism as well as liver health biomarkers. In addition, a targeted electrospray ionization-liquid chromatography-MS-based metabolomics approach was carried out on the plasma samples obtained at the end of the Baseline and SARA 1 phase. Data showed that supplementing the cows’ diet with CM improved ruminating chews per regurgitated bolus by 16% in SARA 1 (P=0.01) and enhanced the dry matter intake during the Recovery phase (P=0.05). Moreover, the SARA-induced decreases in several amino acids and phosphatidylcholines were less pronounced in cows receiving CM (P≤0.10). The CM-supplemented cows also had lower concentrations of lactate (P=0.03) and biogenic amines such as histamine and spermine (P<0.01) in the blood. In contrast, the concentration of acylcarnitines with key metabolic functions was increased in the blood of treated cows (P≤0.05). In SARA 2, the CM-cows had lower concentrations of the liver enzymes aspartate aminotransferase and γ-glutamyltransferase (P<0.05). In conclusion, the data suggest that supplementation of CM holds the potential to alleviate the negative effects of high-grain feeding in cattle by counteracting multiple SARA-induced perturbations in the systemic metabolism and liver health.     

High concentrate-induced subacute ruminal acidosis (SARA) increases plasma acute phase proteins (APPs) and cortisol in goats

The aim of this study was to investigate changes of stress status in dairy goats induced to subacute ruminal acidosis (SARA). The level of acute phase proteins (APPs) including haptoglobin (HP) and serum amyloid A (SAA) in plasma and their mRNA expression in liver, as well as plasma cortisol and genes expression of key factors controlling cortisol synthesis in adrenal cortex were compared between SARA and control goats. SARA was induced by feeding high concentrate diet (60% concentrate of dry matter) for 3 weeks (SARA, n=6), while control goats (Con, n=6) received a low concentrate diet (40% concentrate of dry matter) during the experimental time. SARA goats showed ruminal pH below 5.8 for more than 3 h per day, which was significantly lower than control goats (pH>6.0). SARA goats demonstrated a significant increase of hepatic HP and SAA mRNA expression (P<0.05), and the level of HP but not SAA in plasma was markedly increased compared with control (P<0.05). The level of cortisol in plasma showed a trend to increase in SARA goats (0.05<P<0.1). In adrenal cortex, mRNA expression of 17α-hydroxylase cytochrome (P450_17α) (P<0.01) and 3β-hydroxysteroid dehydrogenase (3β-HSD) (P<0.05) was significantly increased in SARA goats. The contents of 3β-HSD and P450 side-chain cleavage protein were increased by 58.6% and 39.4%, respectively, but did not reach the statistical significance (P>0.05). These results suggested that SARA goats experienced a certain stress status, exhibiting an increase in HP production and cortisol secretion.     

Combined effects of an antioxidant and caspase inhibitor on the reversal of hepatic fibrosis in rats

We sought to determine the hepatic fibrosis-reversal effects upon simultaneous administration of lithospermate B (LAB), an anti-oxidant, and nivocasan, a caspase inhibitor, to rats compared with each compound alone. Liver fibrosis was induced in Sprague–Dawley rats by thioacetamide (TAA). Rats were treated with TAA and then given LAB and (or) nivocasan. Fibrotic areas were evaluated quantitatively by computerized morphometry. Apoptosis was assessed using a TUNEL assay, and immunohistochemical staining for malondialdehyde (MDA) and 4-hydroxy-2-nonenal (4HNE) was performed to assess oxidative stress levels. Real-time quantitative PCR was used to quantify expression of fibrosis-related genes. The degree of hepatic fibrosis was significantly reduced in rats treated with LAB and nivocasan compared to either treatment alone (P < 0.001). Treatment with each compound significantly decreased expression of fibrosis-related genes, such as type I collagen α1 (col1α1), α-SMA and TGF-β1 (P < 0.05). Co-treatment with LAB and nivocasan further reduced col1α1 expression compared to treatment with either compound. A TUNEL assay revealed that hepatocyte apoptosis was significantly decreased in the group treated with nivocasan compared to other groups (P < 0.01). Immunohistochemistry showed a decrease in MDA and 4HNE, reflecting amelioration of oxidative stress, when LAB or LAB+nivocasan was administered compared to nivocasan alone (P < 0.01). Nivocasan was found to inhibit caspase-1, -3, -7, -9 and gliotoxin-induced death of rat-derived hepatic stellate cells was inhibited by nivocasan administration without overexpression of α-SMA. Conclusions: Co-incidental administration of LAB and nivocasan suppressed oxidative stress and apoptosis, resulting in enhanced reversal of hepatic fibrosis in rat.     

Effects of feed withdrawal duration on animal behaviour,rumen microbiota and blood chemistry in feedlot cattle: implications for rumen acidosis

Feed withdrawal (FW) is a frequent issue in open outdoor feedlot systems, where unexpected circumstances can limit the animals’ access to food. The relationship among fasting period, animal behaviour during feed reintroduction (FR) and acidosis occurrence has not been completely elucidated. Twenty steers fitted with rumen catheters were fed a high-concentrate diet (concentrate : forage ratio 85 : 15) and were challenged by a protocol of FW followed by FR. The animals were randomly assigned to one of the four treatments: FW for 12 h (T12), 24 h (T24), 36 h (T36) or no FW (control group) followed by FR. The steers’ behaviour, ruminal chemistry, structure of the ruminal microbial community, blood enzymes and metabolites and ruminal acidosis status were assessed. Animal behaviour was affected by the FW–FR challenge ( P < 0.05). Steers from the T12, T24 and T36 treatments showed a higher ingestion rate and a lower frequency of rumination. Although all animals were suspected to have sub-acute ruminal acidosis (SARA) prior to treatment, a severe case of transient SARA arose after FR in the T12, T24 and T36 groups. The ruminal pH remained below the threshold adopted for SARA diagnosis ( pH value = 5.6) for more than three consecutive hours (24, 7 and 19 h in the T12, T24 and T36 treatments, respectively). The FW–FR challenge did not induce clinical acute ruminal acidosis even though steers from the T36 treatment presented ruminal pH values that were consistent with this metabolic disorder (pH threshold for acute acidosis = 5.2). Total mixed ration reintroduction after the withdrawal period reactivated ruminal fermentation as reflected by changes in the fermentation end-products. Ruminal lactic acid accumulation in steers from the T24 and T36 treatments probably led to the reduction of pH in these groups. Both the FW and the FR phases may have altered the structure of the ruminal microbiota community. Whereas fibrolytic bacterial groups decreased relative abundance in the restricted animals, both lactic acid producer and utiliser bacterial groups increased ( P < 0.05). The results demonstrated a synchronisation between Streptococcus (lactate producer) and Megasphaera (lactate utiliser), as the relative abundance of both groups increased, suggesting that bacterial resilience may be central for preventing the onset of metabolic disturbances such as ruminal acidosis. A long-FW period (36 h) produced rumen pH reductions well below and lactic acid concentration increased well above the accepted thresholds for acute acidosis without any perceptible clinical signs.     

Rumen prokaryotic communities of ruminants under different feeding paradigms on the Qinghai-Tibetan Plateau

Yak and Tibetan sheep are the major indigenous ruminants on the Qinghai-Tibetan Plateau in China. The aim of this work was to study the differences in ruminal fermentation parameters and rumen prokaryotic community composition between hosts and feeding paradigms. The 16S rRNA genes targeting bacteria and archaea were sequenced using the MiSeq platform. The results showed that the prokaryotic community structure between yak and Tibetan sheep was significantly different (P < 0.01). A significant difference in structure was also found between groups of yaks barn fed with a total mixed ration (TMR) and those naturally grazing (NG) (P = 0.034), as well as for Tibetan sheep of the two groups (P = 0.026). The core prokaryotic populations that existed in the rumen mostly dominated the structure. There was an obvious correlation of the prokaryotic community composition at the phylum and genus levels with the host or the feeding pattern. In addition, Tibetan sheep showed significantly higher yields of volatile fatty acids (VFAs) than yak, as did the NG group compared with the TMR group. In conclusion, both the host and feeding pattern may influence rumen microbial ecology system, with host effects being more important than those of the feeding pattern.     

Garlic skin induces shifts in the rumen microbiome and metabolome of fattening lambs

Garlic (Allium sativum L.) and its constituents have been shown to modify rumen fermentation and improve growth performance. Garlic skin, a by-product of garlic processing, contains similar bioactive components as garlic bulb. This study aimed to investigate the effects of garlic skin supplementation on growth performance, ruminal microbes, and metabolites in ruminants. Twelve Hu lambs were randomly assigned to receive a basal diet (CON) or a basal diet supplemented with 80 g/kg DM of garlic skin (GAS). The experiment lasted for 10 weeks, with the first 2 weeks serving as the adaptation period. The results revealed that the average daily gain and volatile fatty acid concentration were higher (P < 0.05) in lambs fed GAS than those in the CON group. Garlic skin supplementation did not significantly (P > 0.10) affect the α-diversity indices, including the Chao1 index, the abundance-based coverage estimator value, and the Shannon and Simpson indices. At the genus level, garlic skin supplementation altered the ruminal bacterial composition by increasing (P < 0.05) the relative abundances of Prevotella, Bulleidia, Howardella, and Methanosphaera and decreasing (P < 0.05) the abundance of Fretibacterium. Concentrations of 139 metabolites significantly differed (P < 0.05) between the GAS and the CON groups. Among them, substrates for rumen microbial protein synthesis were enriched in the GAS group. The pathways of pyrimidine metabolism, purine metabolism, and vitamin B6 metabolism were influenced (P < 0.05) by garlic skin supplementation. Integrated correlation analysis also provided a link between the significantly altered rumen microbiota and metabolites. Thus, supplementation of garlic skin improved the growth performance of lambs by modifying rumen fermentation through shifts in the rumen microbiome and metabolome.     

Effects of starter feeding and early weaning on GHR mRNA expression in liver and rumen of lambs from birth to 84 days of age

The growth hormone receptor (GHR) is associated with animal growth and development. To investigate such effects on GHR gene expression, a total of 102 Hu lambs were randomly allocated to one of three groups (Group 1: starter diet from 7 d of age, weaning at 56 d of age; Group 2: starter diet from 42 d of age, weaning at 56 d of age; Group 3: starter diet from 7 d of age; weaning at 28 d of age). Six lambs from each group were sacrificed every 14 d to investigate the effects of starter feeding and weaning age on GHR mRNA expression in the liver and rumen. The results revealed that GHR mRNA expression was significantly higher in the liver and rumen (p < 0.05) than in other tissues. Early starter feeding up-regulated hepatic GHR mRNA expression on days 14, 28, 42 and 56 and ruminal GHR mRNA expression on days 28, 42, 70, and 84 (p < 0.05). Early weaning up-regulated hepatic GHR mRNA expression on days 56, 70 and 84 and ruminal GHR mRNA expression on days 42, 56, 70 and 84 (p < 0.05). Dietary and weaning regimes and age affected the hepatic and ruminal GHR mRNA expression.     

Relative reticulo-rumen pH indicators for subacute ruminal acidosis detection in dairy cows

Subacute ruminal acidosis (SARA) is usually characterized by abnormal and intermittent drops in rumen pH. Nevertheless, high individual animal variability in rumen pH and the difference in measurement methods for pH data acquisition decrease the sensitivity and accuracy of pH indicators for detecting SARA in ruminants. The aim of this study was to refine rumen pH indicators in long-term SARA based on individual dairy cow reticulo-rumen pH kinetics. Animal performances and rumen parameters were studied weekly in order to validate SARA syndrome and rumen pH was continuously measured using reticulo-rumen sensors. In total, 11 primiparous dairy cows were consecutively fed two different diets for 12 successive weeks: a control diet as low-starch diet (LSD; 13% starch for 4 weeks in period 1), an acidotic diet as high-starch diet (HSD; 32% starch for 4 weeks in period 2), and again the LSD diet (3 weeks in period 3). There was a 1-week dietary transition between LSD and HSD. Commonly used absolute SARA pH indicators such as daily average, area under the curve (AUC) and time spent below pH<5.8 and pH<6 were processed from absolute (raw) daily kinetics. Then signal processing was applied to raw pH values in order to calculate relative pH indicators by filtering and normalizing data to remove inter-individual variability, sensor drift and sensor noise. Normalized AUC, times spent below NpH<−0.3 and NpH<−0.5, NpH range and NpH standard deviation were calculated. Those relative pH indicators were compared with commonly used pH indicators to assess their ability to detect SARA. This syndrome induced by HSD was confirmed by consistent expected changes in milk quality, dry matter intake and acetate : propionate ratio in the rumen, whereas the ruminal concentration of lipopolysaccharide was increased. Commonly used pH SARA indicators were not able to discriminate SARA syndrome due to high animal variability and sensor drift and noise, whereas relative pH indicators developed in this study appeared more relevant for SARA detection as assessed by receiver operating characteristic tests. This work shows that absolute pH kinetics should be corrected for drift, noise and animal variability to produce relative pH indicators that are more robust for SARA detection. These relative pH indicators could be more relevant for identifying affected animals in a herd and also for comparing SARA risk among studies.     

Impact of subacute ruminal acidosis (SARA) adaptation and recovery on the density and diversity of bacteria in the rumen of dairy cows

Subacute ruminal acidosis (SARA) is characterized by ruminal pH depression and microbial perturbation. The impact of SARA adaptation and recovery on rumen bacterial density and diversity was investigated following high-grain feeding. Four ruminally cannulated dairy cows were fed a hay diet, transitioned to a 65% grain diet for 3 weeks, and returned to the hay diet for 3 weeks. Rumen fluid, rumen solids, and feces were sampled during weeks 0 (hay), 1 and 3 (high grain), and 4 and 6 (hay). SARA was diagnosed during week 1, with a pH below 5.6 for 4.6±1.4 h. Bacterial density was significantly lower in the rumen solids with high grain (P=0.047). Rumen fluid clone libraries from weeks 0, 3, and 6 were assessed at the 98% level and 154 operational taxonomic units were resolved. Week 3 diversity significantly differed from week 0, and community structure differed from weeks 0 and 6 (P<0.0001). Clones belonging to the phylum Firmicutes predominated. Compared with the hay diet, the high-grain diet contained clones from Selenomonas ruminantium and Succiniclasticum ruminis, but lacked Eubacterium spp. SARA adaptation was found to significantly alter bacterial density, diversity, and community structure, warranting further investigation into the role bacteria play in SARA adaptation.     

Using microbial fatty acids to improve understanding of the contribution of solid associated bacteria to microbial mass in the rumen

This study sought to distinguish liquid-(LAB) and detached (SAB₁) and undetached (SAB₂) solid-associated bacteria through their fatty acid (FA) and purine base (PB) profiles. Fatty acids and PB were also evaluated as internal microbial markers for estimating microbial biomass associated with rumen particles. Four merino rams fitted with rumen cannulae and fed dehydrated alfalfa pellets provided rumen contents. In 3 consecutive weeks, rumen contents were collected and samples of LAB and SAB₁, total rumen content (TRC), washed rumen particles (WRP) and rumen particles after SAB₁ extraction (ERP) were obtained and analysed for PB and FA. The SAB₂ biomass composition was estimated from the non-NDF organic matter (OM) remaining in ERP. The concentration of total SAB biomass in particles was estimated using both PB and odd and branched-chain fatty acids (OBCFA). Concentrations of PB and OBCFA were highly correlated among the different rumen fractions. Marked differences between LAB and SAB populations occurred with LAB having higher PB content, lower FA content and a higher proportion (g/100 g fatty acids) of OBCFA than did SAB. The chemical composition of SAB₁ and SAB₂ was similar, except for the 15% higher crude protein content of the latter. The concentration of OBCFA (mg/g microbial OM) did not differ between bacterial fractions. The PB/OBCFA ratio (mg/mg) was higher in LAB (2.08) than in SAB (0.94). The ratio between branched-chain and odd-linear-chain FA was higher in LAB (2.26) than in SAB (1.46). Extraction of PB and OBCFA from WRP with our SAB detachment procedure was 61% and 31%, respectively. Estimated SAB₁ and total SAB biomass (mg OM/g WRP) were 158 and 266, and 47 and 164, respectively, using PB and OBCFA as microbial markers. This study suggests that the OBCFA have potential as internal microbial markers in rumen ecosystem studies.     

Effects of dietary crude protein and tannic acid on rumen fermentation,rumen microbiota and nutrient digestion in beef cattle

The objectives of the trial were to study the effects of dietary crude protein (CP) and tannic acid (TA) on rumen fermentation, microbiota and nutrient digestion in beef cattle. Eight growing beef cattle (live weight 350 ± 25 kg) were allocated in a 2 × 2 crossover design using two levels of dietary CP [111 g/kg dry matter (DM) and 136 g/kg DM] and two levels of TA (0 and 16.9 g/kg DM) as experimental treatments. Each experimental period lasted 19 d, consisting of 14-d adaptation and 5-d sampling. The impacts of dietary CP and TA on ruminal microbiota were analysed using high-throughput sequencing of 16S rRNA gene. Results indicated that no interactions between dietary CP and TA were found on rumen fermentation and nutrient digestibility. Increasing dietary CP level from 111 to 136 g/kg DM increased the ruminal concentrations of ammonia nitrogen (NH₃-N) (p < 0.01) and improved the CP digestibility (p < 0.001). Adding TA at 16.9 g/kg DM inhibited rumen fermentation and decreased the digestibility of dietary CP (p < 0.001), DM (p < 0.05) and organic matter (p < 0.01). Increasing the dietary CP level or adding TA did not affect the relative abundances of the major bacteria Firmicutes and Proteobacteria at the phylum level and Prevotella_1 and Christensenellaceae_R-7_group at the genus level, even though adding TA increased the Shannon index of the ruminal bacterial community. TA was partly hydrolysed to pyrogallol, gallic acid and resorcinol in rumen fluid and the inhibitory effects of TA on rumen fermentation and nutrient digestibility could have been resulted from the TA metabolites including pyrogallol, gallic acid and resorcinol as well as the protein-binding effect.     

Effect of protein supplementation on ruminal parameters and microbial community fingerprint of Nellore steers fed tropical forages

In tropical regions, protein supplementation is a common practice in dairy and beef farming. However, the effect of highly degradable protein in ruminal fermentation and microbial community composition has not yet been investigated in a systematic manner. In this work, we aimed to investigate the impact of casein supplementation on volatile fatty acids (VFA) production, specific activity of deamination (SAD), ammonia concentration and bacterial and archaeal community composition. The experimental design was a 4×4 Latin square balanced for residual effects, with four animals (average initial weight of 280±10 kg) and four experimental periods, each with duration of 29 days. The diet comprised Tifton 85 (Cynodon sp.) hay with an average CP content of 9.8%, on a dry matter basis. Animals received basal forage (control) or infusions of pure casein (230 g) administered direct into the rumen, abomasum or divided (50 : 50 ratio) in the rumen/abomasum. There was no differences (P>0.05) in ruminal pH and microbial protein concentration between supplemented v. non-supplemented animals. However, in steers receiving ruminal infusion of casein the SAD and ruminal ammonia concentration increased 33% and 76%, respectively, compared with the control. The total concentration of VFA increased (P<0.05) in steers receiving rumen infusion of casein. SAD and the microbial protein concentration did not vary significantly among treatments during the feeding cycle, but mean SAD values were greater in steers supplemented in the rumen and rumen/abomasum. Ruminal ammonia concentration was positively correlated with SAD in animals receiving ruminal infusion of casein. Polymerase chain reaction–denaturing gradient gel electrophoresis (PCR-DGGE) analysis revealed low similarity between treatments, animals and time of sample collection. Richness analysis and determination of the Shannon–Wiener index indicated no differences (P>0.05) in species richness and diversity of γ-proteobacteria, firmicutes and archaea between non-supplemented Nellore steers and steers receiving casein supplementation in the rumen. However, species richness and the Shannon–Wiener index were lower (P<0.05) for the phylum bacteroidetes in steers supplemented with casein in the rumen compared with non-supplemented animals. Venn diagrams indicated that the number of unique bands varied considerably among individual animals and was usually higher in number for non-supplemented steers compared with supplemented animals. These results add new knowledge about the effects of ruminal and postruminal protein supplementation on metabolic activities of rumen microbes and the composition of bacterial and archaeal communities in the rumen of steers.     

Reducing protein content in the diet of growing goats: implications for nitrogen balance,intestinal nutrient digestion and absorption,and rumen microbiota

Reducing dietary CP content is an effective approach to reduce animal nitrogen excretion and save protein feed resources. However, it is not clear how reducing dietary CP content affects the nutrient digestion and absorption in the gut of ruminants, therefore it is difficult to accurately determine how much reduction in dietary CP content is appropriate. This study was conducted to investigate the effects of reduced dietary CP content on N balance, intestinal nutrient digestion and absorption, and rumen microbiota in growing goats. To determine N balance, 18 growing wether goats (25.0 ± 0.5 kg) were randomly assigned to one of three diets: 13.0% (control), 11.5% and 10.0% CP. Another 18 growing wether goats (25.0 ± 0.5 kg) were surgically fitted with ruminal, proximate duodenal, and terminal ileal fistulae and were randomly assigned to one of the three diets to investigate intestinal amino acid (AA) absorption and rumen microbiota. The results showed that fecal and urinary N excretion of goats fed diets containing 11.5% and 10.0% CP were lower than those of goats fed the control diet (P < 0.05). When compared with goats fed the control diet, N retention was decreased and apparent N digestibility in the entire gastrointestinal tract was increased in goats fed the 10% CP diet (P < 0.05). When compared with goats fed the control diet, the duodenal flow of lysine, tryptophan and phenylalanine was decreased in goats fed the 11.5% CP diet (P < 0.05) and that of lysine, methionine, tryptophan, phenylalanine, leucine, glutamic acid, tyrosine, essential AAs (EAAs) and total AAs (TAAs) was decreased in goats fed the 10.0% CP diet (P < 0.05). When compared with goats fed the control diet, the apparent absorption of TAAs in the small intestine was increased in goats fed the 11.5% CP diet (P < 0.05) and that of isoleucine, serine, cysteine, EAAs, non-essential AAs, and TAAs in the small intestine was increased in goats fed the 10.0% CP diet (P < 0.05). When compared with goats fed the control diet, the relative richness of Bacteroidetes and Fibrobacteres was increased and that of Proteobacteria and Synergistetes was decreased in the rumen of goats fed a diet with 10.0% CP. In conclusion, reducing dietary CP content reduced N excretion and increased nutrient utilization by improving rumen fermentation, enhancing nutrient digestion and absorption, and altering rumen microbiota in growing goats.     

Wet brewers' grains or bean curd pomance as partial replacement of soybean meal for lactating cows

The purpose of this study was to evaluate the effects of supplementation of the by-product, brewers' grains and the bean curd pomance, on the performance of lactating cows and their ruminal characteristics. Through this, we wanted to increase resource utilization and to eliminate pollution from these by-products. Thirty-two Holstein lactating cows were allocated randomly into four dietary treatment groups. The experimental diets were formulated to be isoenergetic and isonitrogenous according to National Research Council (1989), and containing 35% corn silage, 20% alfalfa hay and 45% concentrates on DM basis. The dietary treatments consisted of the inclusion of different by-products sources. These included soybean bean (as the control), brewers' grains, bean curd pomance, and the mixed by-products (containing the same amount of brewers' grains and bean curd pomance). The experimental feeding lasted for eight weeks after one week of adaptation. In addition, four rumen cannulated Holstein cows were used in a 4×4 Latin square with 10-day period for collecting rumen samples. Results showed that cows that were fed the bean curd pomance diet produced significantly more milk than those that were fed the brewers' grains diet or the mixed by-product diet. The cows on the control diet produced significantly less milk than the other treatment groups (P<0.05). Both the control and brewers' grains groups consumed more feed than those in the bean curd pomance and mixed by-products diet groups (P<0.05). The cows that were fed the mixed by-products diet produced a significantly lower percentage of milk fat, total solids, milk protein and milk lactose than the others (P<0.05). The milk lactose percentage was significantly higher for cows that were fed the bean curd pomance than those fed the brewers' grains diet (P<0.05). Cows that were fed the bean curd pomance produced significantly more milk fat, total solids, milk protein and milk lactose than the others (P<0.05). Cows that were fed the mixed by-products diet produced significantly lower amount of milk fat, total solids and milk protein than the control and brewers' grains group (P<0.05). These diets did not significantly influence the body weight of the cows. It, however, significantly influenced the ruminal characteristics (P<0.05).     

Influence of feeding sunflower seed and meal protected against ruminal fermentation on ruminal fermentation,bacterial composition and in situ degradability in sheep

ABSTRACT

The effects of treating sunflower seed (SS) and meal (SM), as well as of a mixture of both feeds (SSM; 45:55) with a solution of malic acid (1 M; 400 ml/kg feed) and heating for protection against ruminal degradation were studied. Four rumen-fistulated sheep were fed two mixed diets composed of oat hay and concentrate (40:60) and differing only in the concentrate, that contained either a mixture of untreated SS and SM (control diet) or treated SS and SM (MAH diet). A crossover design with two 24-d experimental periods was used, and each period included 10 d of diet adaptation, 9 d for in situ incubations of SS, SM and SSM, and 5 d for measuring ruminal fermentation characteristics and rumen emptying. From day 6 onwards a solution of (¹⁵NH₄)₂SO₄ was continuously infused into the rumen of each sheep to label ruminal bacteria. Feeding the MAH diet did not affect either ruminal pH or concentrations of total volatile fatty acids and NH₃-N, but decreased (p ≤ 0.01) the molar proportions of acetate and propionate and increased those of butyrate (p< 0.001). Organic matter and lipid contents of ruminal bacteria were lower whereas both N content and ¹⁵N enrichment were greater (p ≤ 0.05) in MAH-fed sheep. The in situ effective degradability (ED) of different fractions of SS, SM and SSM were calculated from the ruminal rates of particle comminution and passage, and values were corrected for microbial contamination. The MAH treatment decreased the ED of most fractions for all feeds and increased the supply of by-pass crude protein (CP) by 19.1% and 120% for SS and SM, respectively, and that of fat by 34% for SS. The MAH treatment also increased the in vitro intestinal digestibility of the by-pass CP for both SS (from 60.1% to 75.4%) and SM (from 83.2% to 91.0%). The simultaneous heating of both feeds (SSM) reinforced the protective effect of the MAH treatment and increased the by-pass CP without altering its intestinal digestibility, increasing the intestinally digested CP content by 16.8% compared with the value estimated from the results obtained for MAH-treated SS and SM incubated independently. These results indicate that the MAH treatment was effective to protect sunflower protein against rumen degradation and increased its intestinal digestibility.     

The Oxidative Damage and Disbalance of Calcium Homeostasis in Brain of Chicken Induced by Selenium Deficiency

Dietary selenium (Se) deficiency can influence the function of the brain. Our objective was to investigate the effects of Se deficiency on oxidative damage and calcium (Ca) homeostasis in brain of chicken. In the present study, 1-day-old chickens were fed either a commercial diet (as control group) with 0.15 mg/kg Se or a Se-deficient diet (as L group) with 0.033 mg/kg Se for 75 days. Then, brain injury biomarkers were examined, including histological analysis, ultrastructure assay, and apoptosis assay. We also examined the effect of Se deficiency on the Se-containing antioxidative enzyme glutathione peroxidase (GSH-Px), the level of glutathione (GSH), and the Ca homeostasis in brain of chicken. The results showed that the levels of Se and GSH and activity of GSH-Px are seriously reduced by 33.8–96 % (P?<?0.001), 24.51–27.84 % (P?<?0.001), and 20.70–64.24 % (P?<?0.01), respectively. In the present study, we also perform histological analysis and ultrastructure assay and find that Se deficiency caused disorganized histological structure, damage to the mitochondria, fusion of nuclear membrane and nucleus shrinkage, higher apoptosis rate (P?<?0.001), and increase of Ca homeostasis (P?<?0.05 or P?<?0.01 or P?<?0.001) in the brain of chicken. In conclusion, the results demonstrated that Se deficiency induced oxidative damage and disbalance of Ca homeostasis in the brain of chicken. Similar to mammals, chickens brain is also extremely susceptible to oxidative damage and selenium deficiency.