Metatranscriptomic analyses reveal ruminal pH regulates fiber degradation and fermentation by shifting the microbial community and gene expression of carbohydrate-active enzymes.

BACKGROUND: Volatile fatty acids (VFA) generated from ruminal fermentation by microorganisms provide up to 75% of total metabolizable energy in ruminants. Ruminal pH is an important factor affecting the profile and production of VFA by shifting the microbial community. However, how ruminal pH affects the microbial community and its relationship with expression of genes encoding carbohydrate-active enzyme (CAZyme) for fiber degradation and fermentation are not well investigated. To fill in this knowledge gap, six cannulated Holstein heifers were subjected to a continuous 10-day intraruminal infusion of distilled water or a dilute blend of hydrochloric and phosphoric acids to achieve a pH reduction of 0.5 units in a cross-over design. RNA-seq based transcriptome profiling was performed using total RNA extracted from ruminal liquid and solid fractions collected on day 9 of each period, respectively. RESULTS: Metatranscriptomic analyses identified 19 bacterial phyla with 156 genera, 3 archaeal genera, 11 protozoal genera, and 97 CAZyme transcripts in sampled ruminal contents. Within these, 4 bacteria phyla (Proteobacteria, Firmicutes, Bacteroidetes, and Spirochaetes), 2 archaeal genera (Candidatus methanomethylophilus and Methanobrevibacter), and 5 protozoal genera (Entodinium, Polyplastron, Isotricha, Eudiplodinium, and Eremoplastron) were considered as the core active microbes, and genes encoding for cellulase, endo-1,4-beta- xylanase, amylase, and alpha-N-arabinofuranosidase were the most abundant CAZyme transcripts distributed in the rumen. Rumen microbiota is not equally distributed throughout the liquid and solid phases of rumen contents, and ruminal pH significantly affect microbial ecosystem, especially for the liquid fraction. In total, 21 bacterial genera, 4 protozoal genera, and 6 genes encoding CAZyme were regulated by ruminal pH. Metabolic pathways participated in glycolysis, pyruvate fermentation to acetate, lactate, and propanoate were downregulated by low pH in the liquid fraction. CONCLUSIONS: The ruminal microbiome changed the expression of transcripts for biochemical pathways of fiber degradation and VFA production in response to reduced pH, and at least a portion of the shifts in transcripts was associated with altered microbial community structure.

Ruminal volatile fatty acid absorption is affected by elevated ambient temperature.

The objective of this study was to investigate the effect of short-term elevated ambient temperature on ruminal volatile fatty acid (VFA) dynamics and rumen epithelium gene expression associated with the transport and metabolism of VFA. Eight ruminally cannulated Holstein heifers (200 kg) were used in a factorial, repeated measures experiment with two treatments and two periods. During the first period, animals were provided with feed ad libitum and housed at 20 °C. During the second period, one group (HS) was housed at 30 °C and fed ad libitum. The other group (PF) was housed at 20 °C and pair-fed to match the intake of the HS group. During each period, animals were kept on treatment for 10 day, with sample collection on the final day. In the second period, indicators of heat stress were significantly different between PF and HS animals (P < 0.05). There was a thermal environment effect on butyrate production (P < 0.01) that was not associated with feed intake (P = 0.43). Butyrate absorption decreased in HS animals (P < 0.05) but increased in PF animals (P < 0.05) from period 1 to period 2. There was a feed intake effect on BHD1 expression (P = 0.04) and a tendency for a thermal environment effect (P = 0.08), with expression increasing in both cases. Expression of MCT4 was affected by feed intake (P = 0.003) as were all NHE genes (NHE1, NHE2, and NHE3; P < 0.05). These results indicate that with low feed intake and heat stress, there are shifts in rumen VFA dynamics and in the capacity of the rumen epithelium to absorb and transport VFA.

Okara as a protein supplement affects feed intake and milk composition of ewes and growth performance of lambs.

Evaluating the feeding value of wet okara as a protein supplement for lactating ewes with twin lambs was the objective. A 4 × 4 Latin square replicated 2× (4 sheep, 4 treatments, 4 periods per square; 2 squares) was conducted to examine the influence of concentrate mix (okara or not) and type of forage (silage or hay) on ewe milk composition and growth of their lactating lambs. Treatment periods were 14 days (7 days adaptation and 7 days collection). Ewes (55 to 74.8 kg BW) were fed 1 of 4 diets: wheat middling and corn concentrate with mixed grass hay (TSH), okara and corn with mixed grass hay (OSH), soybean and wheat middlings with hay crop silage (TSS), and okara and corn with hay crop silage (OSS). Ewes fed hay diets had lower forage dry matter intakes than ewes fed silage. Intake of okara supplement was higher (P < 0.05) with OSH (3.64 kg/d) than with OSS (1.70 kg/d). There was no difference in supplement intake between TSH and TSS. There were no differences among diets for lamb daily gains or in ewe milk compositions among the diets. Okara is an effective source of protein for lactating ewes and their twin lambs.

The effect of nutritional status and myogenic satellite cell age on turkey satellite cell proliferation, differentiation, and expression of myogenic transcriptional regulatory factors and heparan sulfate proteoglycans syndecan-4 and glypican-1.

Posthatch satellite cell mitotic activity is a critical component of muscle development and growth. Satellite cells are myogenic stem cells that can be induced by nutrition to follow other cellular developmental pathways, and whose mitotic activity declines with age. The objective of the current study was to determine the effect of restricting protein synthesis on the proliferation and differentiation, expression of myogenic transcriptional regulatory factors myogenic determination factor 1, myogenin, and myogenic regulatory factor 4, and expression of the heparan sulfate proteoglycans syndecan-4 and glypican-1 in satellite cells isolated from 1-d-, 7-wk-, and 16-wk-old turkey pectoralis major muscle (1 d, 7 wk, and 16 wk cells, respectively) by using variable concentrations of Met and Cys. Four Met concentrations-30 (control), 7.5, 3, or 0 mg/L with 3.2 mg/L of Cys per 1 mg/L of Met-were used for culture of satellite cells to determine the effect of nutrition and age on satellite cell behavior during proliferation and differentiation. Proliferation was reduced by lower Met and Cys concentrations in all ages at 96 h of proliferation. Differentiation was increased in the 1 d Met-restricted cells, whereas the 7 wk cells treated with 3 mg/L of Met had decreased differentiation. Reduced Met and Cys levels from the control did not significantly affect the 16 wk cells at 72 h of differentiation. However, medium with no Met or Cys suppressed differentiation at all ages. The expression of myogenic determination factor 1, myogenin, myogenic regulatory factor 4, syndecan-4, and glypican-1 was differentially affected by age and Met or Cys treatment. These data demonstrate the age-specific manner in which turkey pectoralis major muscle satellite cells respond to nutritional availability and the importance of defining optimal nutrition to maximize satellite cell proliferation and differentiation for subsequent muscle mass accretion.

The effect of syndecan-4 and glypican-1 expression on age-related changes in myogenic satellite cell proliferation, differentiation, and fibroblast growth factor 2 responsiveness.

Satellite cells are multipotential stem cells responsible for muscle growth and regeneration. Satellite cell proliferation, differentiation, and responsiveness to fibroblast growth factor 2 (FGF2) is, in part, regulated by the heparan sulfate proteoglycans syndecan-4 and glypican-1. Syndecan-4 and glypican-1 expression declines with satellite cell age and may be associated with decreased satellite cell activity. The objective of the current study was to determine if overexpression of syndecan-4 and glypican-1 would increase proliferation, differentiation and FGF2 responsiveness in satellite cells isolated from pectoralis major muscle from 16-wk-old turkeys. Overexpression of syndecan-4 and glypican-1 did not have a significant effect on proliferation and differentiation in 1d, 7 wk, and 16 wk satellite cells, and did not affect FGF2 responsiveness during proliferation. Expression of syndecan-4 and glypican-1 increased differentiation at 48 h in 1d, 7 wk, and 16 wk cells treated with FGF2. Expression of myogenic regulatory factors MyoD, myogenin, and MRF4 was affected by the overexpression of syndecan-4 and glypican-1. However, changes in myogenic regulatory factor expression did not have a significant effect on proliferation or differentiation. These data demonstrate that syndecan-4 and glypican-1 are likely not directly associated with the age related decrease in satellite cell activity.

Changes in proliferation, differentiation, fibroblast growth factor 2 responsiveness and expression of syndecan-4 and glypican-1 with turkey satellite cell age.

Myogenic satellite cells are heterogeneous multipotential stem cells that are required for muscle repair, maintenance, and growth. The membrane-associated heparan sulfate proteoglycans syndecan-4 and glypican-1 differentially regulate satellite cell proliferation, differentiation, fibroblast growth factor 2 (FGF2) signal transduction, and expression of the myogenic regulatory factors MyoD and myogenin. The objective of the current study was to determine the effect of age on syndecan-4 and glypican-1 satellite cell populations, proliferation, differentiation, FGF2 responsiveness, and expression of syndecan-4, glypican-1, MyoD, and myogenin using satellite cells isolated from the pectoralis major muscle of 1-day-old, 7-week-old and 16-week-old turkeys. Proliferation was significantly reduced in the 16-week-old satellite cells, while differentiation was decreased in the 7-week-old and the 16-week-old cells beginning at 48 h of differentiation. Fibroblast growth factor 2 responsiveness was highest in the 1-day-old and 7-week-old cells during proliferation; during differentiation there was an age-dependent response to FGF2. Syndecan-4 and glypican-1 satellite cell populations decreased with age, but syndecan-4 and glypican-1 were differentially expressed with age during proliferation and differentiation. MyoD and myogenin mRNA expression was significantly decreased in 16-week-old cells compared to the 1-day-old and 7-week-old cells. MyoD and myogenin protein expression was higher during proliferation in the 16-week-old cells and decreased with differentiation. These data demonstrate an age-dependent effect on syndecan-4 and glypican-1 satellite cell subpopulations, which may be associated with age-related changes in proliferation, differentiation, FGF2 responsiveness, and the expression of the myogenic regulatory factors MyoD and myogenin.