Short- to medium-term effects of consumption of quebracho tannins on saliva production and composition in sheep and goats

Eight Merino sheep (49.4 ± 4.23 kg BW) and 8 Alpine goats (53.2 ± 2.51 kg BW) were used to study the effect of ingestion of quebracho tannins on salivation. Four sheep and 4 goats were individually fed a daily allotment of 20 g DM of alfalfa hay/kg BW (Control). Another 4 sheep and 4 goats were also given 20 g DM of alfalfa hay/kg BW supplemented with 50 g of quebracho/kg DM (Tannin) for a period of 64 d. The saliva secretion from the left parotid gland was collected by insertion of a polyvinyl chloride catheter into the parotid duct and the amount of parotid saliva produced recorded over three 48-h periods on d 1 and 2 (P1), d 31 and 32 (P2), and d 61 and 62 (P3) after the tannin feeding was initiated. The total amount of saliva produced was estimated from rumen water kinetics determined on d 4, d 34, and d 64 of the experiment. Experimental design was completely randomized, with repeated measures on each experimental unit, performing separate analysis for sheep and goats. Parotid saliva production was not affected by the sampling period in either animal species receiving the Control diet. Corresponding values for sheep were 2.04, 2.12, and 2.27 L/d (P = 0.89) and for goats 1.65, 1.79, and 1.86 L/d (P = 0.95). Sheep fed the Tannin diet produced 55, 73, and 107% of the amount of saliva recorded in sheep fed the Control diet on P1, P2, or P3, respectively. Corresponding values in goats were 88, 130, and 134% on P1, P2, or P3, respectively. Estimated total saliva production was not affected (P = 0.50 for sheep and P = 0.97 for goats) by the ingestion of quebracho. There was no difference (P > 0.10) in osmotic pressure, P, Mg, Ca, urea, and protein concentrations in parotid saliva. There were, however, differences in Na and K concentrations in response to the ingestion of quebracho tannins, with Na concentrations increasing (P = 0.05) and K concentrations decreasing (P = 0.04) in sheep saliva and pH increasing (P = 0.05) in goat saliva. In conclusion, the inclusion of quebracho at 50 g/kg DM for 64 d does not appear to alter saliva production in sheep and goats

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

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

Normalization of <i>RCC2</i> mRNA expression at 0 and 14 days after butyrate infusion using 1 single reference gene.

<p>The X-axis shows the internal gene that was used for estimating each ratio. Y-axis is shown in logarithmic scale.</p

Boxplot of Cq values.

<p>Variations of the quantification cycle are shown for all examined reference genes sorted by expression range (n = 30). Each box represents the 50% quartile; the median is figured as the line in each box; whiskers depict the 25% minimum and maximum range of the Cq value.</p

Description of reference genes and primer sequences.

<p>Primer PCR efficiency and PCR Tm product data represent mean values ± SE (n = 30). PCR efficiencies (E) calculated according to the equation (1 + E) = 10^slope. Free energy for potential secondary structures in the amplicon is shown (ΔG, kcal/mole).</p

Evaluation of reference genes for bovine rumen.

<p>The relative quantities of genes <i>ACTBF</i> and <i>UXTF</i> were normalized for each sample against the 2 best-scored (NF₁) and the 2 worst-scored references (NF₂). Y-axis is shown in logarithmic scale.</p

Normalized relative quantities of <i>RCC2</i> over days 0 and 14.

<p>Data were normalized for each sample against the 2 best-scored (NF₁) and the 2 worst-scored references (NF₂). NRQ were rescaled to day 0 that was arbitrarily set as 1.</p