Effect of rumen and fecal inocula from calves fed either milk replacer or whole milk fed on intestinal cells and digestive tract microbiota

Abstract Text:

The objective of this study was to evaluate cell viability and inflammatory status of intestinal cells incubated with rumen and fecal inocula obtained from Holstein calves fed either a milk replacer or whole milk. Liquid rumen and fecal samples were obtained from 10 calves at 5 wk of age. Calves were either fed a milk replacer supplemented with Enterococcus faecium at the rate of 1.5x10⁹ cfu/kg (MR), or whole milk (WM). Samples from each group of animals (5 animals per group) were pooled, centrifuged, and suspended with PBS at 3%. Lactobacillus and Bifidobacteria were quantified by qPCR from the rumen and fecal-pooled samples. The experiment was repeated twice with ten other calves fed similarly. Jejunum tissue from 12-mo old bulls was collected at a slaughterhouse to obtain jejunum primary cells and perform further in vitro experiments. Jejunum cells were cultured during 6 h with the fecal and ruminal inocula in 24-well plates at 37ºC under a 5% CO₂ atmosphere. After the incubation, the supernatant was recovered to determine LDH activity as a cell damage marker. Cells were washed and lysed with TriZol (Invitrogen) to extract RNA and quantify, by qPCR, the expression of TNFα as an inflammation marker. Data were analyzed with a mixed-effects model considering sample and milk type as fixed effects, and period as random effect. Bifidobacteria and Lactobacillus contents were greater (P<0.05) in feces (15.6±1.04 and 14.1±0.73 Ct, respectively) than in rumen (21.8±1.04 and 21.8±0.73 Ct, respectively) samples, and Bifidobacteria contents tended (P=0.10) to be greater in samples from MR than in those from WM calves (17.1 vs 20.2±1.04 Ct, respectively). Viability of jejunum cells and the expression of TNFα were similar when cultured with rumen inoculum from MR and WM calves. However, LDH activity was greater (P<0.05) in jejunum cells (less cell viability) when incubated with fecal inoculum from WM (0.50±0.296 mU/mL) than from MR (0.14±0.296 mU/mL) calves. The expression of TNFα was greater (P<0.05) when cells were incubated with MR (8.8x10⁵±8.89x10⁶) than with WM (3.2x10⁵±8.89x10⁶) fecal inoculum. In conclusion, fecal inoculum from MR calves improve cell viability and trigger the inflammatory status of intestinal jejunum cells when compared with WM fecal inoculum. These effects might be mediated by the greatest amount of Bifidobacteria in the fecal samples from MR calves, which might be favored, by the presence of Enterococcus faeciumin the milk replacer.

Keywords:

jejunum primary cells, milk replacer, whole milk