| Rumen papillae, the finger like projection covering the entire rumen wall provide surface area for the absorption of nutrients mainly short chain fatty acids (SCFA) produced in the rumen through microbial fermentation of diet. Butyrate, a four carbon containing SCFA is a well known molecule for its strong stimulatory effects on papillae growth thus increases surface area and proposed to increase SCFA absorption. Molecular mechanisms have been studied in detail through several in vitro studies, which show that butyrate modulates cell growth through proliferation. Though a limited number of in vivo studies in monogastrics have shown that butyrate acts as mitogenic factor and induce intestinal growth but none of the studies have investigated the cellular and molecular effects of butyrate on rumen epithelial proliferation. In addition, it has been suggested that butyrate increases surface area and thus SCFA absorption but the effects of butyrate induced surface area enlargement on SCFA absorption from rumen has not been investigated so far. Therefore, the present research is aimed to investigate the cellular and molecular effects of butyrate on rumen epithelial proliferation and to test the hypothesis that weather the butyrate-induced papillae growth increases SCFA absorption from the rumen.Two experiments were conducted on twenty-three goats (Boer x Yangtze River Delta White) approximately 4 months old at the commencement of experiment. Goats were fed concentrates (200 g/d) into two equal portions (0800 and 1700 h) and hay ad-libitum. In Exp.1 fifteen goats were assigned into two groups, butyrate (B, n=8) and control (C, n=7), received potassium phosphate buffer (50ml) intraruminally with and without sodium butyrate (0.3g.kg-1.d"’),1 h before morning feeding for 28 days. The goats were slaughtered on d 28, eight hour after last butyrate infusion. Relative gene expression was quantified by qRT-PCR and SCFA absorption from rumen was estimated by pulse dosing of a complex marker, valerate and cobalt (H-Val-Co) in to the rumen. In Exp.2 Eight goats were fed similarly as in Exp.1. All goats received ruminal infusion of butyrate (0.3g.kg’.d=1) dissolved in 50 ml of potassium phosphate buffer (designated as butyrate or B group) and after an interval of one month the same goats received ruminal infusion of buffer without butyrate (designated as butyrate or B group). The infusates were infused at 0700 every day and lasted for 28 days. SCFA passage and absorption rates were estimated during both infusion periods by pulse dosing of a complex marker, valerate and cobalt (H-Val-Co) into the rumen. During the butyrate infusion period the rumen papillae were biopsied from rumen before (0 h) and 3 h and 8 h after infusion and analyzed for quantitative PCR.The results in Exp.1 results showed that molar and molar proportional concentration of butyrate in rumen significantly increased (P< 0.05) in B at 30 min after infusion and remained elevated for 3.5 h. As a result molar proportion of acetate and propionate decreased significantly (P< 0.05) for 1.5 h and 1 h respectively, in B compared to C and than gradually returned to the pre-infusion value. Morphometry revealed that the papillae length was increased by 22.46%,27.59%, and 26.95%(P< 0.05), width by 33.55%, 22.60%, and 19.20%(P< 0.05); and density by 17.73%,14.80%, and 17.61%(P< 0.05) in atrium ruminis, ruminis ventralis and ventral blind sac, respectively in B compared to C. The overall increase in papillae dimensions (length and width) and densities led to increase in surface area by 90.34,79.24 and 76.2%(P< 0.01) in atrium ruminis, ruminis ventralis and ventral blind sac, respectively, in B compared to C. The number of cell layers forming stratum germinativum, SGv (4.25±0.125) in B was significantly higher than in C (2.68± 0.27; P< 0.05). The cell densities in SGv (9602.1±538.18) and stratum basale, SB (298.58 ±11.38) in B were significantly higher than in C (5322.34±22.58 and 210.55±2.96; P< 0.01 respectively). The fractions of cells in various phases of cell cycle and the mRNA expression of cyclin D1 and CDK4 in rumen epithelium of goats did not differ between the groups. The fractional rate of SCFA passage from the rumen of goats estimated in both%/h (8.29±0.91 in B vs 8.66±0.46 in C); and mmol/h (17.62±1.51in B vs 20.73±.11 in C) did not differ (P> 0.05) between the groups. The fractional rate of total SCFA absorption estimated as%/h increased (P< 0.05) in B (37.03±1.23) than in C (32.13±0.80); however estimated in mmol/h (97.68±6.74 in B vs 98.32±6.43 in C) did not differ (P> 0.05) between the groups. Estimation of individual SCFA absorption rate (expressed as% total SCFA/h) showed that the acetate absorption in B (58.79±.17) did not differ (P= 0.29) than in C (52.98±.77), the butyrate absorption tended to be higher (P= 0.09) in B (13.31±.71) compared to C (11.75±0.36), and the propionate absorption increased (P< 0.05) in B (17.25±0.85) than in C (14.31±.48). In Exp.2 the results showed that mRNA expression of cyclin D1 increased (P< 0.05) 3 h following infusion compared to the pre-infusion value and then subsided to pre-infusion value at 8 h following infusion where as the mRNA expression of CDK4 remained unchanged between the groups. The fractional rate of total SCFA absorption (expressed in both%/h and mmol/h) increased significantly (P< 0.05) in B compared to C group. The fractional absorption rates (mmol/h) of individual SCFA showed that the acetate absorption was not significant between the groups, the butyrate absorption tended to increase (P= 0.06) and the propionate absorption increased (P< 0.05) in B compared to C. Concurrent with higher absorption MCT-4 mRNA expression in rumen epithelium was higher in B compared to C.Analysis of hay intake, performance and rumen growth parameters in Exp.1 showed that the mean hay intake throughout the experimental trial tended to increase (P= 0.083) in B than in C. Weekly observations showed that hay intake was not significant for first two weeks, tended to be higher in third week (607.14±29.01 g in B vs 538.93±3 g in C; P= 0.1) and progressively increased in the fourth week of the experiment (675.36±8.86 g in B vs 588.57±7.66 g in C; P< 0.05) in goats of B compared to that of C. Final body weight and the average daily weight gain were not significant (P> 0.05) between the groups however, the average daily weight was increased by 20.98% in goats of B compared to that of C group. Rumen weight expressed as percent of whole stomach increased in goats of B (71.79±0.57%) compared to that of C (70.10 ±0.36%; P< 0.05). Expressed as percent of full stomach weight, the digesta weight in rumen increased in B (91.72±0.59%) compared to C (89.81±0.54%; P= 0.06). Ruminal volume (2.12±0.09 1), liquid turn over (0.083± 0.01/h) and liquid outflow (0.36±0.021/h) in B were not different than in C (2.03±0.06,0.087±0.04, and 0.35±.02; P> 0.05). Time spent on eating, ruminating and idling was not different (P> 0.05) between the groups, however the total number of chews/min (during eating and ruminating) tended to be higher in B (171.26 ±1.14) than in C (164.03±3.46; P= 0.094).It was concluded that butyrate induced epithelial proliferation through modulation of cyclin D1 gene which showed time-dependent changes in the mRNA expression. Butyrate-induced epithelial growth increased SCFA absorption from the rumen, Ruminal infusion of butyrate increased hay intake, and improved chewing behavior, rumen efficiency and growth performance of goats. |
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