Introduction
Previous studies (Busquet et al., 2005; Calsamiglia et al., 2007) have shown that garlic oil (GO) supplementation to in vitro cultures of ruminal micro-organisms leads to reduced methane production and acetate:propionate ratios, but the effects are related to the dose. Our hypothesis was that GO effects might depend on the rumen microbial populations, and thus on both the incubated substrate and the type of diet fed to donor animals.
The aim of this study was therefore to analyze the effects of different doses of GO on in vitro fermentation of two diets in batch cultures of mixed rumen micro-organisms from the rumen of sheep fed the same diets.
Material and methods
Eight Merino sheep (56.1 ± 2.80 kg) fitted with permanent ruminal cannulas were used as rumen fluid donors. Four sheep received a medium-concentrate (MC; 500:500 alfalfa hay:concentrate) and the other 4 were fed a high-concentrate (HC; 150:850 barley straw:concentrate) diet for 10 days before starting the in vitro incubations.
Samples (300 mg) of MC and HC diets were incubated with 30 mL buffered ruminal fluid in batch cultures. GO was added to the cultures at 0, 20, 60, 180 or 540 mg/L of incubation medium. After 16 h of incubation at 39ºC, the main fermentation variables were determined. Incubations were repeated on four non-consecutive days. Data were analyzed as a mixed model using the MIXED procedure (SAS Inst. Inc., Cary, NC).
Five concentrations of GO, diet, and the interaction of GO x diet were included in the model as fixed effects, and incubation day was considered as a random effect. Nonorthogonal polynomial contrasts were used to test for linear effects of GO. Differences among treatments were declared at P < 0.05, and P values of 0.05 to 0.10 were considered as trends.
Results
There were no effects (P = 0.16 to 0.86) of GO on pH or concentration of ammonia-N and total lactate (results not shown). As shown in Table 1, GO x diet interactions (P = 0.06 to <0.001) were observed for most of the measured parameters. Total VFA production was not affected by any dose of GO with HC diet, but it was reduced (P = 0.02) by GO540 for MC diet indicating inhibition of rumen fermentation.
Increasing doses of GO reduced linearly (P < 0.001) the proportion of acetate and acetate:propionate ratio, and increased (P < 0.001) the proportion of propionate; whereas GO showed significant effects at 60 mg/L with MC diet, a minimum concentration of 180 mg/L was required with HC diet. The addition of GO to HC did not modify the proportion of butyrate, but in agreement with previous results (Busquet et al., 2005) GO at 60, 180 and 540 mg/L increased (P < 0.05) butyrate proportion with diet MC. For diet HC, GO20 reduced CH4 and CH4/VFA ratio by 9.6 and 12.1%, respectively, but no effects (P > 0.05) were observed for diet MC. GO at 60, 180 and 540 mg/L decreased CH4 production to 87, 58 and 36% of the CON values for MC diet, and to 91, 75 and 38% of CON values for HC diet.
Hydrogen recovery, calculated from the stoichiometric relationships between the end products formed (Demeyer, 1991), was linearly lowered (P < 0.001) by increasing GO supplementation. This could indicate accumulation of hydrogen or a reduced end product other than CH4, hydrogen, propionate, butyrate and valerate, since these are involved in the calculation of the hydrogen balance.
Table 1. Effects of five doses of garlic oil (GO; 0, 20, 60, 180 and 540 mg/L for CON, GO20, GO60, GO180 and GO540, respectively) on total volatile fatty acid production (VFA; =mol), molar proportions (mol/100 mol) of acetate (C2), propionate (C3) and butyrate (C4), acetate:propionate ratio (C2:C3; mol/mol), CH4 production (=mol), CH4/VFA ratio (mol/mol) and hydrogen recovery (HR; %) after in vitro fermentation of diets (300 mg) with medium (MC) and high (HC) concentrate content by mixed rumen micro-organisms for 16 h (n = 4)
Conclusion
The results of this study would indicate that GO may show different effects on in vitro rumen fermentation depending on the administered dose and the substrate composition and microbial populations in the inoculum, which could help to explain the variation in the response observed in different studies.
by C. Kamel, H.M.R. Greathead, M.J. Ranilla, M.L. Tejido, S. Ramos and M.D.Carro
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