Two trials were conducted to identify the optimal levels of essential oil active components (EOAC) and their combination with fumarate on in vitro rumen fermentation. In trial 1, eugenol, carvacrol, citral and cinnamaldehyde were mixed at ratios of 1:2:3:4, 2:1:4:3, 3:4:1:2, 4:3:2:1 and 1:1:1:1 to make up five combinations (EOAC1, EOAC2, EOAC3, EOAC4 and EOAC5 respectively). The mixtures were supplied at levels of 0, 50, 200 or 500 mg/l to identify the optimal combination for methane reduction. Methane production and ammonia nitrogen were decreased by adding EOAC, irrespective of component compounds, but the production of gas and total volatile fatty acids (VFA) were also decreased. Hydrogen balance analysis indicated that the ratio of hydrogen consumed via methane to hydrogen consumed via VFA was lowest at 200 mg/l of EOAC5 treatment, from which the proportional change in methane was more than the change in VFA, with 31.5% of methane reduction and 12.9% of VFA reduction. In trial 2, 200 mg/l of EOAC5 was added with 0, 5, 10 and 15 mm monosodium fumarate to see whether fumarate had a further effect on rumen fermentation. The addition of fumarate had no influence on gas production, but it further decreased methane and increased the total VFA in comparison with EOAC added solely, with the greatest decrease occurring in methane (78.1%) from 10 mm of fumarate. Quantification of the microbial populations in rumen fluids by RT-PCR showed that methanogen, protozoa, fungi, Fibrobacter succinogenes and Ruminococcus flavefaciens populations were significantly decreased by EOAC5, but were not influenced by fumarate. In summary, the addition of EOAC had consistent effects on rumen fermentation parameters, but high levels of EOAC would induce the inhibition of rumen fermentation. Adding fumarate can enhance the methane-inhibiting effect of EOAC, and the decrease was higher than that calculated stoichiometrically.
This study aims to determine the effects of alfalfa supplementation on the pre- and postweaning performance, rumen development, and feed transition in starter diet-fed lambs. Six of 66 male Hu lambs were slaughtered at the age of 10 d to serve as a control. The other 60 lambs were randomly allocated to 2 dietary treatments: milk replacer and starter pellets without (STA) or with free-choice chopped alfalfa (S-ALF). The animals were offered 300 g/d of the concentrate mixture and had free access to alfalfa after weaning at the end of wk 4 (age 38 d). The alfalfa inclusion in the S-ALF group tended to increase the starter intake before weaning, significantly increased the concentrate intake soon after weaning ( < 0.05), and increased the BW ( < 0.01) and ADG ( < 0.10) in pre- and postweaning lambs. The S-ALF group had heavier carcasses ( < 0.05), rumens ( < 0.05), reticula ( < 0.05), omasums ( < 0.10), abomasums ( < 0.05), and visceral organs ( < 0.10) than the STA lambs after weaning. Alfalfa supplementation increased ( < 0.05) the rumen papillae length and the ratio of the duodenal villus height to the crypt depth; it also decreased ( < 0.05) the concentration and molar proportion of propionate in wk 1 and 5. The STA lambs had higher ( < 0.01) blood concentrations of globulin and blood urea nitrogen and lower β-hydroxybutyrate after weaning. The STA group also had a higher incidence of feed plaque. From the above results, we infer that the free-choice addition of chopped alfalfa to starter diets is beneficial to rumen development, relieves weaning stress, and improves the performance of lambs.
This study was conducted to investigate effects of disodium fumarate (DF) on fermentation characteristics and microbial populations in the rumen of Hu sheep fed on high-forage diets. Two complementary feeding trials were conducted. In Trial 1, six Hu sheep fitted with ruminal cannulae were randomly allocated to a 2 3 2 cross-over design involving dietary treatments of either 0 or 20 g DF daily. Total DNA was extracted from the fluid-and solid-associated rumen microbes, respectively. Numbers of 16S rDNA gene copies associated with rumen methanogens and bacteria, and 18S rDNA gene copies associated with rumen protozoa and fungi were measured using real-time PCR, and expressed as proportion of total rumen bacteria 16S rDNA. Ruminal pH decreased in the DF group compared with the control (P , 0.05). Total volatile fatty acids increased (P , 0.001), but butyrate decreased (P , 0.01). Addition of DF inhibited the growth of methanogens, protozoa, fungi and Ruminococcus flavefaciens in fluid samples. Both Ruminococcus albus and Butyrivibrio fibrisolvens populations increased (P , 0.001) in particle-associated samples. Trial 2 was conducted to investigate the adaptive response of rumen microbes to DF. Three cannulated sheep were fed on basal diet for 2 weeks and continuously for 4 weeks with supplementation of DF at a level of 20 g/day. Ruminal samples were collected every week to analyze fermentation parameters and microbial populations. No effects of DF were observed on pH, acetate and butyrate (P . 0.05). Populations of methanogens and R. flavefaciens decreased in the fluid samples (P , 0.001), whereas addition of DF stimulated the population of solid-associated Fibrobacter succinogenes. Population of R. albus increased in the 2nd to 4th week in fluid-associated samples and was threefold higher in the 4th week than control week in solid samples. Analysis of denaturing gradient gel electrophoresis fingerprints revealed that there were significant changes in rumen microbiota after adding DF. Ten of 15 clone sequences from cut-out bands appearing in both the 2nd and the 4th week were 94% to 100% similar to Prevotella-like bacteria, and four sequences showed 95% to 98% similarity to Selenomonas dianae. Another 15 sequences were obtained from bands, which appeared in the 4th week only. Thirteen of these 15 sequences showed 95% to 99% similarity to Clostridium sp., and the other two showed 95% and 100% similarity to Ruminococcus sp. In summary, the microorganisms positively responding to DF addition were the cellulolytic bacteria, R. albus, F. succinogenes and B. fibrisolvens as well as proteolytic bacteria, B. fibrisolvens, P. ruminicola and Clostridium sp.
Two trials were conducted to investigate the effect of a combination of essential oils (CEO) along with fumarate on in vitro rumen fermentation. In trial 1, the essential oil (EO) from thyme, oregano, cinnamon and lemon were mixed at five different ratios. The CEO were applied at levels of 0-500 mg/l. Addition of CEO decreased gas, methane, total volatile fatty acid (VFA) production at 24 h incubation in a dose-dependent manner. Methane tended to decrease much more than total VFA and gas at the same EO level. The mixture of oils at an equal ratio at 500 mg/l that decreased methane much more than VFA was chosen as the optimal combination. In trial 2, the optimal combination was used with 0, 5, 10 or 15 mmol/l of monosodium fumarate. Addition of fumarate further decreased methane production, with 10 mmol/l fumarate resulting in the largest reduction (80.2%) and the smallest decrease in total VFA (5.7%) and gas production (16.7%). Quantification of several ruminal microbe populations by RT-PCR showed that the optimal combination sharply decreased ruminal protozoa; the populations of fungi and fibrolytic bacteria were also decreased. In summary, at an appropriate level, CEO can inhibit methane production. Inclusion of fumarate can further decrease it, which is attributed mainly to inhibition of protozoa and methanogens.
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