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Effect of Tea Saponin on Rumen Fermentation and Methane Production in Vitro and Toxicity of Tea Saponin

Author: ZhangZuoZuo
Tutor: YangZaiBin
School: Shandong Agricultural University
Course: Animal Nutrition and Feed Science
Keywords: tea saponin rumen fermentation methane toxicity
CLC: S816.7
Type: Master's thesis
Year: 2011
Downloads: 32
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Abstract


Abstract: The study was carried out to find the best methods to determine the content of the tea saponin and investigate the effects of the saponin fractions extracted from CTS with 1-butanol （BTS）, other bioactive components in the CTS （OBC） and pure tea saponin （PTS） on methane production, rumen fermentation and microbial populations. Microbiology mechanism for decreased methane production by tea saponins was discussed. The toxicity mechanism of the tea saponin was also carried out.The experiment 1 found the best methods to determine the content of the tea saponin. The result found that saponin contents were 400 and 700 g/kg （w/w） for the CTS and BTS, and sugar contents were 160 and 800 g/kg （w/w） for CTS and OBC, respectively.Crude tea saponin （CTS）, saponin fractions extracted from CTS with 1-butanol （BTS）, other bioactive components in the CTS （OBC） and pure tea saponin （PTS） were used in in vitro gas test to evaluate their effects on methane emission, fermentation and microbial communities in the rumen in vitro. In Trial 1, BTS was added at levels of 0, 1.71, 3.43 and 6.86 mg, respectively, against 200 mg mixture of corn meal and grass meal （1/1, w/w） in the rumen fluid, and OBC was tested at four doses （0, 0.6, 1.2 and 2.4 mg against 200 mg of substrates）. After incubation for 24 h, rumen fluid was sampled for analysis of rumen fermentation products. The microbial DNA was also extracted to study the effect on microbial communities. Numbers of gene copies associated with rumen methanogens （mcrA gene）, specific bacteria （16S rDNA gene） and rumen protozoa and fungi （18S rDNA gene） were measured using real-time quantitative ploymerase chain reaction. The abundance of marker gene-copy number for the methanogens, protozoa, fungi, Ruminococcus flavefaciens and Fibrobacter succinogenes were expressed relative to the copy number of total rumen bacterial 16S rDNA as a reference gene. Six milligram of CTS was added as a positive control. No significant effects were observed on pH, gas value and total volatile fatty acids by addition of CTS, BTS or OBC, while propionate proportion was enhanced （P < 0.05） and ammonia nitrogen were decreased （P < 0.05） by addition of CTS or BTS. Methane production showed a linear and quadratic decrease （P < 0.05） with increasing levels of BTS. In contrast, inclusion of OBC linearly （P = 0.009） increased the methane production. No significant effect was observed on the relative abundance of the fungi and R. flavefaciens by addition of BTS and OBC. The abundance of protozoa relative to total bacterial 16S rDNA showed a linear and quadratic decrease （P < 0.05） with increasing levels of BTS. The relative abundance of the methanogens were not affected by addition of BTS, but increased when the OBC was included. In Trial 2, pure tea saponin （PTS） was added at levels of 0, 1.9, 3.8 and 7.6 mg against 200 mg of substrates. Beside the measurement of rumen fermentation parameters, the microbial DNA was also extracted to study the effect on microbial communities. Addition of PTS did not significantly affect pH and total volatile fatty acids （P > 0.05）. Gas value, methane production and ammonia nitrogen showed a linear and quadratic decrease （P < 0.05） with increasing levels of PTS, while proportion of propionate and butyrate increased in the PTS-added group. The abundance of protozoa and F. succinogenes marker genes, relative to total bacterial 16S rDNA, showed a linear and quadratic effect by PTS, but no significant effects were observed on the relative abundance of the methanogens and fungi genes （P > 0.05）. Compared to the control, the relative abundance of R. flavefaciens was linearly （P = 0.008） and quadratically （P = 0.017） inhibited when the PTS was included. It is suggested that the saponin fractions in the CTS modify the fermentation and inhibit methane production in the rumen, probably mediated by direct action against the rumen microbes involved in methane formation.The experiment 3 evaluated the tea saponin acute toxicity when administered intragastrically and determined LD50 for laboratory mice. Based on the pretest results, seven dosages （7.0, 5.95, 5.06, 4.3, 3.65, 3.1and 2.64g/kg body mass） were established with each group comprised of 20 mice using random block design （average body mass 18–20 g）.Tea saponin LD50 and its 95% confidence limits for male mice were 3.24g/kg body mass and 2.86-3.54g/kg body mass; and LD5 and LD95 were 1.87 and 5.60 g/kg body mass, respectively. The regression equation between the probits of mortalities （Y） and the log of doses （D） was derived as: Y （Probit） = 1.48+6.9002 Log（D）. Tea saponin had toxicity on the lung, liver, kidney, brain, heart and intestines of the mice. But it had no toxicity on the spleen.

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CLC: > Agricultural Sciences > Livestock, animal medicine,hunting,silkworm,bee > General Animal Science > Feed > Feed additives
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