| Methane production in the rumen represents as a loss of energy for the host animal,and, in addition, methane emission by ruminants may contribute to a greenhouse effect orglobal warming. Scientists have been recently paying much attention to studymethnogen-inhibitors with the global concerning on greenhouse gas emission. The grosssaponin of Tribulus terrestris（GSTT）were selected to systematically study its effects onrumen fermentation, methane production, digestion and utilization of nutriment,antioxidant capacity and immune function of dairy cows by using artificial gas productiontrial, dual outflow continuous culture trial and feeding ruminally cannulated trial.Real-Time PCR technique to monitor ruminal microbial population to discuss themicrobiology mechanism for decreased methane production by GSTT.Exp.1The objective of this study was to determine preliminarily the effect of GSTTon ruminal fermentation and methane production in in vitro gas production test by usingMenke’s syringe system.The GSTT were added at levels of0,0.15,0.30,0.60and0.90g/Lin30mL culture medium. Substrate was200mg mixture of corn grain and Chinese wildryeon an equal mass basis.Each treatment （every dose of GSTT） was incubated inquadruplicate, and fermentations were repeated on two separate days. The set free of GSTTbut containing of the substrate was used as control. After24h of incubation, methaneproduction were reduced with increasing levels of GSTT, however, it was similar to that ofthe control treatment when addited with the lowest level（0.15g/L）.Compared to control,methane production were decreased by26.67%,28.89%,31.11%, addited with0.15,0.30,0.60and0.90g/L of GSTT, respectively. GSTT had little effect on pH and theconcentration of volatile fatty acids（VFA）in culture fluid（P>0.05）, while NH3-Nconcentrations were decreased（P<0.05）, by addition of GSTT. Molar proportion of acetateand butyrate was reduced and propionate was increased with a corresponding reduction inA/P ratio（P<0.05）. Total protozoal counts showed a linear decrease（P<0.01）withincreasing levels of GSTT. Compared to control, total protozoal counts were reduced by27.03%,37.84%,60.36%and72.07%for0.15,0.30,0.60and0.90g/L of GSTT（P<0.05）,respectively. Excluding the lower levels（0.15and0.30g/L）of GSTT, IVDDM wasdecreased（P<0.05）at higher levels（0.60and0.90g/L）.Exp.2The objective of this study was to further research on ruminal fermentation, methane production and microbial community in in vitro gas production test by usingANKOM RFS gas measurement system. The GSTT were added at levels of0,0.15,0.30,0.60and0.90g/L in100mL culture medium. The substrate was composed on a dry matter（DM）basis of340g/kg corn grain,160g/kg soybean meal, and500g/kg Chinese wildryegrass hay.Each treatment（every dose of GSTT）was incubated in quadruplicate, andfermentations were repeated on two separate days. The set free of GSTT but containing ofthe substrate was used as control. Results showed that methane emission decreased（P<0.01）in a line manner at all incubation times.Addition with0.15,0.30,0.60and0.90g/L of GSTT decreased methane production by8.72%（P>0.05）,15.44%（P<0.05）,21.48%（P<0.05）and24.16%（P<0.05）relative to controls at24h, respectively. GSTT hadlittle effect on pH and the concentration of total VFA in culture fluid（P>0.05）. However,the fermentation patterns were modified, with higher propionate molar proportion, andlower acetate molar proportion, A/P ratio as well as NH3-N concentration. In addition,GSTT also altered the population of rumen microbes relative to total bacterial16SrDNA.The abundance of rumen protozoa were significantly decreased by addition ofGSTT,in general, linear and quadratic（P<0.01）. The abundance of methanogens and fungidecreased by GSTT addition with linear dose–response effects.However, Fibrobactersuccinogenes and Ruminococcus falvefaciens were not affected. In summary, there were nosignificant difference in in vitro rumen fermentation, gas production or methane productionbetween Menke’s syringe system and ANKOM RFS gas measurement system,indicatedthat both systems could be used to simulate the rumen fermentation.Exp.3The effects of different levels of GSTT on rumen fermentation characteristicwere examined in DFCOS－Ⅱdual-flow continuous culture system. Furethermore, thepoper adding levels of GSTT in vitro was estabilished to provide reference basis forsubsequent experiment in vivo study. The GSTT were added at levels of0,0.15,0.30and0.60g/L in culture medium. Each treatment（every dose of GSTT）was incubated intriplicate. The entire experimental period was7days, including5days for adjustment andthe last2days for sample collection. It was concluded that addition with0.15,0.30and0.60g/L of GSTT had little effect on ruminal pH and the concentration of total VFA（P>0.05）, while molar proportion of acetate, A/P ratio as well as NH3-N concentrationsignificantly decreased（P<0.05）,molar proportion of propionate significantly increased（P<0.05）. Theses results indicated that GSTT has a potential to be used as a rumenmodulator to improve the utilization efficiency of energy and protein.Exp.4The experiment was conducted to study the effects of GSTT on rumenmicrobial fermentation,methane production, digestion and utilization of nutriment,antioxidant capacity and immune function of dairy cows. Four healthy ruminally fistulateddairy cows were randomly assigned in a4×4Latin square design. The animals receivedthe same basal diets and added with the GSTT at levels of0（control）,5,10and15g d1,respectively.The experiment lasted for four periods, each feeding period lasting for28days. The results showed as follows：（1）Addition with different levels of GSTT had little effect on dry matter intakes（DMI）, ruminal pH and the concentration of total VFA（P>0.05）. Compared with thecontrol treatment,methane emission of dairy cows,added with the GSTT at levels of10and15g d1,were decreased（P<0.05）,but there were no difference between the twotreatments（P>0.05）.When expressed on the basis of DMI，methane production was foundlowest（14.94g/kg DMI）for dairy cows with GSTT supplement at the dose of10g d1.Compared with the control treatment,rumen NH3-N concentration and A/P ratio was lowerfor dairy cows with GSTT supplement at the dose of10and15g d1（P<0.05）.However,addition with GSTT significantly enhanced molar proportion of propionate（P>0.05）.Inaddition, feeding GSTT decreased significantly rumen total protozoal counts,the totalprotozoal counts for dairy cows added with the GSTT at levels of10and15g d1werelower than those of dairy cows in control treatment（P<0.05）.Moreover, The rumenmethanogens and fungi counts of dairy cows tend to decrease linearly with increasinglevels of GSTT,but there were no significant differences between different treatments（P>0.05）. The rumen Fibrobacter succinogenes and Ruminococcus falvefaciens were notaffected significantly by addition with GSTT（P>0.05）.（2）Feeding GSTT did not affect the apparent degradabity or the effectivedegradation（ED）of dry matte（rDM）（P>0.05）.However, the ED of DM tend to decrease.Addition with GSTT declined the apparent degradabity and ED of CP at36h and48h,andthe numerical value of dairy cows added with the GSTT at levels of15g d1was thelowest,which was lower significantly than that of the control treatment（P<0.05）.FeedingGSTT had little effect on the ED of NDF and（P>0.05）.In addition, the apparent total tractdigestibilities of DM,OM,GE,CP,NDF and ADF were unaffected by the supplementationwith GSTT（P>0.05）. There were no significant differences in rumen cellulase, CMCaseor salicinase activites of dairy cows between different groups（P>0.05）.However, thexylanase activities of dairy cows added with the GSTT at levels of15g d1were lowerthan those of the control treatment（P<0.05）.（3）The serum concentration of total proteins（TP）, albumin（ALB）, globulin（GLB）and ratio of albumin to globulin （A/G） ratio was unaffected significantly by thesupplementation with GSTT（P>0.05）,but the serum concentration of TP tend to enhancewith the increasing levels of GSTT（P=0.09）. Addition with GSTT reduced the serumconcentration of serum urea nitrogen（SUN）（P<0.05）, triacylglycerol（TG）（P<0.01）and cholesterol（TC）（P<0.05）.Moreover, The serum concentration of HDL cholesterol（HDL-C）was increased linearly with the increasing levels of GSTT（P=0.03）,however,there were no significant differences among groups（P>0.05）.There was no significantdifference in the enzyme activties of glutamic-oxalacetic transaminase （GOT）, glutamic-pyruvic transaminase（GPT）, alkaline phosphatase（ALP）and in the serumconcentration of creatinine, glueose, Ca and P among treatments（P>0.05）.（4）Compared to the control treatment, the enzyme activities of serum T-AOC andGSH-Px for dairy cows added with the GSTT at levels of10and15g d1were improved（P<0.05）, the serum concentration of MDA was declined（P<0.05）,but there were nodifferences between the two treatments（P>0.05）.The enzyme activities of serum SODtend to improve with the increasing levels of GSTT（P=0.06）.It was concluded thataddition with GSTT contribute to improve the antioxdtion capability of dairy cows.（5）The serum concentrations of IgA, IL-2or TNF-a did not differ significantlyamong the gruops（P>0.05）. However, the serum concentrations of IgG and IgM of dairycows added with the GSTT at levels of10and15g d1were higher than those of thecontrol treatment（P<0.05）.In addition,the serum concentrations of IgA and IL-2wereimproved in a linear manner with the increasing levels of GSTT（P=0.05）.It was indicatedthat the supplement of GSTT could improve immune functions of dairy cows.In summary, proper leves of GSTT may moderate rumen fermentation, includinginhibited rumen protozoa,decreased rumen NH3-N concentrations moderately and methaneemission of dairy cows. Moreover, antioxidant capacity and immune function of dairycows were enhanced. Ovell, under the study conditions, the optimum adding level ofGSTT to the diet in dairy cows was10g/d. |
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