In vitro
Fermentation and Bacterial Protein Synthesis in the
Different Diets Supplemented with Lerak
Extract plus Mineral (Ca, P, Mg, S)
S. Suharti1,*, N. Aizah1, D. M. Suci1, D.A. Astuti1 & E. Wina2
1Department of Nutrition and Feed Technology, Faculty of Animal Science,
Bogor Agricultural University, Bogor, 16680, Indonesia,
*e-mail: harti_ss@yahoo.com
2Indonesian Research Institute for Animal Production, Bogor,
Jl.Veteran III, Banjarwaru PO Box 221, Bogor 16002, Indonesia
Abstract
Bacterial protein supply is usually low in animals fed with high forage ration because of the lack of sources of minerals in the rumen. The aim of this study was to evaluate the use of lerak extract plus mineral (Ca, P, Mg, S) on fermentation and bacterial protein synthesis in the in vitro fermentation with different ratios of forage and concentrate. The design of experiment was factorial block design (3x3) with 2 factors which were: ratio of forage and concentrate (70:30, 50:50, 30:70) as the first factor and the type of supplements (0, 1mg/ml lerak extract and 1mg/ml lerak extract + minerals) as the second factor. Total volatile fatty acid (VFA), NH3 concentration, population of protozoa, and bacterial protein synthesis were measured at 4 h incubation. Dry matter and organic matter digestibility were evaluated after 48 h incubation. The result showed that there was no interaction effect between ratio of forage to concentrate and the type of supplements. The different ratios of forage and concentrate had no effect on dry matter and organic matter digestibility, and NH3 concentration. The increase of concentrate ratio in the diet reduced population of protozoa, but increased total VFA and bacterial protein synthesis. The addition of 1 mg/ml lerak extract without minerals significantly decreased (P<0.05) population of protozoa and increased (P<0.05) bacterial protein synthesis but no effect on dry matter and organic matter digestibility, NH3 concentration, and total VFA production compared to the control. However, the addition of lerak extract plus mineral (Ca, P, Mg, S) had no effect on all parameters measured. In conclusion, bacterial protein synthesis increased by supplementation of lerak extract without mineral addition.
Introducton
Rumen mcrobal populaton has an mportant role n the dgeston of feed fber by rumnants. The rumen has a lot of varety of mcrobal communtes such as protozoa, bactera, fung, and vruses. Interacton between protozoa and bactera sometme has dsadvantage because of predaton of bactera by protozoa. Ths pre-daton can cause the reducton of bacteral populaton and affect the growth of ru-mnants. It has been known that mcrobal proten n the rumen, especally bacteral proten, s the major good qualty proten resource for rumnants (Pathak, 2008).
Bacteral proten supply s usually low n anmals fed by hgh forage raton because of the ntrogen/proten defcency n the det. In addton, forage based dets often lack of some mnerals requred for the synthess of mcrobal proten n the ru-men. To overcome ths problem, a strategy s requred to mprove the qualty of for-age wth mneral supplementaton and to reduce the populaton of rumen protozoa.
Defaunaton by usng whole frut lerak (Sapindus rarak) extract modfed bacteral
composton and ncreased the growth of some bactera, especally Prevotella
ru-minicola and Ruminococcus albus, and stmulated proponate producton (Suhart
et al., 2011). Our prevous in vivo study showed that the use of lerak extract for beef cattle wth hgh forage dets ncreased VFA producton wthout a sgnfcant
ncrease n mcrobal proten synthess (Suhart et al., 2010) whch may be due to
mneral defcency n the hgh forage based det, especally Calcum (Ca), Phosphor (P), Magnesum (Mg) and Sulfur (S).
Ths study was conducted to evaluate the use of lerak extract plus mneral (Ca,
P, Mg, S) on fermentaton and bacteral proten synthess n the in vitro fermentaton
wth dfferent ratos of forage and concentrate.
Materals and Methods
Whole Fruit Lerak Extract Preparation
The lerak fruts (ncludng seed) were harvested from Central Java Indonesa.
Whole frut lerak extract was extracted by usng maceraton method (Wna et al.,
2006). The extract was freeze-dred the freeze-dred extract was dssolved n dstllate water just before beng used as extract of whole frut lerak
In vitro Fermentation
In vitro fermentaton was conducted accordng to Tlley and Terry method (1963). The rumen flud for ths experment was obtaned before mornng feed-ng from the rumen of fstulated Ofeed-ngole crossbred beef cattle fed commercal con-centrate and elephant grass (50:50, DM). The rumen flud was squeezed through a
double-layer cheesecloth for in vitro experment. The commercal concentrate
and wheat pollard. Elephant grasses were harvested from the Faculty of Anmal Scence Farm, Bogor Agrcultural Unversty, and then were dred n the oven and mlled. The desgn of experment was factoral block desgn (3x3) wth 2 factors .e., rato of elephant grass and concentrate (70:30, 50:50, 30:70) as the frst factor and type of supplements (0, 1mg/ml lerak extract and 1mg/ml lerak extract + mner-als mx) as the second factor. Mneral mx was composed of Ca 0.54%, P 0.37%, Mg 0.23%, and S 0.1%. The levels of lerak extract used n ths experment were based on our prevous study showng that the addton of 1 mg/ml lerak extract n-creased fermentaton actvtes. Fve hundred mllgrams of substrate (Concentrate, elephant grass and supplement) accordng to the treatments were put nto a 100 ml fermentaton tube. Forty mlllters of McDougall buffer was added, followed by 10 ml of rumen flud. The McDougall buffer contaned, per 6 lters, NaHCO
3 (58.8 g),
Na2HPO4.7H2O (42 g), KCL (3.42 g), NaCl (2.82 g), MgSO4.7H2O (0.72 g), CaCl2
(0.24 g) and H2O. The mxture was strred and flushed wth O2-free carbon doxde.
The tubes were then sealed wth rubber corks ftted wth the gas release valve. All
fermentaton tubes were ncubated n a shaker water bath at 39oC.
Sampling and measurement
Total volatle fatty acd (VFA), NH3 concentraton, protozoal populaton, and
bacteral proten synthess were measured from lqud sample taken at 4 h ton. Dry matter and organc matter dgestblty were evaluated after 48 h ncuba-ton. After 4 h of ncubaton, samples of rumen alquot were taken for protozoa countng under a mcroscope (Ogmoto & Ima, 1981). The contents of fermentaton tubes were shaken and 0.5 ml alquot was mxed wth 0.5 ml methyl green formal-dehyde salne soluton contanng 35% formalformal-dehyde, dstlled water, methyl green and NaCl. The staned sample was kept at room temperature and the populaton of protozoa was counted drectly by usng a countng chamber under a mcroscope
(40×). Ammona concentraton was analyzed by usng mcro dffuson Conway and
total volatle fatty acd producton was analyzed by steam dstllaton (General Lab-oratory Procedure).
Mcrobal proten synthess was determned based on Lowry assay accordng
to Makkar et al. (1981). The straned rumen lquor was shaken n a magnetc strrer
(400 rpm) for 45 s to remove the mcrobes adsorbed on the feed partcles. It then was centrfuged at 408 × g for 5 mn for removng protozoa and remanng feed partcles. Alquots of 10 ml of rumen lquor, obtaned after removng the feed partcles and protozoa, were taken, and 2.5 ml of 64.5% TCA and 3.8 ml of 2/3 N sulfurc acd were added to each sample. Alquots were then centrfuged at 15000 rpm for 20 mn. Supernatants were dscarded, and cells obtaned were washed wth McDougall’s buffer and the mxtures were then centrfuged. The precptates obtaned after wash-ng wth dstlled water were suspended n 30 ml of .25 N NaOH, heated n bol-ng water bath for 10 mn, and proten was estmated accordbol-ng to Lowry method
Statistical Analysis
Statstcal analyss of the data was carred out by ANOVA usng General Lnear Procedure. Computaton was performed usng SPSS 13.0 for wndows evaluaton verson.
Results and Dscusson
Population of Protozoa
The populaton of protozoa were sgnfcantly reduced (P<0.05) wth the addton of lerak extract. Supplementaton of mneral mx to the lerak extract dd not affect the populaton of protozoa as compared to the control treatment at 4 h ncubaton. The dfferent ratos of forage to concentrate dd not affect the populaton of protozoa (Table 1). There was no nteracton effect between the addton of supplement (lerak extract or lerak extract plus mneral supplementaton) and rato of forage to concentrate on the populaton of protozoa.
The reducton of protozoal populaton was caused by saponn content n the lerak extract. It s known that saponn could nhbt the growth of protozoa due to
bndng actvty of saponn to sterol that composed protozoal membrane (Patra et
al., 2006). Among all rumen mcrobes, protozoa are almost susceptble to
saponn-nduced changes n cell membrane propertes (Moss et al., 2000). Hu et al. (2005)
reported the reduced protozoal numbers was due to the saponn treatment. The addton of mneral mx (Ca, P, Mg, & S) to lerak extract dd not sgnfcantly reduce protozoal populaton compared wth lerak extract alone. These may be due to the presence of mneral that could stmulate the growth of protozoa and mprove protozoal survval.
Bacterial Protein Synthesis (BPS)
There was no nteracton effect between the addton of lerak extract supple-mentaton and the dfferent ratos of forage to concentrate on bacteral proten syn-thess. The addton of lerak extract or lerak extract + mneral ncreased bacteral proten synthess sgnfcantly (P<0.05). The dfferent ratos of forage to concen-trate also affected bacteral proten synthess sgnfcantly (P<0.05). Bacteral pro-ten synthess ncreased when the level of concentrate was ncreased n the raton (Table 1).
The dfferent dets also affected the bactera proten synthess. Bacteral proten synthess tended to ncrease n lne wth the ncrease n concentrate rato n the rumen. Ths may be due to the suffcency of energy proten rato (C/N) n the hgher concentrate rato that stmulates bacteral proten synthess.
Fermentation Characteristic
Ammonia Concentration. At 4 h of ncubaton, there was no nteracton between the addton of supplement (lerak extract or lerak extarct plus mneral supplementaton) and the rato of forage to concentrate on ammona concentraton. Ammona concentraton was not affected by ether lerak extract or lerak extract + mneral supplementaton or dfferent ratos of forage to concentrate (Table 1). The addton of saponn from lerak extract dd not affect proten degradaton n the rumen.
Total VFA Production. The addton of lerak extract or lerak extract + mneral dd not affect total VFA producton. However, total VFA producton was sgnfcantly affected by forage:concentrate rato n the det. Total VFA producton ncreased when the level of concentrate n the raton was 70%. There was no nteracton effect between lerak extract supplementaton and dfferent ratos of forage to concentrate (Table 1). The hgher concentrate rato n the dets could stmulate VFA producton by rumen mcrobe as concentrate feed contans hgh carbohydrate that a major substrate for VFA producton.
Dry Matter and Organic Matter Digestibilities. There was no nteracton effect between the addton of lerak extract supplementaton and the dfferent ratos of forage to concentrate on dry matter and organc matter dgestbltes. The addton
Table 1. Protozoal populaton, bacteral proten synthess and fermentaton characterstc wth the addton of lerak extract or lerak extract + mneral n dfferent dets
Parameters Type of Supplementaton Det Rato (F:C)
0 lerak extract lerak extract +
mneral mx 70:30:00 50:50:00 30:70 Protozoa
(Log 10 CFU)
4.37±0.09a 4.18±0.15b 4.24±0.05a 4.34±0.06 4.27±0.09 4.19±0.06
BPS* (mg/10 ml)
95.90± 9.57a 137.49±20.92b 114.39±24.61ab 92.38±29.12a 119.66±24.14ab 135.70±12.18b
NH3 (mM) 8.76±1.07 9.82±0.78 10.08±1.22 8.60±0.29 9.58±1.11 10.48±1.22 Total VFA (mM) 158.08±14.16 148.55±8.87 138.51±15.27 148.88±5.84a 128.81±7.98b 167.45±8.87a
DMD (%) 57.86±1.73 55.48±3.11 55.26±1.4 54.16±3.27 56.90±1.14 57.53±3.36 OMD (%) 60.75±1.44 60.20±2.23 54.27±0.88 54.89±3.39 55.99±0.91 59.23±1.28
*BPS=Bacteral Proten Synthess, F= Forage, C= Concentrate
of lerak extract or lerak extract + mneral dd not affect dry matter and organc matter dgestbltes. The dfferent ratos of forage to concentrate also dd not affect dry matter and organc matter dgestbltes (Table 1). These results showed that the reducton of protozoa caused by saponn dd not alter the dgestblty of feed n the rumen. Although protozoa have a role n feed degradaton, nhbton of protozoa by
saponn dd not affect in vitro dry matter and organc matter dgestbltes.
Concluson
There was no nteracton effect between rato of forage to concentrate and the type of supplements on protozoal populaton and bacteral proten synthess. The addton of 1 mg/ml lerak extract wth or wthout mnerals decreased protozoal populaton and ncreased bacteral proten synthess wthout effect on dry matter
and organc matter dgestbltes, NH3 concentraton, and total VFA producton as
compared to control. The dfferent ratos of forage and concentrate had no effect on dry matter and organc matter dgestbltes and NH
3 concentraton. The ncreased
concentrate ratos n the det reduced protozoal populaton but ncreased total VFA producton and bacteral proten synthess. Bacteral proten synthess ncreased by supplementaton of lerak extract wthout mneral addton.
References
Guterrez, J. 2007. Observatons on Bacteral Feedng by the Rumen Clate
Isot-richa prostoma. J. Eukaryotc Mcrobol.5:122-126
Hu, W., J. Lu, J. Ye, Y. Wu and Y. Guo. 2005. Effect of tea saponn on rumen
fer-mentaton in vitro. Anm. Feed Sc. Technol. 120:333-339
Lowry O. H, N. J. Rosenbrough, A. L. Farr, and R. J. Randall. 1951. Proten Mea-surement wth the foln phenol reagent. J. Bol. Chem. 193: 265-275
Makkar, H.P.S., O.P. Sharma, R.K. Dawra and S. Neg. 1981. Smple Determna-ton of Mcrobal Proten n Rumen Lquor. J. Dary Sc. 65: 2170-2173 Moss, A.R., J.P. Jouany, and J. Newbold. 2000. Methane producton by rumnants:
Its contrbuton to global warmng. Annal Zootechnology, 49:231-253. Pathak, A.K. 2008. Varous factors affectng mcrobal proten synthess n the
ru-men. Veternary World, 1: 186-189
Patra, A.K., D.N. Kamra, and N. Agarwal. 2006. Effect of plant extract on n vtro methanogeness, enzyme actvtes and fermentaton of feed n rumen lquor of
buffalo. Anm. Feed Sc. Tech. 128:276-291.
Suhart, S. 2010. Modfcaton of Rumen Mcrobe Dversty and Fermentaton of
Cattle Usng Lerak (Sapindus rarak) Saponn. Ph.D Thess. Bogor
Agrcul-tural Unversty. Indonesa.
Popula-ton n the In vitro Fermentaton of Dfferent Ratos of Forage and Concentrate
n the Presence of Whole Lerak (Sapindus rarak) Frut Extract.
Asan-Austra-lan J. Anmal Scence (AJAS). 24:1086-1091.
Tlley, J.M.A and R.A. Terry. 1963. A two stage technque for the in vitro dgeston
of forage. J. Brtsh Grassland Socety. 18: 104–111.
Wna, E., S. Muetzel, and K. Becker. 2006. The dynamcs of major fbrolytc m-crobes and enzyme actvty n the rumen n response to short-and long-term
feedng of Sapindus rarak saponns. Journal of Appled Mcrobology 100:
