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ISSN : 2287-5824(Print)
ISSN : 2287-5832(Online)
Journal of The Korean Society of Grassland and Forage Science Vol.32 No.4 pp.379-386

Effect of Different Silages for TMR on In vitro Rumen Simulative Fermentation

Nag-Jin Choi, David Tinotenda Mbiriri, Seong Jin Oh
Department of Animal Science, Chonbuk National University
(Received November 7, 2012/Accepted December 3, 2012)


In this study, the in vitro fermentation parameters of whole crop barley (WCBS-TMR) and Italianryegrass (IRGS-TMR) silage total mixed rations were compared. A rice straw based diet (RSBD), whichwas a mixture of rice straw and concentrate (60:40), was used as the control. The feeds were incubated inbuffered rumen fluid for 3, 6, 9, 12, 24, 48 and 72 hours at 39°C. At the end of each incubation periodthe following parameters were determined, total gas, pH, ammonia nitrogen (NH3-N), volatile fatty acids(VFA) and then the acetate to propionate ratio (A/P) was calculated. The dietary treatments did not affect(p>0.05) the overall production of NH3-N, gas, total VFA and all the individual VFA, with the exceptionof n-butyrate (p<0.001). The treatment diets significantly affected the A/P ratio (p<0.01). The control dietresulted in the lowest A/P ratios, followed by WCBS-TMR and lastly IRGS-TMR had the highest ratios.Gas production was not different between treatments, suggesting a probable similar level of digestibilitywhen treatments are fed to animals. It can therefore be concluded from the present study that WCBS andIRGS are of almost an equivalent nutritional value when incubated in a TMR form. WCBS-TMR howeverresulted in lower A/P ratios than IRGS-TMR, which is indicative of a more energy efficient diet.


Feed costs in an intensive beef production account for a critical percentage of the production costs. Provision of productively efficient feed is therefore of greatest importance in sustaining a viable beef production entity. Key to achieving this is the choice of forage source. Forages are the basis for good feed formulation (Van Saun, 2007). Rice is the staple crop of Korea and consequently, rice straw has been widely used as a forage source for ruminants. However, in an effort to improve feed quality whole crop barley (WCB) and Italian ryegrass (IRG) have gained popularity as forage sources among beef producers. These roughage sources are either provided alone or as constituents of total mixed rations (TMR). They are used as straws or silages. Ensiling is one way of preserving forages whilst maintaining their high nutritional value. Silages also make more palatable straws (Gao et al., 2008). In our previous study (Mbiriri et al., 2012), we investigated the in vitro fermentation patterns of the forages being investigated in the present study when incubated alone. From the results we concluded that there were no significant differences in total and individual VFA production between forages. However, WCBS was reported a better forage source on the basis of a significantly higher NH3-N output than the other treatment diets. There is need, however, to investigate on the fermentation parameters when these forages are incorporated into TMRs. The objective of this research was to compare in vitro fermentation patterns of WCBS-TMR and IRGS-TMR. 


1. Diets

Three diets, WCBS-TMR, IRGS-TMR and RSBD (the control diet) were used as substrates. The control diet was a mixture of rice straw and a concentrate in a 60:40 ratio. Whole crop barley silage TMR and IRGS-TMR were freezedried, ground and passed through 1 mm screen. The nutritional information of the diets was shown in Table 1. 

Table 1. Chemical composition of experimental diets

2. Rumen fluid

Rumen fluid was collected before morning feeding from a rumen cannulated Hanwoo steer weighing about 350 kg. The steer was being fed a commercial TMR (forage:concentrate was 50:50) individually in a metabolic pen twice a day, at 0900 and 1700 h. The fluid was strained through 4 layers of cheese cloth into a flask previously filled with N2 gas. The flask was then transported to the laboratory in less than 1 hour. The rumen fluid was further strained through another 4 layers of cheese cloth on arrival at the laboratory. The strained rumen fluid was then mixed with McDougall’s buffer prepared according to Troelsen and Donna (1966), at a rate of 1:4 (rumen fluid:buffer). The buffer (pH 6.5) contained 9.8 g of NaHCO3, 4.62 g of Na2HPO4 · 2H2O, 0.57 g KCl, 0.47 g NaCl, 0.12 g MgSO4 · 7H2O and 4 g CaCl2 per 1 L of distilled water. 

3. Incubation procedure

In vitro rumen simulated fermentation was performed in 250 mL serum bottles under the stream of N2 gas to maintain anaerobic condition. 0.5 g of each diet was mixed with 50 mL of buffered rumen fluid in the serum bottles whilst gassing with N2 gas. The bottles were then sealed and capped using aluminium sealed rubber stoppers. They were then placed in an incubator at 39°C. All treatments were performed in triplicate. The procedure followed the method outlined by Tilley and Terry (1963). At each sampling time, total gas production was measured by way of displacing a glass syringe. The bottles were then uncapped and the pH measured using a pH meter. A cell and diet particle free supernatant was prepared by centrifugation (10,000 rpm for 15 min at 4°C) and stored at -20°C for further analysis. Ammonia nitrogen was analysed using a spectrophotometer according to the method of Chaney and Marbach (1962). Volatile fatty acids were determined using gas chromatography (Agilent 6890, Agilent Technology Inc., USA)according to Erwin et al. (1961). 

4. Statistical analysis

Data on the measured fermentation parameters were subjected to ANOVA using SPSS program (version 18, IBM, New York, USA). The following model was used for all observed patterns 


Yijk is response variable (pH, gas e.t.c). μ and Ti are overall mean common to all observations and the ith treatment diet, respectively. Hj and (T×H)ij are the jth incubation time and interaction between the ith treatment diet and the jth time, respectively. eijk is random residual error. The model used to evaluate overall effect of treatment diets did not include time effects. 


Significantly different fermentation patterns (p<0.01) were observed for all the measured fermentation parameters. The observed pH profiles were shown in Table 2. The RSBD resulted in the lowest pH values throughout the incubation period. The pH values, for all diets, decreased with increasing incubation time, which is an expected trend as VFA accumulate with time. Between 9 and 48 h, IRGS-TMR had higher pH values than RSBD and WCBS-TMR. 

Table 2. Effect of treatment diets on pH patterns

Gas production trends differed significantly (p<0.001) between treatments (Table 3). Gas output increased with time although after 72 h it either decreased or tended to show smaller increment than at the 48 h sampling time. The observation suggests an almost complete degradation of the substrate. Since there was no significant difference (p>0.05) in the overall gas output, it indicates that the diets would most likely to be of the same in vivo digestibility when fed to animals (Menke et al., 1979). 

Table 3. Effect of treatment diets on gas production (mL) patterns

Ammonia nitrogen values for all treatments started-off higher than at 6 to 12 h incubation (Table 4). This observation is a result of the breaking down of the rapidly degraded protein in the concentrate portion of the diets. Mbiriri et al. (2012) who incubated 4 forage sources that included WCBS, IRGS and rice straw, without a concentrate, did not observe such a trend. However, they noted a steeper rise in NH3-N output after 12 h incubation of the forages. The same pattern is apparent in this study, although in this case the overall NH3-N for the diets did not differ significantly (p>0.05). At the 72 h mark, WCBS-TMR registered a lower value than at the 48 h mark, as is shown in Table 4. This suggests an almost complete degradation of the substrate. Contrary to NH3-N values out of the optimal range (15~20 mg/100 mL) (Perdok and Leng, 1989) observed when the same forages were incubated alone, when incubated in a TMR form, the NH3-N reached the optimal values after 48 h of incubation. 

Table 4. Effect of treatment diets on ammonia nitrogen (mg/100 mL) patterns

There was no significant difference between treatments in overall VFA production except for n-butyrate (p<0.001) (Table 5). Volatile fatty acid production trends over time (Table 6) were however significantly different between treatments diets used in this experiment. The RSBD consistently produced significantly lower (p<0.05) total VFA for all incubation periods with the exception of 24 and 72 h incubations where total VFA did not significantly differ (p>0.05) between treatments. This is an almost similar result as in the previous incubations where the forages were incubated alone (Mbiriri et al., 2012). Italian ryegrass silage and WCBS-MRs exhibited a similar production trend although WCBS-TMR tended to tapper-off after 72 h incubation. The RSBD showed a steady rise in acetate. Although it resulted in lower production from 3 to 12 h incubation, it matched the other 2 treatments after 24 h incubation. This is indicative of a higher content of slowly degradable fiber than IRGS and WCBS-TMRs. A similar pattern was observed in propionate production, but in this case the control diet actually tended to result in more propionate than either  WCBSTMR or IRGS-TMR. The significantly different (p<0.001) A/P ratios reflect the different acetate and propionate production patterns. RSBD had the lowest A/P ratios followed by WCBS-TMR and lastly IRGS-TMR had the highest values. This suggests that the control diet could prove the most energy efficient to animal production and IRGS-TMR will most likely be the least energy efficient diet to the animal (Wolin, 1960).

Table 5. Effect of treatment diets on overall mean volatile fatty acid production

Table 6. Volatile fatty acid production patterns of the dietary treatments


In the present in vitro study WCBS and IRGS TMRs, resulted in different fermentation patterns. The fermentation of WCBS-TMR resulted in lower overall A/P ratios than IRGSTMR, making it a more likely energy efficient diet to the animal. However, the overall pH, gas, NH3-N, total VFA and individual VFA, with the exception of n-butyrate were not affected significantly by the dietary treatments. 


This work was supported by “Cooperative Research Program for Agriculture Science & Technology Development (Project No. PJ906934)” Rural Development Administration, Republic of Korea. 



1.Chaney, A.L. and Marbach, E.P. 1962. Modified reagents for the determination of urea and ammonia. Clinical Chemistry. 8:130-132.
2.Erwin, E.S., Marco, J. and Emery, E.M. 1961. Volatile fatty acid analysis of blood and rumen fluid by gas chromatography. Journal of Dairy Science. 44:1768-1771.
3.Gao, L.J., Yang, H.Y., Huang, X.F. Ishii, M.Y., Igarashi, Y. and Cui, Z. 2008. Rice straw fermentation using lactic acid bacteria. Bioresource Technology. 99:2742-2748.
4.Mbiriri, D.T., Oh, S.J., Lee, A.R., Chae, J.I., Choi, C.W. and Choi, N.J. 2012. In vitro rumen fermentation patterns of environmental friendly whole crop barley, Italian ryegrass and rice straw silages. Korean Journal of Organic Agriculture.20:221-230.
5.Menke, K.H., Raab, L., Salewski, A., Steingass, H.,Fritz, D. and Schneider, W. 1979. The estimation of digestibility and metabolisable energy content of ruminant feeding-stuffs from the gas production when they are incubated with rumen liquor in vitro. Journal of Agricultural Science (Cambridge)93:217-222.
6.Perdok, H. and Leng, R.A. 1989. Rumen ammonia requirements for the efficient digestion and intake of straw by cattle. In: The role of protozoa and fungi in ruminant digestion (Ed. J. V. Nolan and R. A. Leng), Penambul Books, Armidale, Australia.
7.Tilley, J.M.A. and Terry, R.A. 1963. A two stage technique for the in vitro digestion of forage crops. Journal of the British Grassland Society. 18:104-111.
8.Troelsen, J.M. and Donna, H.J. 1966. Ruminant digestion in vitro as affected by inoculums donor collection day, and fermentation time. Canadian Journal of Animal Science. 46:149-156.
9.Van Saun, R.J. 2007. Forage testing: How to translate numbers into actions. In: Proceedings of 2006 Mid-Atlantic States Conference for Bovine Practitioners, Fredrick, Maryland, March 30-31, 2006, pp. 13.
10.Wolin, M.J. 1960. A theroretical rumen ferment- ionbalance. Journal of Dairy Science. 3:1452- 1459.