1 The use of the in vitro gas production technique to study the fermentation characteristics of tropical grasses at different roughage to concentrate ratios Chaowarit Mapato a* and Metha Wanapat a a Tropical Feed Resources Research and Development Center (TROFREC), Department of Animal Science, Faculty of Agriculture, Khon Kaen University, Khon Kaen, Thailand * Corresponding email:chaowarit.map@gmail.com Abstract The aim of this study was to use an in vitro gas production technique to study gas production kinetics, and in vitro degradability of tropical grasses at different roughage to concentrate ratios. Two male, rumen fistulated dairy steers were used as rumen fluid donors. The treatments were in a 5 x 4 Factorial arrangement in a Completely Randomized Design with five roughage sources namely Ruzi grass (Brachiaria ruziziensis), Guinea grass (Panicum maximum), Napier Pakchong1 grass (Pennisetum purpureum x Pennisetum americanum), dwarf Napier grass (Pennisetum purpureum cv. Mott) and Sweet grass (Pennisetum purpureum cv. Mahasarakham) with four R:C ratios at 80:20, 60:40, 40:60 and 20:80, respectively. Under this investigation, the results revealed that the short or dwarf variety of Napier grass especially Sweet grass has the highest nutritive values (15.2 % CP, 12.1% NFC), and lowest fiber contents (34.9 % ADF and 59.5 %NDF). Sweet grass produced the highest gas production, and in vitro degradability increased up to 80:20 R:C (P<0.01). Furthermore, decreasing ratio of R:C could increase gas production and in vitro degradability (P<0.001). Based on this study, it could be concluded that the nutrient compositions of the diets highly influenced nutrient degradability, rumen fermentation, especially associated with higher quality of roughage. Keywords: Tropical grasses, Degradability, Nutrient, Rumen fermentation Introduction Roughages are the main feed source and therefore important for ruminant production. The quality of the roughage could affects feed intake, rumen fermentation and performance of the animal. Moreover, using high quality roughages can reduce the use of dietary concentrates for lactating dairy cows as they allow rumen microbes to increase the digestion of roughage,
2 providing more nutrients to the host animal, and hence, decrease the concentrate supplementation (Wanapat et al., 2006). In tropical areas, especially during the summer, the productivity of cattle is limited due to the low quality of the forage crop species that grow in this area, among other factors. However, some tropical grasses, such as elephant or napier grass (Pennisetum purpureum) and napier pakchong 1 (Pennisetum purpureum x Pennisetum americanum) produce large yields per area (Wangchuk et al., 2015). Those grasses are the tall varieties of napier grass. Moreover, the short or dwarf varieties of napier grass have a higher overall nutritive quality compared to the taller varieties mainly due to having a higher leaf-tostem ratio which is about 1.4 in dwarf and less than 0.8 in tall varieties (Halim et al., 2013). Sweet grass (Pennisetum purpureum cv. Mahasarakham) is a perennial bunch grass which is of potentially high nutritive value. Sweet grass is a short variety similar to dwarf napier grass (Pennisetum purpureum cv. Mott). However, knowledge of the intrinsic values of this forage and exactly how they affect the rumen digestion and fermentation efficiency are still need to be determined. Therefore, the aim of this experiment was to investigate the effects of grass s quality of tropical grasses combined with difference of roughage to concentrate ratios on rumen degradation, gas production kinetic, and rumen fermentation using the in vitro gas production technique. Materials and Methods The five roughage sources were ruzi grass (Brachiaria ruziziensis), guinea grass (Panicum maximum), napier pakchong1 grass (Pennisetum purpureum x Pennisetum americanum), dwarf napier grass (Pennisetum purpureum cv. Mott) and sweet grass (Pennisetum purpureum cv. Mahasarakham). These were collected sample at 45±3 days of regrowth. All grass sample were collected to be analyzed for dry matter (DM), crude protein (CP), ether extract (EE), and ash by using technique the of AOAC, (2012). Neutral detergent fiber (NDF) and acid detergent fiber (ADF) were analyzed by the technique of Van Soest et al. (1991). Non-fiber carbohydrate (NFC) was estimated by the equation of NRC (2001). A subsample of each grass was mixed with concentrate at 4 ratios of R:C for use in the in vitro gas technique. The feed ingredients of the concentrate are shown in Table1. A 5 x 4 factorial arrangement in a Completely Randomized Design (CRD) with five roughage sources and four roughage to concentrate ratios (R:C ratios; 80:20, 60:40, 40:60, 20:80) were used determine gas production using the technique of Menke and Steingass (1988) with some modification. Cumulative gas production data were fitted to the model of Orskov and McDonald (1979). At 24 and 48 h post inoculation, a set of samples were tested for in vitro degradability using the technique of Tilley and Terry (1963). All obtained data were subjected to Statistical Analysis System Institute (SAS, 2004) according to a 5 4 factorial arrangement in a completely randomized design. The statistical model included roughage sources, R:C ratios, roughage sources R:C ratios interactions. For all parameters, differences between treatments means were contrasted by Duncan s new multiple range test (Steel and Terrie, 1980).
3 Results and discussion Chemical composition of experimental diets At 45 day of growth, the moisture, CP and NFC content were highest in sweet grass (86.6, 15.2 and 12.1 %, respectively) and lowest in ruzi grass (74.7, 8.9 and 7.2 %, respectively). ADF and NDF content were highest in guinea grass (39.7 and 69.2 %, respectively) and lowest in sweet grass (34.9 and 59.5 %, respectively). Ruzi and guinea grass were similar in CP content (8.9 and 9.2 %, respectively). Ash content was highest in ruzi grass and lowest in sweet grass. Table 1 Feed ingredients and chemical compositions of concentrate and roughages. Ingredients Concentrate Napier dwarf Ruzi Guinea (% of DM) pakchong1 Napier Sweet Cassava chip 60.1 Rice bran 1.8 Soybean meal 19.7 Palm kernel meal 4.5 Coconut meal 7.2 Urea 1.5 Molasses 3.7 Salt 0.5 Sulfur 0.5 Mineral mixture 0.5 Total 100 Chemical composition Dry matter (DM), % 91.1 25.3 20.1 18.2 14.8 13.4 -------------------------------% of dry matter--------------------------- Crude protein 17.9 8.9 9.2 11.1 13.4 15.2 Ether extract 3.2 2.2 2.0 2.0 2.1 2.2 Acid detergent fiber 8.9 36.2 39.7 38.3 36.4 34.9 Neutral detergent fiber 17.7 67.8 69.2 64.4 61.4 59.5 Non fiber carbohydrate 55.5 7.2 8.0 9.8 10.4 12.1 Ash 5.7 14.1 12.6 12.7 10.7 10.0 Effects on gas production kinetics and in vitro degradability Gas production patterns are shown in Table 2. They were significantly affected by roughage sources and R:C ratios. Gas production level was highest (P<0.001) in the diets with the highest concentrate proportions at 80 %. For the roughage, it was highest (P<0.001) in diets containing sweet grass. However, there was no by interaction between roughage sources and R:C ratios (P>0.05). IVDMD was significantly different between the roughage sources (P<0.01) and R:C ratios (P<0.001). Sweet grass diets were highest in IVDMD especially at 60 and 80 % of roughage. The lowest IVDMD was guinea grass in the diets. Moreover, IVDMD and IVOMD
4 were highly significantly different between R:C ratios (P<0.001). Diet combinations with the highest proportion of concentrate were highest in IVDMD and IVOMD. There were no significant interactions between roughage sources and R:C ratios (P>0.05). Table 2 Gas production kinetics and in vitro degradability. Roughages R:C ratios Gas production kinetics Gas d In vitro degradability (%) a b c a+b IVDMD IVOMD Ruzi grass 80:20-1.0 79.0 0.06 77.9 77.7 48.3 67.9 (Brachiaria ruziziensis) 60:40-1.7 79.9 0.07 78.2 78.1 53.8 74.4 40:60-2.4 81.7 0.09 79.3 79.3 57.3 76.6 20:80-0.1 79.6 0.08 82.0 81.9 60.8 80.1 Guinea grass 80:20-2.6 74.6 0.08 72.0 71.9 45.0 67.9 (Panicum maximum) 60:40-1.9 75.3 0.08 73.3 73.3 49.5 69.3 40:60-1.0 76.5 0.09 75.5 75.5 54.0 76.6 20:80-1.4 80.2 0.09 78.8 78.8 58.2 81.3 Napier pakchong1 grass 80:20-0.2 75.3 0.06 75.1 74.7 43.9 67.5 (Pennisetum purpureum 60:40-0.8 77.0 0.06 76.2 76.0 48.6 70.7 x Pennisetum 40:60-1.9 78.6 0.07 76.7 76.6 53.1 76.9 americanum) 20:80-2.9 83.6 0.09 80.7 80.7 57.7 80.1 Dwarf Napier grass 80:20-0.4 76.2 0.06 75.8 75.6 49.2 70.0 (Pennisetum purpureum 60:40-0.8 79.2 0.07 78.4 78.3 53.4 73.1 cv. Mott) 40:60-1.3 83.0 0.08 81.7 81.6 56.7 75.8 20:80-3.8 84.5 0.09 80.7 80.7 61.3 80.3 Sweet grass 80:20-1.8 80.2 0.07 78.4 78.3 51.3 70.6 (Pennisetum purpureum 60:40-2.7 81.7 0.08 79.0 79.0 55.1 74.0 cv. Mahasarakham) 40:60-1.3 82.2 0.08 80.9 80.9 58.7 78.5 20:80-1.8 85.3 0.09 83.5 83.5 62.6 80.9 SEM 0.16 0.51 0.001 0.46 0.47 1.29 1.15 Comparison Roughages ns *** *** *** *** ** ns R:C ratios ns *** *** *** *** *** *** Interaction ** ns *** ns ns ns ns a, the gas production from the immediately soluble fraction; b, the gas production from the insoluble fraction; c, the gas production rate constant for the insoluble fraction (b); a+b, the gas potential extent of gas production; d, Cumulative gas production at 96 h (ml/0.2 g DM substrate); SEM, Standard error of the means; *, P<0.05; **, P<0.01; ***, P<0.001; ns, nonsignificant (P>0.05); R:C ratios, roughage to concentrate ratios; IVDMD, In vitro dry matter degradability; IVOMD, In vitro organic matter degradability.
5 Conclusions It can be concluded that the diets with greater nutritive values resulted in the highest nutrient degradability and gas production. Especially, the use of the diet contained sweet grass at the high ratio of roughage. It may be suggested that this is a good quality roughage could reduce the need for concentrate supplementation. Further research using sweet grass in high roughage diets to improve rumen fermentation and feed efficiency in ruminants are recommended References AOAC. 2012. Official methods of Analysis, 19 th edition. Animal Feed: Association of Official Analytical Chemists, VA. Halim, R. A., S. Shampazurini, and A. B. Idris. 2013. Yield and nutritive quality of nine Napier grass varieties in Malaysia. Malaysian Journal of Animal Science., 16:37 44. Menke, K. H., and H. Steingass. 1988. Estimation of the energetic feed value obtained from chemical analysis and gas production using rumen fluid. Anim. Res. Dev., 28: 7-55. NRC (National Research Council). 2001. Nutrient Requirements of Dairy Cattle. 6 th Rev. Ed. National Academy of Sciences, Washington, DC. Orskov, E. R., and I. McDonal. 1979. The estimation of protein degradability in the rumen from incubation measurements weighted according to rate of passage. J. Agric. Sci., 92: 499-503. SAS. 2004. What s New in SAS 9.0, 9.1, 9.1.2, and 9.1.3. Cary, NC: SAS Institute Inc. Steel, R. G. D. and J.H. Terrie. 1980. Principle and procedures of statistic: A biomaterial approach. 2 nd ed. Mc Graw Hill, New york. pp631. Tilley, J. M. A. and R. A. Terry. 1963. A two-stage technique for the in vitro digestion of forage crops. Journal of the British Grassland Society., 18: 104-111. Van Soest, P. J., J. B. Robertso, and B. A. Lewis. 1991. Methods for dietary fiber, neutral detergent fiber and non-starch polysaccharides in relation to animal nutrition. J. Dairy Sci., 74: 3583-3597. Wanapat, M., C. Promkot, and S. Khampa. 2006. Supplementation of cassava hay as a protein replacement for soybean meal in concentrate supplement for dairy cows. Pakistan Journal of Nutrition., 6: 68-71. Wangchuk, K., K. Rai, H. Nirola, C. Dendup, and D. Mongar. 2015. Forage growth, yield and quality responses of Napier hybrid grass cultivars to three cutting intervals in the Himalayan foothills. Tropical Grass lands Forrajes Tropicales., 3: 142 150.