DOI: 10.1111/jpn.12253
ORIGINAL ARTICLE
Improving the quality of rice straw by urea and calcium hydroxide on rumen ecology, microbial protein synthesis in beef cattle S. Polyorach1,2 and M. Wanapat1 1 Tropical Feed Resources Research and Development Center (TROFREC), Faculty of Agriculture, Khon Kaen University, Muang, Thailand, and 2 Department of Animal Science, Faculty of Natural Resources, Rajamangala University of Technology-Isan, Phang Khon, Thailand
Summary Four rumen-fistulated beef cattle were randomly assigned to four treatments according to a 4 9 4 Latin square design to study the influence of urea and calcium hydroxide [Ca(OH)2] treatment of rice straw to improve the nutritive value of rice straw. Four dietary treatments were as follows: untreated rice straw, 50 g/kg urea-treated rice straw, 20 g/kg urea + 20 g/kg calcium hydroxide-treated rice straw and 30 g/kg urea + 20 g/kg calcium hydroxide-treated rice straw. All animals were kept in individual pens and fed with concentrate at 0.5 g/kg of BW (DM), rice straw was fed ad libitum. The experiment was conducted for four periods, and each period lasted for 21 days. During the first 14 days, DM feed intake measurements were made while during the last 7 days, all cattle were moved to metabolism crates for total faeces and urine collections. The results revealed that 20 g/kg urea + 20 g/kg calcium hydroxide-treated rice straw improved the nutritive value of rice straw, in terms of dry matter intake, digestibility, ruminal volatile fatty acids, population of bacteria and fungi, nitrogen retention and microbial protein synthesis. Based on this study, it could be concluded that using urea plus calcium hydroxide was one alternative method to improve the nutritive value of rice straw, rumen ecology and fermentation and thus a reduction of treatment cost. Keywords rice straw, urea, calcium hydroxide [Ca(OH)2], rumen ecology, microbial protein, ruminants Correspondence M. Wanapat, Department of Animal Science, Faculty of Agriculture, Khon Kaen University, P.O. Box 40002, Muang, Khon Kaen, Thailand. Tel: +66 43 202368; Fax: +66 43 202368; E-mail: [email protected] Received: 23 February 2014; accepted: 18 August 2014
Introduction Rice straw is an important crop residue practically used by farmers for ruminant feeding especially during the long dry season (Ørskov et al.,1999). However, rice straw is low in nutritive value with low level of protein (20–50 g/kg DM), high fibre and lignin content (NDF > 650 g/kg), low DM digestibility ( 0.05). Rumen micro-organism population
Rumen micro-organisms in the treated groups are presented in Table 3 and were found significantly higher than those in the control group. It was significantly (p < 0.05) higher than in the control group and was
the highest in the 50 g/kg urea treatment, 30 g/kg urea + 20 g/kg Ca(OH)2-treated groups for bacteria, fungi and cellulolytic bacteria, while protozoal population was significantly lower (p < 0.05) than in the control group. In addition, the population of proteolytic and amylolytic bacteria in treated group were significantly (p < 0.05) higher than in control group. Effect on feed intake and digestibility
Table 4, shows effects of various treatments of rice straw on voluntary feed intake and nutrient digestibility in beef cattle. The results of this experiment using 50 g/kg urea, 20 g/kg urea + 20 g/kg Ca(OH)2 and 30 g/kg urea + 20 g/kg Ca(OH)2 for the straw treatment can significantly (p < 0.05) enhance DM intake and digestibility. Apparent digestibility of DM, OM, CP, NDF and ADF in the 30 g/kg urea + 20 g/kg Ca (OH)2-treated rice straw was the highest (p < 0.05), followed by the 50 g/kg urea-treated rice straw, 20 g/ kg urea + 20 g/kg Ca(OH)2-treated rice straw and untreated rice straw respectively. Urinary excretion of purine derivatives and microbial nitrogen supply
The effect of dietary treatments on urinary excretion of purine derivatives and microbial nitrogen supply are presented in Table 5. The findings under this investigation revealed significant (p < 0.05) improvement in N balance in terms of N intake, faecal N, urinary N, N absorption and N retention and urinary purine derivatives. As a result of this study, beef cattle fed 50 g/kg urea, 20 g/kg urea + 20 g/kg Ca(OH)2 and 30 g/kg urea + 20 g/kg Ca(OH)2-treated rice straw resulted in higher N retention, microbial protein supply and efficiency of microbial N synthesis (EMNS)
Table 2 Effect of rice straw treatment on ruminal pH, temperature, NH3-N and volatile fatty acid (VFA) concentrations in beef cattle
Item
Untreated rice straw
Ruminal pH 6.2a Ruminal temperature (°C) 39.1 NH3-N, mg/100 ml 13.5a BUN, mg/100 ml 15.2a Total VFA, mmol/l 105.0 Molar proportion of VFA (mol/100 mol) Acetic acid (C2) 73.9a 16.3a Propionic acid (C3) Butyric acid (C4) 9.7 C2:C3 ratio 4.7a
50 g/kg urea-treated rice straw
20 g/kg urea + 20 g/kg Ca(OH)2-treated rice straw
30 g/kg urea + 20 g/kg Ca(OH)2-treated rice straw
SEM
p-Value
6.6bc 39.2 16.8c 15.5a 111.0
6.6b 39.1 15.3b 13.8b 108.8
6.8c 39.0 16.1cb 14.6ab 112.1
0.03 0.09 0.24 0.30 1.73
** ns ** * ns
67.7b 22.2b 10.1 3.1b
0.90 1.02 0.73 0.30
* * ns *
69.7b 21.2b 9.7 3.4b
70.4b 19.1ab 10.5 3.7ab
NH3-N, ammonia nitrogen; BUN, blood urea nitrogen; SEM, standard error of the mean; ns, no significance; *p < 0.05; **p < 0.01; a,b,cValues on the same row with different superscripts differ (p < 0.05).
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Table 3 Effect of rice straw treatment on microbial population in the rumen of beef cattle
Item
Untreated rice straw
Total direct count, cell/ml 3.6a Bacteria, 9109 Protozoa, 9105 8.2a 5 Fungal zoospores, 910 3.8a Ruminal bacterial groups, CFU/ml Total viable bacteria, 9109 3.5a Cellulolytic bacteria, 9108 5.7a Amylolytic bacteria, 9107 3.9a 7 Proteolytic bacteria, 910 3.9a
50 g/kg urea-treated rice straw
20 g/kg urea + 20 g/kg Ca(OH)2-treated rice straw
30 g/kg urea + 20 g/kg Ca(OH)2-treated rice straw
SEM
p-Value
5.7c 5.9b 5.5c
4.9b 6.3b 4.7b
5.8c 5.3b 5.7c
0.19 0.53 0.19
** * **
5.0b 8.2c 5.7b 7.2b
4.9b 7.2b 5.5b 5.9b
5.9b 9.3d 5.9b 6.9b
0.32 0.22 0.41 0.42
* ** * **
SEM, standard error of the mean; ns, no significance; *p < 0.05; **p < 0.01; a,b,c,dValues on the same row with different superscripts differ (p < 0.05).
Table 4 Effect of rice straw treatment on voluntary feed intake and nutrient digestibility in beef cattle
Item Concentrate DM intake kg/day g/kg BW0.75 daily Roughage DM intake kg/day g/kg BW0.75 daily Total DM intake kg/day g/kg BW0.75 daily Apparent digestibility, g/kg Dry matter Organic matter Crude protein Neutral detergent fibre Acid detergent fibre
Untreated rice straw
50 g/kg urea-treated rice straw
20 g/kg urea + 20 g/kg Ca(OH)2-treated rice straw
30 g/kg urea + 20 g/kg Ca(OH)2 treated rice straw
SEM
p-Value
1.3 20.1
1.3 20.0
1.4 19.9
1.3 20.2
0.07 0.90
ns ns
3.8a 60.0a
5.4b 85.2b
5.1b 79.6b
5.2b 79.8b
0.17 2.84
** **
5.1a 80.1a
6.7b 105.2b
6.5b 99.5b
6.5b 100.0b
0.21 2.75
** **
549.3a 589.0a 629.3a 486.0a 450.0a
591.0ab 627.0b 672.3b 543.8bc 491.3bc
577.3ab 615.5ab 652.3ab 532.5b 478.0ab
605.8b 643.8b 681.3b 571.5c 518.5c
14.06 14.55 11.89 10.75 9.99
* * * ** **
SEM, standard error of the mean; ns, no significance; *p < 0.05; **p < 0.01; a,b,cValues on the same row with different superscripts differ (p < 0.05).
when compared with the control group (p < 0.05). However, there were not significant (p > 0.05) differences among treated groups.
Characteristics of ruminal fermentation and blood metabolites
When rice straw was treated with urea, it resulted in improving the nutritional quality of the straw in terms of nitrogen content, palatability and digestibility. During the treatment process, ammonia generated from urea, in the presence of water formed an alkali (ammonium hydroxide), which impacted on lingocellulosic bonding of the rice straw (Wanapat et al., 1996). Trach et al. (2001b) reported that the result was higher when Ca(OH)2 (20–30 g/kg DM of straw) and urea (20 g/kg DM of straw) were used in combination.
Under this experiment, BUN ranged from 13.1 to 16.8 mg/dl and was similar to the values reported by Wanapat and Pimpa (1999). The ruminal NH3-N result obtained under this study was closer to 15–30 mg/dl (Wanapat and Pimpa, 1999), these concentrations could improve rumen fermentation and feed intake in swamp buffaloes offered urea-treated rice straw. In addition, Preston et al. (1965) reported that the concentration of BUN produced was closely correlated to the level of ammonia production in the rumen. Furthermore, Preston (1995) suggested that the quantity of ammonia absorbed from the rumen reflected in the circulating BUN. Diets which are balanced in protein (P)/energy (E) could result in BUN produced concentrations of approximately
Journal of Animal Physiology and Animal Nutrition © 2014 Blackwell Verlag GmbH
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Discussion Chemical composition of feeds and ruminal parameters
Urea and calcium hydroxide for improve rice straw quality
S. Polyorach and M. Wanapat
Table 5 Effect of rice straw treatment on allantoin excretion, purine derivative excretion and purine derivative absorption and microbial protein synthesis
Item Nitrogen balance (g/day) N intake Faecal N Urinary N N absorption N retention Purine derivatives (PD) (mmol/day) Allantoin excretion Allantoin absorbtion Microbial protein supply, g N/day† EMNS (g N/kg of OMDR)‡
Untreated rice straw
50 g/kg urea-treated rice straw
20 g/kg urea + 20 g/kg Ca(OH)2-treated rice straw
30 g/kg urea + 20 g/kg Ca(OH)2-treated rice straw
SEM
p-Value
53.5a 24.8a 21.5a 31.5a 10.0a
104.2b 41.9b 37.3b 65.3b 28.0b
99.4b 40.5b 37.7b 61.0b 23.3b
103.2b 41.4b 38.3b 66.4b 28.1b
5.92 1.28 0.83 3.51 3.66
** ** ** ** *
58.5a 41.2a 30.0a 15.5a
96.5b 85.4b 62.1b 22.7b
92.4b 79.8b 58.0b 22.8b
97.4b 86.8b 63.1b 23.4b
3.89 4.58 3.33 1.60
* ** ** *
SEM, standard error of the mean; ns, no significance; *p < 0.05; **p < 0.01; Values on the same row with different superscripts differ (p < 0.05). †Microbial crude protein (MCP) (g/day) = 3.99 9 0.856 9 mmoles of purine derivatives excreted (Galo et al., 2003). ‡Efficiency of microbial N synthesis (EMNS, g/kg of OM digested in the rumen (OMDR) = [(MCP (g/day) 9 1000)/DOMR (g)], assuming that rumen digestion was 650 g/kg OM of digestion in total tract.
a,b
15 mg/100 ml, while BUN levels lower or higher than this could be due to an insufficiency or exceeding in CP per unit of digestible energy (Wanapat and Pimpa, 1999). Volatile fatty acids concentration in this experiment were similar to those reported by Wanapat et al. (2009) who reported that the effects of various treated rice straw on VFA concentrations especially increasing those of C3 were increased while those of C2 were decreased, thus C2/C3 was subsequently lowered in cows fed with 30 g/kg urea + 20 g/kg Ca(OH)2 treated rice straw, 50 g/kg urea-treated rice straw and 20 g/kg urea + 20 g/kg Ca(OH)2-treated rice straw respectively. Moreover, Ørskov et al. (1999) discussed that, when high fibre diets were offered, VFAs in ruminal fermentation shifted from 65:25:10 to 70:20:10 (acetate: propionate: butyrate, in molar percentage) ratios. Rumen micro-organism population
The results of this experiment agreed with Wanapat et al. (2009), who revealed that 50 g/kg urea-treated rice straw and 20 g/kg urea + 20 g/kg Ca(OH)2-treated rice straw increased total viable bacteria and cellulolytic bacteria in lactating dairy cows. Moreover, Vinh et al. (2011) showed that total ruminal bacteria, Fibrobacter succinogenes, Ruminococcus albus, cellulolytic bacteria and fungal population were greater by dietary treatment with urea–lime (20 g/kg urea + 20 g/kg lime)-treated rice straw while Ruminococcus flavefaciens, protozoal population were significantly reduced (p < 0.05). Amylolytic, proteolytic bacterial groups, total bacteria were not changed 454
among treatments in swamp buffalo. Furthermore, Khejornsart et al. (2011) showed that when feeding urea–lime (20 g/kg urea + 20 g/kg lime)-treated rice straw could enhance the nutritive value of rice straw and had a significantly higher (p < 0.05) in bacterial population than those in untreated group, showing buffaloes utilized feeds more efficiently with higher rumen fermentation. Concentration of alkaline agents (urea and Ca(OH)2) can chemically change structural fibre swollen and easily digested by rumen microbes (Wanapat et al., 2009). This effect avails the rumen microbes to attack the structural carbohydrates more easily, improve digestibility, as well as the palatability of treated straw (Bod’a, 1990). Moreover, Goto et al. (1993) reported that alkaline treatment can attack the lignin–polysaccharide bond which solublizes hemicellulose and lignin in the straw and hence exposes the cellulose for the microbial population to be digested. Chen et al. (2008) also reported that chemical treatments enhanced the nutritive value of rice straw through increasing the number of accessible sites of microbial attachment on the surface of the particles, increasing fibrolytic microbe quantity and hence fibrolytic enzyme activities and improving the overall rumen fermentation characteristics. Effect on feed intake and digestibility
Wanapat et al. (1996) reported that alkaline agenttreated rice straw significantly improve digestibility and feed intake by the ruminants. The concentrated alkaline agents can chemically break the ester bonds between lignin hemicellulose and cellulose, and Journal of Animal Physiology and Animal Nutrition © 2014 Blackwell Verlag GmbH
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physically changed the fibrous structure. In finding a good combination level of urea and lime, Sirohi and Roi (1995) revealed that a combination of 3 0 g/kg urea + 40 g/kg lime was the most effective treatment for improving the digestibility of rice straw. In addition, Zaman (1994) also reported that a level of at least 15 g/kg urea was required to prevent mould in the straw treated with lime. Trach et al. (2001b) earlier reported the effective level was 20 g/kg urea and 30 g/kg lime in treatment of the rice straw in in vitro and in sacco trials. Urinary excretion of purine derivatives and microbial nitrogen supply
As a result of this study, beef cattle fed 50 g/kg urea, 20 g/kg urea + 20 g/kg Ca(OH)2 and 30 g/kg urea + 20 g/kg Ca(OH)2-treated rice straw resulted in higher N retention, microbial protein supply and EMNS which could be explained that NH3-N produced in the rumen was used for microbial growth. In addition, urea and Ca(OH)2 treatment affected on lingocellulosic bonding, increased the fragility of inside and outside cell wall structure and decreased the content of phenolic, uronic acid and acetyl group of cell wall polysaccharides. As a result, it resulted in an increase in accessibility of the rumen micro-organisms to the cell wall especially for the fungal zoospores and cellulolytic bacteria (Table 3) which were the main microbes for structural carbohydrate utilization. The increase in microbial growth observed with treated group in this study may be due to increased availability of carbon skeletons for protein synthesis in combination with ammonia. This result was in agreement with Cherdthong et al. (2011) who reported that increase in microbial N synthesis was related to the increase in diet digestion. Microbial protein synthesis in the rumen could provide the majority of protein supplied to the small intestine of ruminants, References AOAC, 1997: Official Methods of Analysis. 16th edn. Association of Official Analytical Chemists, Gaithersburg, MD, USA. Bod’a, K., 1990: Non Conventional Feedstuffs in the Nutrition of Farm Animals. Developments in Animal and Veterinary 23. Elsevier, Amsterdam-Oxford-New YorkTokyo. Calabr o, S.; Moniello, G.; Piccolo, V.; Bovera, F.; Infascelli, F.; Tudisco, R.; Cutrignelli, M. I., 2008: Rumen fermentation and degradability in buffalo
accounting for 50% to 80% of total absorbable protein (Firkins et al., 2007). Moreover, Koenig et al. (2000) reported that ruminal bacteria obtained from 630 to 800 g/kg of their N requirements from the ruminal NH3-N pool and thus the remaining 200 to 370 g/kg of their N requirements from non-NH3-N (NAN), primarily in the form of amino acid and peptides. However, the total amount of microbial protein flowing to the small intestine depends on nutrient availability and efficiency of use of these nutrients by the ruminal bacteria (Cutrignelli et al., 2007; Calabr o et al., 2008; Maeda et al., 2012). Conclusions and recommendations Based on this study, it could be concluded that the use of urea plus calcium hydroxide could improved the nutritive value of rice straw in terms of dry matter intake, digestibility, ruminal volatile fatty acids, ruminal microbial population, N retention and microbial protein synthesis in beef cattle. Combination of urea plus Ca(OH)2 was an important alternative to only urea treatment which reduced feed cost in ruminant feeding. Acknowledgements The authors would like to express their most sincere thanks to all who have assisted and supported the research in this study, particularly the Tropical Feed Resources Research and Development Center (TROFREC), Department of Animal Science, Faculty of Agriculture, Khon Kaen University (KKU), Thailand for their kind facilitation and permission to use the research facilities. The Thailand Research Fund (TRF) through the Royal Golden Jubilee Ph.D. Scholarship Programme awarded to the first author is gratefully appreciated.
and cattle using the in vitro gas production technique. Journal of Animal Physiology and Animal Nutrition 92, 356–362. Chen, X. B.; Gomes, M. J., 1995: Estimation of Microbial Protein Supply to Sheep and Cattle Based on Urinary Excretion of Purine Derivatives-an Overview of the Technical Details. Occasional Publication 1992, International Feed Resources Unit, Rowel Research Institute, Aberdeen, UK. Chen, X. L.; Wang, J. K.; Wu, Y. M.; Liu, J. X., 2008: Effects of chemical treat-
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Koenig, K. M.; Newbold, C. J.; McIntosh, F. M.; Rode, L. M., 2000: Effects of protozoa on bacterial nitrogen recycling in the rumen. Journal of Animal Science 78, 2431–2445. Maeda, E. M.; Zeoula, L. M.; Jobim, C. C.; Cecato, U.; Rigolon, L. P.; Kazama, R.; Jacobi, G.; Carvalho, A. F. G., 2012: Intake, digestibility, rumen characteristics and microbial protein synthesis efficiency in bovine and bubaline fed sugar cane silage with additives. Revista Brasileira de Zootecnia 41, 707–716. Napasirth, V.; Wanapat, M.; Berg, J., 2012: Assessment of urea and/or lime treatment on rice straw quality using in vitro gas fermentation technique. Journal of Animal and Veterinary Advances 11, 295–299. Ørskov, E. R.; Meehan, D. E.; Macleod, N. A.; Kyle, D. J., 1999: Effect of glucose supply on fasting nitrogen excretion and effect of level and type of volatile fatty acid on response to protein infusion in cattle. British Journal of Nutrition 81, 389–393. Preston, T. R., 1995: Tropical Animal Feeding — A Manual for Research Worker. FAO Animal Production and Health, Rome, Italy. Preston, R. L.; Schnakanberg, D. D.; Pander, W. H., 1965: Protein utilization in ruminants. I. Blood urea nitrogen as affected by protein intake. Journal of Nutrition 86, 281–288. Samuel, M.; Sagathewan, S.; Thomas, J.; Mathen, G., 1997: An HPLC method for estimation of volatile fatty acids of ruminal fluid. Indian Journal of Animal Sciences 67, 805–811. Sarnklong, C.; Cone, J. W.; Pellikaan, W.; Hendriks, W. H., 2010: Utilization of rice straw and different treatments to improve its feed value for ruminants: a Review. Asian-Australasian Journal of Animal Sciences 23, 680–692. SAS, 1998: User’s Guide: Statistic, Version 6, 12th edn. SAS Inst., Cary, NC. Sirohi, S. K.; Roi, S. N., 1995: Associative effect of lime plus urea treatment of paddy straw on chemical composition and in vitro digestibility. Indian Journal of Animal Sciences 65, 1346–1351.
Steel, R. G. D.; Torrie, J. H., 1980: Principles and Procedures of Statistics. McGraw Hill Book, New York, NY, USA. Trach, N. X.; Mo, M.; Dan, C. X., 2001a: Effect of treatment of rice straw with lime and/or urea on its chemical composition, in-vitro gas production and in-sacco degradation characteristics. Livestock Research for Rural Development 13, 1–16. Trach, N. X.; Mo, M.; Dan, C. X., 2001b: Effect of treatment of rice straw with lime and/or urea on responses of growing cattle. Livestock Research for Rural Development 13, 1–9. Van Soest, P. J.; Robertson, J. B.; Lewis, B. A., 1991: Methods for dietary fiber neutral detergent fiber, and nonstarch polysaccharides in relation to animal nutrition. Journal of Dairy Science 74, 3583–3597. Vinh, N. T.; Wanapat, M.; Khejornsart, P.; Kongmun, P., 2011: Study of diversity of rumen microorganisms and fermentation in swamp buffalo fed different diet. Journal of Animal and Veterinary Advances 10, 406–414. Wanapat, M.; Pimpa, O., 1999: Effect of ruminal NH3-N levels on ruminal fermentation, purine derivatives, digestibility and rice straw intake in swamp buffaloes. Asian-Australasian Journal of Animal Sciences 12, 904–907. Wanapat, M.; Chenost, M.; Munoz, F.; Kayouii, C., 1996: Methods to improving the nutritive value of fibrous feed: treatment and supplementation. Annales de Zootechnie 45, 69–103. Wanapat, M.; Polyorach, S.; Boonnop, K.; Mapato, C.; Cherdthong, A., 2009: Effects of treating rice straw with urea or urea and calcium hydroxide upon intake, digestibility, rumen fermentation and milk yield of dairy cows. Livestock Science 125, 238–243. Zaman, S. M. H., 1994: Deepwater Rice, Wetlands of Bangladesh. Bangladesh Centre for Advanced Studies and Nature Conservation Movement, Dhaka, Bangladesh, pp. 34.
Journal of Animal Physiology and Animal Nutrition © 2014 Blackwell Verlag GmbH
ORIGINAL ARTICLE
Improving the quality of rice straw by urea and calcium hydroxide on rumen ecology, microbial protein synthesis in beef cattle S. Polyorach1,2 and M. Wanapat1 1 Tropical Feed Resources Research and Development Center (TROFREC), Faculty of Agriculture, Khon Kaen University, Muang, Thailand, and 2 Department of Animal Science, Faculty of Natural Resources, Rajamangala University of Technology-Isan, Phang Khon, Thailand
Summary Four rumen-fistulated beef cattle were randomly assigned to four treatments according to a 4 9 4 Latin square design to study the influence of urea and calcium hydroxide [Ca(OH)2] treatment of rice straw to improve the nutritive value of rice straw. Four dietary treatments were as follows: untreated rice straw, 50 g/kg urea-treated rice straw, 20 g/kg urea + 20 g/kg calcium hydroxide-treated rice straw and 30 g/kg urea + 20 g/kg calcium hydroxide-treated rice straw. All animals were kept in individual pens and fed with concentrate at 0.5 g/kg of BW (DM), rice straw was fed ad libitum. The experiment was conducted for four periods, and each period lasted for 21 days. During the first 14 days, DM feed intake measurements were made while during the last 7 days, all cattle were moved to metabolism crates for total faeces and urine collections. The results revealed that 20 g/kg urea + 20 g/kg calcium hydroxide-treated rice straw improved the nutritive value of rice straw, in terms of dry matter intake, digestibility, ruminal volatile fatty acids, population of bacteria and fungi, nitrogen retention and microbial protein synthesis. Based on this study, it could be concluded that using urea plus calcium hydroxide was one alternative method to improve the nutritive value of rice straw, rumen ecology and fermentation and thus a reduction of treatment cost. Keywords rice straw, urea, calcium hydroxide [Ca(OH)2], rumen ecology, microbial protein, ruminants Correspondence M. Wanapat, Department of Animal Science, Faculty of Agriculture, Khon Kaen University, P.O. Box 40002, Muang, Khon Kaen, Thailand. Tel: +66 43 202368; Fax: +66 43 202368; E-mail: [email protected] Received: 23 February 2014; accepted: 18 August 2014
Introduction Rice straw is an important crop residue practically used by farmers for ruminant feeding especially during the long dry season (Ørskov et al.,1999). However, rice straw is low in nutritive value with low level of protein (20–50 g/kg DM), high fibre and lignin content (NDF > 650 g/kg), low DM digestibility ( 0.05). Rumen micro-organism population
Rumen micro-organisms in the treated groups are presented in Table 3 and were found significantly higher than those in the control group. It was significantly (p < 0.05) higher than in the control group and was
the highest in the 50 g/kg urea treatment, 30 g/kg urea + 20 g/kg Ca(OH)2-treated groups for bacteria, fungi and cellulolytic bacteria, while protozoal population was significantly lower (p < 0.05) than in the control group. In addition, the population of proteolytic and amylolytic bacteria in treated group were significantly (p < 0.05) higher than in control group. Effect on feed intake and digestibility
Table 4, shows effects of various treatments of rice straw on voluntary feed intake and nutrient digestibility in beef cattle. The results of this experiment using 50 g/kg urea, 20 g/kg urea + 20 g/kg Ca(OH)2 and 30 g/kg urea + 20 g/kg Ca(OH)2 for the straw treatment can significantly (p < 0.05) enhance DM intake and digestibility. Apparent digestibility of DM, OM, CP, NDF and ADF in the 30 g/kg urea + 20 g/kg Ca (OH)2-treated rice straw was the highest (p < 0.05), followed by the 50 g/kg urea-treated rice straw, 20 g/ kg urea + 20 g/kg Ca(OH)2-treated rice straw and untreated rice straw respectively. Urinary excretion of purine derivatives and microbial nitrogen supply
The effect of dietary treatments on urinary excretion of purine derivatives and microbial nitrogen supply are presented in Table 5. The findings under this investigation revealed significant (p < 0.05) improvement in N balance in terms of N intake, faecal N, urinary N, N absorption and N retention and urinary purine derivatives. As a result of this study, beef cattle fed 50 g/kg urea, 20 g/kg urea + 20 g/kg Ca(OH)2 and 30 g/kg urea + 20 g/kg Ca(OH)2-treated rice straw resulted in higher N retention, microbial protein supply and efficiency of microbial N synthesis (EMNS)
Table 2 Effect of rice straw treatment on ruminal pH, temperature, NH3-N and volatile fatty acid (VFA) concentrations in beef cattle
Item
Untreated rice straw
Ruminal pH 6.2a Ruminal temperature (°C) 39.1 NH3-N, mg/100 ml 13.5a BUN, mg/100 ml 15.2a Total VFA, mmol/l 105.0 Molar proportion of VFA (mol/100 mol) Acetic acid (C2) 73.9a 16.3a Propionic acid (C3) Butyric acid (C4) 9.7 C2:C3 ratio 4.7a
50 g/kg urea-treated rice straw
20 g/kg urea + 20 g/kg Ca(OH)2-treated rice straw
30 g/kg urea + 20 g/kg Ca(OH)2-treated rice straw
SEM
p-Value
6.6bc 39.2 16.8c 15.5a 111.0
6.6b 39.1 15.3b 13.8b 108.8
6.8c 39.0 16.1cb 14.6ab 112.1
0.03 0.09 0.24 0.30 1.73
** ns ** * ns
67.7b 22.2b 10.1 3.1b
0.90 1.02 0.73 0.30
* * ns *
69.7b 21.2b 9.7 3.4b
70.4b 19.1ab 10.5 3.7ab
NH3-N, ammonia nitrogen; BUN, blood urea nitrogen; SEM, standard error of the mean; ns, no significance; *p < 0.05; **p < 0.01; a,b,cValues on the same row with different superscripts differ (p < 0.05).
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Table 3 Effect of rice straw treatment on microbial population in the rumen of beef cattle
Item
Untreated rice straw
Total direct count, cell/ml 3.6a Bacteria, 9109 Protozoa, 9105 8.2a 5 Fungal zoospores, 910 3.8a Ruminal bacterial groups, CFU/ml Total viable bacteria, 9109 3.5a Cellulolytic bacteria, 9108 5.7a Amylolytic bacteria, 9107 3.9a 7 Proteolytic bacteria, 910 3.9a
50 g/kg urea-treated rice straw
20 g/kg urea + 20 g/kg Ca(OH)2-treated rice straw
30 g/kg urea + 20 g/kg Ca(OH)2-treated rice straw
SEM
p-Value
5.7c 5.9b 5.5c
4.9b 6.3b 4.7b
5.8c 5.3b 5.7c
0.19 0.53 0.19
** * **
5.0b 8.2c 5.7b 7.2b
4.9b 7.2b 5.5b 5.9b
5.9b 9.3d 5.9b 6.9b
0.32 0.22 0.41 0.42
* ** * **
SEM, standard error of the mean; ns, no significance; *p < 0.05; **p < 0.01; a,b,c,dValues on the same row with different superscripts differ (p < 0.05).
Table 4 Effect of rice straw treatment on voluntary feed intake and nutrient digestibility in beef cattle
Item Concentrate DM intake kg/day g/kg BW0.75 daily Roughage DM intake kg/day g/kg BW0.75 daily Total DM intake kg/day g/kg BW0.75 daily Apparent digestibility, g/kg Dry matter Organic matter Crude protein Neutral detergent fibre Acid detergent fibre
Untreated rice straw
50 g/kg urea-treated rice straw
20 g/kg urea + 20 g/kg Ca(OH)2-treated rice straw
30 g/kg urea + 20 g/kg Ca(OH)2 treated rice straw
SEM
p-Value
1.3 20.1
1.3 20.0
1.4 19.9
1.3 20.2
0.07 0.90
ns ns
3.8a 60.0a
5.4b 85.2b
5.1b 79.6b
5.2b 79.8b
0.17 2.84
** **
5.1a 80.1a
6.7b 105.2b
6.5b 99.5b
6.5b 100.0b
0.21 2.75
** **
549.3a 589.0a 629.3a 486.0a 450.0a
591.0ab 627.0b 672.3b 543.8bc 491.3bc
577.3ab 615.5ab 652.3ab 532.5b 478.0ab
605.8b 643.8b 681.3b 571.5c 518.5c
14.06 14.55 11.89 10.75 9.99
* * * ** **
SEM, standard error of the mean; ns, no significance; *p < 0.05; **p < 0.01; a,b,cValues on the same row with different superscripts differ (p < 0.05).
when compared with the control group (p < 0.05). However, there were not significant (p > 0.05) differences among treated groups.
Characteristics of ruminal fermentation and blood metabolites
When rice straw was treated with urea, it resulted in improving the nutritional quality of the straw in terms of nitrogen content, palatability and digestibility. During the treatment process, ammonia generated from urea, in the presence of water formed an alkali (ammonium hydroxide), which impacted on lingocellulosic bonding of the rice straw (Wanapat et al., 1996). Trach et al. (2001b) reported that the result was higher when Ca(OH)2 (20–30 g/kg DM of straw) and urea (20 g/kg DM of straw) were used in combination.
Under this experiment, BUN ranged from 13.1 to 16.8 mg/dl and was similar to the values reported by Wanapat and Pimpa (1999). The ruminal NH3-N result obtained under this study was closer to 15–30 mg/dl (Wanapat and Pimpa, 1999), these concentrations could improve rumen fermentation and feed intake in swamp buffaloes offered urea-treated rice straw. In addition, Preston et al. (1965) reported that the concentration of BUN produced was closely correlated to the level of ammonia production in the rumen. Furthermore, Preston (1995) suggested that the quantity of ammonia absorbed from the rumen reflected in the circulating BUN. Diets which are balanced in protein (P)/energy (E) could result in BUN produced concentrations of approximately
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Discussion Chemical composition of feeds and ruminal parameters
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Table 5 Effect of rice straw treatment on allantoin excretion, purine derivative excretion and purine derivative absorption and microbial protein synthesis
Item Nitrogen balance (g/day) N intake Faecal N Urinary N N absorption N retention Purine derivatives (PD) (mmol/day) Allantoin excretion Allantoin absorbtion Microbial protein supply, g N/day† EMNS (g N/kg of OMDR)‡
Untreated rice straw
50 g/kg urea-treated rice straw
20 g/kg urea + 20 g/kg Ca(OH)2-treated rice straw
30 g/kg urea + 20 g/kg Ca(OH)2-treated rice straw
SEM
p-Value
53.5a 24.8a 21.5a 31.5a 10.0a
104.2b 41.9b 37.3b 65.3b 28.0b
99.4b 40.5b 37.7b 61.0b 23.3b
103.2b 41.4b 38.3b 66.4b 28.1b
5.92 1.28 0.83 3.51 3.66
** ** ** ** *
58.5a 41.2a 30.0a 15.5a
96.5b 85.4b 62.1b 22.7b
92.4b 79.8b 58.0b 22.8b
97.4b 86.8b 63.1b 23.4b
3.89 4.58 3.33 1.60
* ** ** *
SEM, standard error of the mean; ns, no significance; *p < 0.05; **p < 0.01; Values on the same row with different superscripts differ (p < 0.05). †Microbial crude protein (MCP) (g/day) = 3.99 9 0.856 9 mmoles of purine derivatives excreted (Galo et al., 2003). ‡Efficiency of microbial N synthesis (EMNS, g/kg of OM digested in the rumen (OMDR) = [(MCP (g/day) 9 1000)/DOMR (g)], assuming that rumen digestion was 650 g/kg OM of digestion in total tract.
a,b
15 mg/100 ml, while BUN levels lower or higher than this could be due to an insufficiency or exceeding in CP per unit of digestible energy (Wanapat and Pimpa, 1999). Volatile fatty acids concentration in this experiment were similar to those reported by Wanapat et al. (2009) who reported that the effects of various treated rice straw on VFA concentrations especially increasing those of C3 were increased while those of C2 were decreased, thus C2/C3 was subsequently lowered in cows fed with 30 g/kg urea + 20 g/kg Ca(OH)2 treated rice straw, 50 g/kg urea-treated rice straw and 20 g/kg urea + 20 g/kg Ca(OH)2-treated rice straw respectively. Moreover, Ørskov et al. (1999) discussed that, when high fibre diets were offered, VFAs in ruminal fermentation shifted from 65:25:10 to 70:20:10 (acetate: propionate: butyrate, in molar percentage) ratios. Rumen micro-organism population
The results of this experiment agreed with Wanapat et al. (2009), who revealed that 50 g/kg urea-treated rice straw and 20 g/kg urea + 20 g/kg Ca(OH)2-treated rice straw increased total viable bacteria and cellulolytic bacteria in lactating dairy cows. Moreover, Vinh et al. (2011) showed that total ruminal bacteria, Fibrobacter succinogenes, Ruminococcus albus, cellulolytic bacteria and fungal population were greater by dietary treatment with urea–lime (20 g/kg urea + 20 g/kg lime)-treated rice straw while Ruminococcus flavefaciens, protozoal population were significantly reduced (p < 0.05). Amylolytic, proteolytic bacterial groups, total bacteria were not changed 454
among treatments in swamp buffalo. Furthermore, Khejornsart et al. (2011) showed that when feeding urea–lime (20 g/kg urea + 20 g/kg lime)-treated rice straw could enhance the nutritive value of rice straw and had a significantly higher (p < 0.05) in bacterial population than those in untreated group, showing buffaloes utilized feeds more efficiently with higher rumen fermentation. Concentration of alkaline agents (urea and Ca(OH)2) can chemically change structural fibre swollen and easily digested by rumen microbes (Wanapat et al., 2009). This effect avails the rumen microbes to attack the structural carbohydrates more easily, improve digestibility, as well as the palatability of treated straw (Bod’a, 1990). Moreover, Goto et al. (1993) reported that alkaline treatment can attack the lignin–polysaccharide bond which solublizes hemicellulose and lignin in the straw and hence exposes the cellulose for the microbial population to be digested. Chen et al. (2008) also reported that chemical treatments enhanced the nutritive value of rice straw through increasing the number of accessible sites of microbial attachment on the surface of the particles, increasing fibrolytic microbe quantity and hence fibrolytic enzyme activities and improving the overall rumen fermentation characteristics. Effect on feed intake and digestibility
Wanapat et al. (1996) reported that alkaline agenttreated rice straw significantly improve digestibility and feed intake by the ruminants. The concentrated alkaline agents can chemically break the ester bonds between lignin hemicellulose and cellulose, and Journal of Animal Physiology and Animal Nutrition © 2014 Blackwell Verlag GmbH
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physically changed the fibrous structure. In finding a good combination level of urea and lime, Sirohi and Roi (1995) revealed that a combination of 3 0 g/kg urea + 40 g/kg lime was the most effective treatment for improving the digestibility of rice straw. In addition, Zaman (1994) also reported that a level of at least 15 g/kg urea was required to prevent mould in the straw treated with lime. Trach et al. (2001b) earlier reported the effective level was 20 g/kg urea and 30 g/kg lime in treatment of the rice straw in in vitro and in sacco trials. Urinary excretion of purine derivatives and microbial nitrogen supply
As a result of this study, beef cattle fed 50 g/kg urea, 20 g/kg urea + 20 g/kg Ca(OH)2 and 30 g/kg urea + 20 g/kg Ca(OH)2-treated rice straw resulted in higher N retention, microbial protein supply and EMNS which could be explained that NH3-N produced in the rumen was used for microbial growth. In addition, urea and Ca(OH)2 treatment affected on lingocellulosic bonding, increased the fragility of inside and outside cell wall structure and decreased the content of phenolic, uronic acid and acetyl group of cell wall polysaccharides. As a result, it resulted in an increase in accessibility of the rumen micro-organisms to the cell wall especially for the fungal zoospores and cellulolytic bacteria (Table 3) which were the main microbes for structural carbohydrate utilization. The increase in microbial growth observed with treated group in this study may be due to increased availability of carbon skeletons for protein synthesis in combination with ammonia. This result was in agreement with Cherdthong et al. (2011) who reported that increase in microbial N synthesis was related to the increase in diet digestion. Microbial protein synthesis in the rumen could provide the majority of protein supplied to the small intestine of ruminants, References AOAC, 1997: Official Methods of Analysis. 16th edn. Association of Official Analytical Chemists, Gaithersburg, MD, USA. Bod’a, K., 1990: Non Conventional Feedstuffs in the Nutrition of Farm Animals. Developments in Animal and Veterinary 23. Elsevier, Amsterdam-Oxford-New YorkTokyo. Calabr o, S.; Moniello, G.; Piccolo, V.; Bovera, F.; Infascelli, F.; Tudisco, R.; Cutrignelli, M. I., 2008: Rumen fermentation and degradability in buffalo
accounting for 50% to 80% of total absorbable protein (Firkins et al., 2007). Moreover, Koenig et al. (2000) reported that ruminal bacteria obtained from 630 to 800 g/kg of their N requirements from the ruminal NH3-N pool and thus the remaining 200 to 370 g/kg of their N requirements from non-NH3-N (NAN), primarily in the form of amino acid and peptides. However, the total amount of microbial protein flowing to the small intestine depends on nutrient availability and efficiency of use of these nutrients by the ruminal bacteria (Cutrignelli et al., 2007; Calabr o et al., 2008; Maeda et al., 2012). Conclusions and recommendations Based on this study, it could be concluded that the use of urea plus calcium hydroxide could improved the nutritive value of rice straw in terms of dry matter intake, digestibility, ruminal volatile fatty acids, ruminal microbial population, N retention and microbial protein synthesis in beef cattle. Combination of urea plus Ca(OH)2 was an important alternative to only urea treatment which reduced feed cost in ruminant feeding. Acknowledgements The authors would like to express their most sincere thanks to all who have assisted and supported the research in this study, particularly the Tropical Feed Resources Research and Development Center (TROFREC), Department of Animal Science, Faculty of Agriculture, Khon Kaen University (KKU), Thailand for their kind facilitation and permission to use the research facilities. The Thailand Research Fund (TRF) through the Royal Golden Jubilee Ph.D. Scholarship Programme awarded to the first author is gratefully appreciated.
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