Effect of urea

Proceedings Buffalo Workshop December  2001


Effect of urea-molasses-mineral supplementation on in vivo, in situ
and in vitro feed digestibility of swamp buffaloes 

Nguyen van Thu

Dept. of Animal Husbandry, Faculty of Agriculture,
Cantho University, Cantho city, Vietnam 


Two experiments were carried out in young swamp buffaloes to investigate effect of supplementation of urea-molasses-mineral mixture on feed digestibility in vivo, in situ and in vitro. In Exp. 1 four young buffaloes were used in a 4x4 Latin square experiment to investigate effects of a molasses-mineral mixture combined urea, soybeans or both urea and soybeans on rumen pH, NH3-N, microbial population and feed digestibility. The animals were all fed a basal diet of rice straw. The treatments were: no supplements (R), molasses-urea mixture with 265 g molasses, 53.2 g urea, 120 g  whole soya bean, 26.6 g salt, 26.6 g bone meal and 2.1 g trace minerals (RMUS), the same mixture without the soya bean (RMU), and the mixture without urea (RMS). In Exp 2 twelve buffaloes were allocated in a factorial design experiment, the first factor was roughage including rice straw and maize stover, and the second one was the supplementation of the mixture used for Exp 1, but it was higher in amount of 180g/day. In in situ and in vitro digestibility studies, incubated rice straw was used with or without the supplementation. These mixtures were given once daily at 7:00h and rumen fluid was collected 3h post-feeding for measuring rumen parameters and in vitro digestibility studies.

In Exp 1 results showed that ruminal pH was similar for the different diets, while NH3-N was higher (p<0.001) for the RMUS and RMU diets compared to the R and RMS diets. The bacteria and protozoa populations were higher (p<0.05) for the RMUS diet compared to the others. Total volatile fatty acid concentrations differed among the treatments (p<0.05) and were 92.7, 123, 113 and 95.5mM for the R, RMUS, RMU and RMS diets, respectively. No differences were found among treatments in either in sacco or in vitro rice straw degradation. However, improvements (p<0.01) were found in in vivo dry matter, organic matter and neutral detergent fiber digestibility in the supplemented diets. Findings in Exp 2 were similar to Exp 1 in pH, NH3-N concentration and feed digestibility as effected by supplementation. Rice straw DM, NDF digestibilities were higher than these of maize stover. However no difference was found in digestibility of incubated rice straw with or without supplemented mixture for both in situ and in vitro techniques. Supplementation with the urea-molasses-mineral mixture improved ruminal NH3-N, rumen microbial populations and feed digestion of swamp buffaloes. However, in situ and in vitro techniques were not able to detect the improvements of rumen degradability of rice straw due to the supplementation. Results also suggest that soybean meal could be combined with urea to improve buffalo rumen function.

 Key words: Urea-molasses-mineral mixture, Buffalo, rice straw, maize stover, digestibility


Rice straw and maize stover are roughages with a low nutrient content and low digestibility and usually fed to buffaloes as a main diet during the dry season in many Asian countries. These diets result in low performance and poor health. It has been suggested that urea-molasses-mineral block supplementation can improve rice straw digestibility by increasing the rumen microbial population (Sansoucy et al 1995). However, positive results of this strategy on digestibility have not been reported (Schiere et al 1989 and Hosmani et al 1998). Supplementation of rice straw with urea-molasses cake (a small soft block) made from urea, molasses, coconut meal, rice bran, and minerals have shown positive effects on performance of working and lactating buffaloes (Thu and Udén 2000). However, effects of each of these components on ruminal parameters and feed intake of buffaloes have not been apparent (Thu and Udén 2001). This suggests that the combination of molasses, N and minerals in the cake would have their main effects on rumen function.

Supplementing cattle with rumen undegradable protein improves performance, presumably by providing more intestinally absorbable protein, although an alternative strategy is to increase microbial production in the rumen by increasing intake of fermentable organic matter (Mullik et al 1998). Wejdemar (1996) found that besides non-protein N, fiber digesting organisms also use peptides and amino acids for growth. Cellulolytic bacteria require polypeptides or true protein for optimal growth and digestibility of cellulose (Huque and Thomsen 1984). Thus, supplementation of  rice straw and maize stover diets with urea-molasses-mineral mixture that includes degradable protein may increase their digestibility by stimulating rumen microbial growth.

The objective of this study was to investigate the possibility of increasing the rumen activities and feed digestibility, by supplementing rice straw or maize stover with urea and/or soybean in molasses-mineral mixtures to swamp buffaloes.

Materials and  Methods

Animals and feeding

Two experiments were carried out at the experimental farm of Cantho University in Vietnam in 1998 and 2000. In Exp 1, four rumen fistulated young swamp buffaloes were used in a 4x4 Latin square design experiment. The animals were all fed a basal diet of rice straw. The treatments were: no supplement (R), urea-molasses mixture with 265g molasses, 53.2g urea, 120g whole ground soya beans, 26.6g salt, 26.6g bone meal and 2.1g trace minerals (RMUS), the same mixture without soya bean meal (RMU) and the same mixture without urea (RMS). Crude protein (CP) supplied was 220, 164 and 66g/d for the RMUS, RMU and RMS diets respectively (see Thu and Udén 2000). For the RMUS, soya bean protein N contribution to the total N content of the mixture was approximately 27%, and the CP and metabolizable energy ratio (g/MJ) was 57.5 (NIAH 1995). Low-fiber soya bean meal was specially produced from soya beans, which were roasted and the cortex and germ removed before grinding.

In Exp 2 twelve buffaloes were allocated in a factorial design experiment, the first factor was roughage including rice straw and maize stover, and the second one was the supplementation of the mixture of Exp 1, but it was higher in amount of 180g/d. In in situ and in vitro digestibility studies, incubated rice straw was used with or without the supplementation. Each experimental period was three weeks, including one week for adaptation. The supplement mixtures were fed in a liquid form once daily at 7:00h, while the rice straw was fed twice a day at 7:15 and 14:15h.

Sampling and chemical analysis

Samples of rumen contents were collected 3 h post-feeding by suction through a tube of about 1 cm internal diameter following the method of Warner (1962) to measure rumen pH, ammonia N (NH3-N), protozoa and total bacteria populations. Rumen pH was measured by pH meter and NH3-N was analyzed according to AOAC (1980). For counting protozoa, the preparation of the rumen content samples followed the procedure of Dehority (1984) using a 0.2mm deep chamber under 100 x magnification. Total bacteria populations were counted in a Neubauer chamber under 1200 x magnification after preparation of rumen content samples following the procedure of Warner (1962). Analysis of total VFA was by a distillation method (Barnett and Reid 1957).

Feeds offered and refusals were collected daily and pooled weekly for analysis of DM to calculate feed intake. Feed and samples were dried at 105oC overnight to determine dry matter (DM). The OM was determined by ashing samples in a furnace at 500oC for 4 h. The CP was determined by a Kjeldahl method (AOAC 1980). Analysis of neutral detergent fiber (NDF) was by a method described by Van Soest et al (1991). Acid detergent fiber (ADF) and acid detergent lignin (ADL) were analyzed by methods suggested by Robertson and Van Soest (1981). The analysis of NDF, ADF and ADL were done with residue ashing.

Rice straw samples were dried at 55°C overnight and ground to pass a 1mm sieve for rumen incubations. In situ incubations were made at 0, 12, 24, 48, 72 and 96 hours in duplicate to measure feed degradability (Ørskov et al 1980). Rice straw samples were also used to determine DM, OM or NDF degradability in vitro at 0, 24, 48, 72 and 96 h using rumen fluid from the buffaloes of the corresponding treatments (Goering and Van Soest,1970 as modified by Mbwile and Udén 1991). In vivo DM, OM and NDF digestibilities were estimated by total fecal collection for 7 d beginning 2 d after the start of feed intake recording (Mc Donald et al 1998).

Biometric analysis

In Exp 1 data was subjected to analysis of variance (ANOVA) using the General Linear Model (GLM) procedure of Minitab (1998), and when the F test was significant (P<0.05), Tukey’s test for paired comparison was used. The data of in situ and in vitro degradability was fitted to a non-linear model (Ørskov et al. (1980): DMD= a + b (1- e-ct ), where, DMD = DM disappeared after time (t), a = y  intercept, c= fractional degradation rate (h-1) and a+b = curve asymptote  representing the potential degradability. The Maximum Likelihood program (MLP) of Ross (1987) was used for curve fitting and also for comparison of the treatment differences among the curves by parallel curves analysis (CPCA). The CPCA and subsequent F-testing was done in three steps. First, individual parameter sets were generated for each treatment, then the parameter sets were derived assuming common rate parameters (c) and finally one parameter set was derived for the pooled treatments. The total residual sum of squares for each step was used for testing of parallelism (common rates) or displacement (common a and/or b) by common F-test.    

In Exp 2 data was subjected to analysis of variance (ANOVA) using the General Linear Model (GLM) procedure of Minitab (1998) The data of in situ and in vitro degradability was first analysed by GLM at different incubated times to compare between maize stover and rice straw diet, supplementation and no supplementation, and incubated rice straw with or without UMM, and then fitted to a non-linear model (Ørskov et al. (1980) by Neway Programme.


Experiment 1

The low NDF and ADF values for soybean meal were probably due to the absence of cortex and germ (Table 1).

Table 1. Chemical composition of rice straw, whole soya beans, bone meal and ‘’B’’ molasses (DM basis except for DM) in Exp.1.









Rice straw








Soya beans








Bone meal








‘’B’’ molasses








a B molasses: molasses after the second sugar extraction
 DM: dry matter, CP: crude protein, NDF: neutral detergent fiber, ADF: acid detergent fiber and EE: ether extraction, ADL: acid detergent lignin 
 Not determined

Ruminal pH was similar for the different diets (Table 2), while NH3-N (mg/100ml) was higher (P<0.05) for the RMUS and RMU diets compared to the R and RMS diets. The bacteria and protozoa populations were highest on the RMUS diets (P<0.05) compared to other diets. The rumen VFA concentration (mM) was highest for the RMUS diet, lowest for the R diet and intermediate for the RMU and RMS diets.

 Table 2. pH, ammonia N, protozoa and bacterial populations as well as total volatile fatty acid (VFA) concentrations of rumen fluid of young swamp buffaloes fed rice straw with or without molasses based  supplements in Exp.1.













NH3-N (mg/100ml)






Bacteria (x10-8)






Protozoa (x10-5)






Total VFA (mM)






a, b Means with different letters within the same rows differ significantly at the 5% level.
R: no supplement, RMUS: 265g molasses, 53.2g urea, 120g whole soya beans, 26.6g salt, 26.6g bone meal and 2.1g trace minerals, RMU: RMUS without soya beans and RMS: RMUS without urea


Table 3. Model parameter values for in sacco dry matter (DM) and in vitro organic matter (OM)  degradation of rice straw as affected by supplements fed to donor buffaloes eating rice straw in Exp1. 






In sacco DM digestibility 


















In vitro OM digestibility 

















model: y = a +b (1-e-ct) fitted to the data, where a represents the immediately soluble fraction, b represents the potentially degr
R: no supplement, RMUS: 265g molasses, 53.2g urea, 120g soybean meal, 26.6g salt, 26.6 g bone meal and 2.1g trace minerals, RMU: RMUS without soybean meal and RMS: RMUS without urea.adable fraction and c represents the fractional degradation rate (h-1).


In situ and in vitro rice straw digestibility showed no differences among treatments (Table 3). However, there were significant improvements (P<0.01) of in vivo DM, OM and NDF digestibility in the supplemented diets (Table 4). Values were numerically higher for the RMUS diet as compared to the RMU and RMS diet, but not significantly so (P>0.05). Assuming that the supplements were 100% digestible, from the in vivo calculation rice straw DM and OM digestibilities were also found to be significantly (P<0.01) higher for the supplemented diets compared to the R diet (Table 4).

Table 4. In vivo digestibility of young swamp buffaloes fed rice straw and supplemented with different molasses based mixtures.







Total digestibility (%)



Dry matter






Organic matter






Neutral detergent fiber






Rice straw digestibility (%) 


Dry matter






Organic matter






ab Means with different letters within rows differ significantly at the 5% level.
R: no supplement, RMUS: 265g molasses, 53.2g urea, 120g soybean meal, 26.6g salt, 26.6 g bone meal and 2.1g trace minerals, RMU: RMUS without soybean meal and RMS: RMUS without urea.

Experiment 2

Crude protein of maize stover was higher than that of rice straw and urea-molasses-mineral (UMM) supplementation to maize stover and rice straw increased their CP content about 3% and NDF content from 4.7-6.1%. There was a reduction of ADF and lignin when UMM was supplemented (Table 5).

Table 5. Chemical composition of roughages and urea-molasses-mineral mixture (UMM) used in Exp. 2 (%DM).









Maize stover








Maize stover + UMM








Rice straw








Rice straw + UMM








Urea-molasses-mineral mixture (UMM)








Table 6 showed there was no difference of rumen pH values between rice straw and maize stover or UMM supplement and no supplement. Ruminal NH3-N was significantly higher in maize stover diet and in UMM supplemented diets. DM, OM and NDF digestibility of rice straw diets were higher than these of maize stover (p<0.01). UMM supplementation increased dietary DM, OM and NDF digestibility (p<0.01) and there was an interaction between roughage and supplementation (p<0.05).


Table 6. Effect of roughage consumed and urea-molasses-mineral supplementation on rumen parameters and total feed digestibility in vivo (%) in Exp. 2.


Roughage  consumed (RC)

Supplement (S)

Significance level


Rice straw

Maize stover














NH3-N (mg/100ml)
































ns non-significant, * significant difference at  5%; ** significant difference at 1%, *** significant difference at 1%
DMD dry matter digestibility, OM organic matter  digestibility, NDFD neutral detergent fiber digestibility.

The results showed that there were significantly higher DM rice straw digestibilities in in situ and in vitro in the rice straw compared to maize stover diets at different incubated times. Similarly, in situ and in vitro NDF rice straw digestibilities were also higher in the rice straw diets  (p<0.01). However, effect of UMM supplementation on rice straw DM digestibility was not apparent. At 12 and 24h incubation there were a significantly higher values in supplemented diets, but after that no difference was found up to 96h in in situ technique. While in in vitro technique there was no significant difference of rice straw DM and NDF degradabilities at different incubation times. There were similar DM and NDF degradable values (p>0.05) in both in situ and in vitro technique between incubated rice straw with or without UMM.


Rumen pH, NH3-N concentration, microbial growth and VFA production

The results of rumen fluid pH of the present study are similar to those of Wanapat et al (1991) when rice straw-fed cattle and buffaloes were supplemented with urea-molasses blocks. They also indicated that the rumen pH condition (around 7) of the buffaloes was optimum for microbial activities in all treatments. Wanapat et al. (1991) found that supplementation of swamp buffaloes and beef cattle with a high quality feed block that included molasses, urea, cassava, oil seed meals, minerals, and sulfur markedly enhanced ruminal NH3-N concentration at 0 h, 3 h and 6 h post-feeding. In the present study, the higher NH3-N concentration for the RMUS and RMU diets  was likely due to the urea in the supplements. By infusing NH4HCO3 to manipulate the rumen NH3-N concentration of swamp buffaloes, Wanapat and Pimpa (1999) concluded that the optimum rumen NH3-N concentration in swamp buffaloes is higher than 13.6 mg/100ml for microbial protein synthesis, digestibility and rice straw intake.

Pimpa et al. (1996) showed that increasing the rumen NH3-N concentration in swamp buffalo increased total bacteria and protozoa populations. Increases in rumen bacteria and protozoa populations were also found when rice straw and grass-fed swamp buffaloes were supplemented by a urea-molasses cake (Thu and Uden 2001). The principle cellulolytic bacteria species utilize ammonia as the main source of nitrogen (Bryant, 1973) whereas for microbes utilizing sugars or starches there is an apparently high requirement for preformed amino acids and peptides (Leng, 1990). Maeng et al (1989) concluded that in continuous culture, the optimum ratio of non-protein N for rumen microbial growth was 75% urea N plus 25% amino acid N. In the present study, a combination of protein N from soybean (27% total N of the supplement), NPN from urea and a readily available energy source in the form of molasses of the RMUS diet supported increased microbial growth compared to the R, RMU and RMS diets. Chowdry and Huque (1998) found that microbial protein synthesis was better in the combination of urea and molasses than that of urea and rice soup. Thus, in the present study energy from molasses and N from urea and soybean of the supplements may be available for microbial protein synthesis. Yan et al. (1997) also found that when a diet contained a high concentration of molasses (248g/kgDM), supplementation with urea and soybean meal increased intake and milk production in dairy cows.

Fadel et al.(1987) indicated that supplementing molasses, urea and starch increased total VFA concentration of rumen fluid in cattle. Total VFA concentration in rumen fluid is also increased when lambs consumed urea-molasses blocks with or without additional by-pass protein (Sansoucy et al. 1995). Similarly, in the present study the RMUS diet enhanced the rumen fermentation as compared to the R due to the additional protein N supplementation. Leng and Nolan (1984) stated that, depending on the efficiency of utilization of ATP, the carbohydrate converted to microbial cells could approach the amount fermented to VFA. Thus, in the RMUS diet of Exp 1 the microbial populations increased with increasing availability of the substrates in the form of readily fermentable carbohydrates and through a enhanced fiber utilization. The latter was evidently brought about by the additional nitrogen supply of the supplementation.

Feed digestion

Pearce (1973) observed that lick blocks were more effective when the quality of the basal ration was poor. In the present study the animals were fed low quality rice straw. There were significant improvements of DM, OM and NDF diet digestibility as well as of DM and OM rice straw digestibility for the supplemented diets compared to the unsupplemented diet in vivo (Table 4 and 6). These may be attributed to the increases of microbial populations caused by supplemented nutrients. Murphy (1990) reported that increases in nitrogen supply or sparing effects of the branched amino acids have led to increases in the numbers of cellulolytic bacteria and fiber digestion. Protozoa may also be responsible for an efficient fiber digestion (Wejdemar, 1996) by themselves or by a higher growth rate of cellulolytic bacteria in presence of protozoa which increases ammonia level in the rumen liquid (Jouany and Ushida, 1999).

Wu and Liu (1996) concluded that urea-mineral lick blocks improve the digestion of fiber in lambs on a low quality roughage, even though the blocks did not greatly influence the rumen degradation of either dry mater or crude protein. Fadel et al. (1987) also found no difference in the degradable fiber fraction of oat straw by the supplementation with urea; molasses, urea and starch; casein; or fish and maize gluten meal. In the present study there were no significant in situ digestibility differences observed among the treatments, despite significant increases in vivo digestibility. These results agree with those reported by Khalili et al. (1993). The lack of effects of supplements on in situ degradability is possibly explained by a lower microbial activity and pH in the bag contents compared to in the rumen (Russel and Dombrowski, 1980 and Nozière and Michalet-Doreau, 1996). Moreover, the variability of results in situ reported in the literature, may also have been caused by different bag porosities affecting the movement of rumen fluid and micro-organism across the bag membrane (Udén and Van Soest, 1984 and Kitessa et al., 1999). The similar lack of treatment effects using the in vitro technique may be attributed to the nutrients supplied by the in vitro medium, which included a nitrogen source from trypticase. Garg and Gupta (1991) found that in vitro studies were unable to demonstrate any fermentation or digestibility effects of supplementing straw fed donor cows. These were also experienced by Fujimaki et al. (1989).


Supplementation with a complete mixture including urea, molasses, soybeans and minerals improved rumen environment and feed digestion in swamp buffaloes fed rice straw or maize stover. However, in situ and in vitro methods were not able to detect improvements of rumen degradability of rice straw due to the supplementation. No differences were found in rumen VFA concentrations, digestibility of swamp buffaloes among the diets of the complete mixture and the mixture without soybean or urea component. The experimental results also suggest that ground soybeans may have a combined effect with urea to enhance rumen microbial population and rumen function.


Financial support of this work was provided by SAREC/SIDA. The authors would like to thank the Department of Animal Husbandry, Faculty of Agriculture, Cantho University, Vietnam and the Department of Animal Nutrition and Management, Swedish Agricultural Sciences, Sweden for use of their facilities. The authors also would like to thank Dr. Brian Ogle and Mr. Börje Ericson for their kind help.


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