Animal Feed Science and Technology 152 (2009) 1220

Contents lists available at ScienceDirect

Animal Feed Science and Technology

journal homepage: www.elsevier.com/locate/anifeedsci

Effects of supplementary inosine on nutrient digestibility, ruminal fermentation and nitrogen balance in goats fed high amount of concentrate
F. Kanyinji a, H. Kumagai b,, T. Maeda a, S. Kaneshima b, D. Yokoi c
a b c

Graduate School of Biosphere Science, Hiroshima University, 1-4-4 Kagamiyama, Higashi-Hiroshima, 739-8528, Japan Graduate School of Agriculture, Kyoto University, Kitashirakawa-Oiwake-cho, Sakyo-ku, Kyoto, 606-8502, Japan Production and Technology Administration Centre, Ajinomoto Co., Inc., 5-8 Kyobashi I-Chome, Chuo-Ku, Tokyo, 104-8315, Japan

a r t i c l e

i n f o

a b s t r a c t
A study was conducted to compare responses of nutrient intake, digestibility, ruminal fermentation, and N balance in goats fed high amounts of concentrate supplemented with inosine or urea. Four Japanese Saneen goats (49.2 5 kg), housed in individual metabolic cages, were blocked by weight into two pairs, and assigned inosine or urea as non-protein N (NPN) supplements to their basal diet at 0.15 g per kg body weight on a urea basis, in a 2 2 switch back experimental design. The basal diet consisted of 0.4 timothy hay, and 0.6 concentrates, and was fed to the goats as a total mixed ration (TMR). The experimental period was 13 days, including 7 days of adaptation, 5 days of sampling of feces, urine and orts, and 1 day of sampling of rumen uid. Intake of dry matter (DM), organic matter (OM), neutral detergent ber (NDF) and crude protein (CP) and digestibility in the total tract, as well as N balance, were assessed by total fecal collection. Rumen uid was sampled at 0, 1, 4 and 7 h after feeding, and analyzed for pH, ammonia and volatile fatty acids (VFA). Additionally, samples collected at 0 and 4 h after feeding were assessed for protozoal counts. Daily intakes of DM, OM, NDF and CP did not differ among treatments. Digestibility in the total tract tended (P=0.07) to be lower for NDF and was numerically lower for DM (P=0.11), OM (P=0.11), and NDF (P=0.12) in

Article history: Received 26 June 2008 Received in revised form 12 March 2009 Accepted 12 March 2009 Keywords: Goats Supplementary inosine Digestibility Ruminal fermentation High concentrate feeding

Abbreviations: ADF, acid detergent ber; BUN, blood urea N; CP, crude protein; DM, dry matter; EE, ether extract; GLM, general linear model; NDF, neutral detergent ber; NEFA, non-esteried fatty acids; NPN, non-protein N; OM, organic matter; RDP, ruminally degradable CP; TMR, total mixed ration; VFA, volatile fatty acids. Corresponding author. Tel.: +81 75 753 6358; fax: +81 75 753 6365. E-mail address: hkuma@kais.kyoto-u.ac.jp (H. Kumagai). 0377-8401/$ see front matter 2009 Published by Elsevier B.V. doi:10.1016/j.anifeedsci.2009.03.004

F. Kanyinji et al. / Animal Feed Science and Technology 152 (2009) 1220

13

goats supplemented with inosine versus urea. Ammonia concentration and ruminal pH were similar among treatments, and protozoal counts 4 h after feeding was lower (P=0.02) in goats supplemented with inosine compared to those on urea. No differences between treatments occurred in concentrations of rumen VFA (i.e., acetate, propionate, valerate, iso-acids, and total VFA) except for butyrate, which was higher (P=0.03) in goats supplemented with inosine versus urea 4 h after feeding. There were no differences in intake, absorption and retention of N between treatments. Thus, responses in nutrient intake, digestibility, ruminal fermentation and N balance in goats fed high amounts of concentrate with supplementary inosine and urea were similar. 2009 Published by Elsevier B.V.

1. Introduction Animal nutritionists have evaluated many non protein N (NPN) compounds as ruminant feeds in order to identify those with a slower release in the rumen in order to optimize efciency of their utilization by rumen microorganisms. These have included isobutylidene diurea, methenamine, acetylurea, starea, tung- and linseed coated urea, biuret, polymer-coated urea and formaldehyde treated urea (Lest et al., 2001; Galo et al., 2003; Golombeski et al., 2006). However, not all of these compounds have been as advantageous as urea because the NPN in many of them leaves the rumen without being completely converted to ammonia, thereby reducing their incorporation into microbial protein. Additionally, the ammonia formation in the rumen from these compounds, though slower than urea, is often still too fast for high capture as microbial protein (Henning et al., 1993). According to McAllan (1982), the ribose ring of a nucleic acid compound is degraded in the rumen, yielding mainly hypoxanthine that is further hydrolyzed to ammonia and carbon dioxide. That the heterocyclic purine in inosine molecule, which is a nucleic acid compound, can be hydrolyzed to ammonia suggests its potential to supply ammonia-N required by rumen microorganisms. However, because use of ammonia-N produced in the rumen is also dependent on energy availability, inclusion of readily fermentable energy sources to forage diets supplemented with an NPN source is essential to increase the efciency with which rumen microorganism capture its ammonia-N and stimulate their growth and adherence to feed particles (Caton and Dhuyvetter, 1997; Jetana et al., 2000). Additionally, some studies (Herrera-Saldana et al., 1990; Matras et al., 1991) have demonstrated increased ruminal microbial protein passage from the rumen and improved N balance when readily fermentable energy sources are supplied to the rumen. In this study, we hypothesized that use of inosine as a supplement to forages fed with readily fermentable energy sources would improve ruminal fermentation, forage utilization and N balance in goats. The objective was to compare responses of nutrient intake, digestibility, ruminal fermentation and N balance in goats fed high amounts of concentrate to supplementary inosine and urea.

2. Materials and Methods 2.1. Animals, Diets and Design Goats used in this study were managed according to guidelines of the Kyoto University Animal Ethics Committee. Four Japanese Saanen male castrated goats with an average body weight (BW) of 49.2 5 kg, and housed in individual metabolic cages, were blocked by weight into two pairs, and assigned inosine (Ajinomoto Inc., Tokyo, Japan) or urea as NPN supplement, in a 2 2 switch back experimental design. The basal diet, which consisted of 0.4 timothy hay and 0.6 concentrates, was fed as a total mixed ration (TMR). The determined chemical composition of the components of the TMR is in Table 1. The ration was fed to the goats twice daily, at 09:00 and 16:00 h, with ad libitum supply of clean drinking water.

14

F. Kanyinji et al. / Animal Feed Science and Technology 152 (2009) 1220 Table 1 Chemical composition of timothy hay and concentrate (on a dry matter basis, g/kg)a . Timothy hay DM OM EE CP ADF NDF Ash
a b

Concentrateb 881.4 0.23 934.3 0.66 32.3 0.51 167.4 0.05 92.5 0.70 352.6 0.96 65.7 0.02

919.4 0.43 945.8 0.67 17.0 0.33 87.3 0.01 381.8 0.96 685.8 0.94 54.2 0.01

Mean SE (n = 9). Composed of 0.55 rolled barley, 0.34 wheat bran, 0.06 alfalfa, 0.04 soybean meal, 0.01 sodium chloride, and 0.01 minerals.

Prior to commencement of the feeding study, the basal diet was fed to the goats for 13 days after putting them in the cages. The NPN supplements were offered to goats once daily during the morning feeding at 0.15 g per kg BW on a urea basis (16.5 g of inosine and 7.4 g of urea), to supply equal amounts of N. Each supplement was rst mixed with a small portion of the concentrate before feeding it to the goats and feeding the rest of the days ration, to ensure complete consumption. The experimental period consisted of 7 days of adaptation, 5 days of sampling of orts, feces and urine, and 1 day of sampling of rumen uid and blood, as well as measuring the BW of the goats.

2.2. Sampling and measurements Total fecal output and orts for each goat were measured daily, and an approximate 100 g representative sample of feces and orts were taken and dried in a forced-air oven at 60 C for 72 h before determining their chemical composition. Daily urine output from each animal was collected in buckets containing 100 g concentrated sulfuric acid per kg water to prevent loss of N through volatilization to ammonia, and the volume was measured before sampling 50 ml for freezing and analysis of N. On the sixth day of the sampling period, 20 ml of rumen liquid was collected from each goat using a stomach tube and a vacuum pump at 0, 1, 4 and 7 h after the morning feeding. The rumen uid samples were ltered using four layered cheesecloth to remove feed particles, and the pH of each ltrate was measured using a portable pH meter (F-72, HORIBA, Kyoto, Japan), and divided into two parts. One part of the ltrate was centrifuged at 1000 g for 10 min at 5 C, and the supernatant was used to determine the concentration of volatile fatty acids (VFA) and ammonia. The other part (not centrifuged) of the samples collected at 0 and 4 h after morning feeding, was used for protozoa counting. Also, before the afternoon feeding (7 h after morning feeding) on the same day, the BW of each goat was measured, and blood samples were collected by jugular puncture into heparinized vacuum tubes. The blood samples were centrifuged at 2500 g for 15 min at 5 C, and the supernatant of each sample was taken and frozen at 20 C before analysis. Concentrations of blood urea N (BUN), non-esteried fatty acids (NEFA), total cholesterol, albumin and glucose were analyzed using commercially available diagnostic kits (L type Wako UN, NEFA-HR, L type Wako CHO-H, Albumin-HRII and Glucose-HRII Wako; Wako Pure Chemical Industries, Ltd., Osaka, Japan). Dried samples of orts and feces were ground through a 1 mm mesh sieve, pooled per goat on a 5 day basis, and analyzed for DM (105 C for 24 h), organic matter (OM) by weight loss upon heating at 450 C for 8 h, ash (AOAC, 1990, I.D. No 942.05), ether extract [(EE) AOAC, 1990, I.D. No 920.39], N (AOAC, 1990, I.D. No 984.13), neutral detergent ber (NDF) and acid detergent ber (ADF) by procedure of Van Soest et al. (1991) without amylase, sodium sulte, or correction for residual ash. Ammonia concentration in rumen uid samples was measured following the Conway and OMalley (1942) method, and VFA concentrations by gas chromatography (GC14B, Shimadzu, Kyoto, Japan). Frozen urine samples were thawed, pooled per goat on 5 day basis, and analyzed for N by a Kjeldahl method (AOAC, 1990, I.D. No 954.01).

F. Kanyinji et al. / Animal Feed Science and Technology 152 (2009) 1220 Table 2 Effects of supplementary inosine or urea on body weight, dry matter intake and total nutrient intake. Supplement Inosine Final BW (kg) Total DM intake (kg/day) Timothy hay Concentrate Total DM intake (g/day/ BWkg0.75 ) Total nutrient intake (kg/day) OM NDF CP 52.57 1.62 0.54 1.08 83.06 1.52 0.71 0.25 Urea 53.52 1.57 0.55 1.06 79.43 1.47 0.69 0.25 SEM 1.619 0.049 0.030 0.030 1.907 0.053 0.031 0.006 P

15

0.64 0.62 0.79 0.53 0.60 0.63 0.98 0.55

2.3. Statistical analysis Statistical analysis of data was by the general linear model (GLM) Procedure of SAS (2002), with treatment and goat as factors, based on the mathematical model; Yijkl = + Tj + Pj + Ak + eijkl

where: Yijkl is the observation; is the overall population mean; Ti is the effect of treatment, Pj is the effect of period, Ak is the effect of animal, and eijkl is random error. Means were compared using Tukey test (SAS Institute, 2002), and results are reported as means. Fermentation parameters (i.e., pH, protozoa counts, ammonia concentration, acetate, propionate, butyrate, total VFA) were analyzed using repeated measures data of MIXED procedure with the model; Yijk = + Ni + Tj + (NT)ij + Aik + ijk

where: is the overall mean, Ni the xed effect of NPN supplement, Tj the xed effect of time after feeding, (NT)ij is the xed interaction effect of Nj and Tj , Aik is the random effect of animal within Ni , and ijk is the random error. Time after feeding was used as repeated measure. Differences among means with P<0.05 were accepted as representing statistically signicant differences. However, differences among means with 0.05<P<0.10 were accepted as representing tendencies to differences. 3. Results 3.1. Intake and digestion Intakes of total DM (timothy + concentrate), timothy hay, concentrate, and DM intake per metabolic weight, as well as intakes of OM, NDF and CP in goats supplemented with inosine did not differ from those fed the urea supplement (Table 2). There were no signicant differences in nutrient digestibility between goats supplemented with inosine and those that received urea supplement (Table 3), but digestibility tended (P=0.07) to be lower for NDF in goats supplemented with inosine versus urea.
Table 3 Effects of supplementary inosine or urea on apparent nutrient digestibility (g/kg). Supplement Inosine Nutrient digestibility DM OM EE CP ADF NDF 673 682 743 789 361 483 Urea 688 696 721 795 368 516 SEM 6.8 6.7 9.8 3.9 19.9 15.2 P 0.11 0.11 0.35 0.72 0.12 0.07

16

Table 4 Effects of supplementary inosine or urea on diurnal patterns of ruminal pH, protozoa counts ruminal ammonia and volatile fatty acids. Supplement Time after feeding, (h) 0 Rumen pH Protozoa counts (106 /ml) Ruminal ammonia (mmol/dl) Acetate (mmol/dl) Propionate (mmol/dl) Butyrate (mmol/dl) Valerate (mmol/dl) Iso-acids2 (mmol/dl) Total volatile fatty acids (mmol/dl)
a,b A,B

Pooled SEM 4 6.12 6.23 6.00b,A 6.85b,B 12.50 12.77 5.52 4.59 1.85 1.64 1.17ab,A 0.68ab,B 0.05 0.04 0.16 0.12 8.78 7.22 7 6.38 6.41 NC NC 9.43 9.42 4.11 3.69 1.26 1.24 0.72a 0.45a 0.03 0.03 0.28 0.29 6.63 5.62 0.054 3.583 0.920 0.217 0.088 0.064 0.004 0.018 0.356

P Supplement 0.898 0.022 0.696 0.329 0.575 0.148 0.915 0.729 0.404 Time 0.002 0.001 0.001 0.005 0.001 0.009 0.158 0.007 0.002 Supplement time 0.500 0.038 0.802 0.739 0.296 0.033 0.998 0.950 0.565 F. Kanyinji et al. / Animal Feed Science and Technology 152 (2009) 1220

1 6.34 6.25 NC1 NC 20.26 23.08 5.08 5.23 1.64 2.05 1.07b 1.08b 0.05 0.05 0.15 0.14 8.02 8.57

Inosine Urea Inosine Urea Inosine Urea Inosine Urea Inosine Urea Inosine Urea Inosine Urea Inosine Urea Inosine Urea

6.62 6.73 3.06a 3.34a 11.46 11.39 3.68 3.51 0.88 1.00 0.61a 0.68a 0.03 0.03 0.15 0.14 5.36 5.34

Means in the same row with different superscripts differ signicantly (P<0.05). Means in the same column within response parameter with different superscripts differ signicantly (P<0.05). Not counted. Includes iso-butyrate and iso-valerate.

1 2

F. Kanyinji et al. / Animal Feed Science and Technology 152 (2009) 1220 Table 5 Effects of supplementary inosine or urea on nitrogen balance. Parameter Supplement Inosine N intake (g/day) Fecal N (g/day) Urinary N (g/day) N retained (g/day) N retained as % of intake N retained as % of digested 52.2 8.4 19.0 24.8 46.1 55.1 Urea 50.8 7.9 18.1 24.9 46.8 55.6 SEM 3.44 0.24 0.89 2.83 2.69 2.64 P

17

0.44 0.48 0.92 0.59 0.74 0.71

Table 6 Effects of supplementary inosine or urea on blood metabolites. Parameter Supplement Inosine BUN (mg/dl) NEFA (mEq/l) Albumin (g/dl) Glucose (mg/dl) Total cholesterol (mg/dl) 18.97 0.23 2.87 84.67 71.00 urea 19.00 0.12 2.88 82.50 71.67 SEM 0.655 0.064 0.028 3.783 2.151 P 0.71 0.58 0.84 0.80 0.32

3.2. Ruminal fermentation Ruminal ammonia concentrations in both treatments (Table 4) were similar and generally increased after feeding, reaching peak concentration 1 h after feeding in both groups of animals. Ammonia concentrations averaged over times were 13.41 mmol/dl for goats receiving inosine and 14.16 mmol/dl for those on urea treatment. There were no signicant differences in ruminal pH between goats supplemented with inosine and those receiving urea. Ruminal pH in both treatments declined, reaching minimum pH levels of 6.12 and 6.23 for inosine and urea groups, respectively, 4 h after morning feeding. Total VFA concentration as well as proportions of acetate, propionate, valerate and the iso-acids (iso-butyrate and iso-valerate) did not differ among the treatments. However, butyrate concentration at 4 h after the morning feeding was higher (P=0.03) in goats supplemented with inosine versus urea. Similarly, the number of protozoa counted per ml of rumen uid prior to feeding was similar between treatments, but was lower (P=0.02) 4 h later in goats supplemented with inosine compared to those that received urea. 3.3. Nitrogen balance There were no differences between treatments in N intake, proportion of N retained from N intake or N digested (Table 5). Fecal and urinary excretion of N in inosine supplemented goats was also not different from those that received urea. Additionally, blood metabolites (i.e., BUN, NEFA, albumin, glucose and total cholesterol) of both groups of animals were similar (Table 6). 4. Discussion The slower rate at which inosine releases its ammonia-N in the rumen, which may synchronize better with the release of energy from ingested forages than urea is a hypothesis to explain benets of inosine versus urea feeding. Assuming their hydrolysis by rumen microorganisms to ammonia differed, levels of ruminal ammonia in goats supplemented with inosine versus urea would be expected to be different at sampling times. However, ruminal concentrations of ammonia in both groups of animals were similar at all sampling times, and peak levels occurred 1 h after the morning feeding (Table 4), suggesting a similar pattern of release from inosine and urea to ammonia. Ruminal ammonia peaking 1 h after the morning feeding is consistent with the observations of McAllan (1982), who noted that

18

F. Kanyinji et al. / Animal Feed Science and Technology 152 (2009) 1220

nucleic acids were converted to ultraltrable bases (mainly xanthine, hypoxanthine and uracil) within 1 h of infusion into the rumen of steers. The similarity in the pattern of release ammonia-N in the rumen between inosine and urea may also explain the lack of signicant differences between the two groups in terms of nutrient intake and digestibility. The impact of lower ruminal pH on ber digestion was commented on in Cheng et al. (1984), who reported that low ruminal pH prevented strong attachment of bacteria to plant cell walls, thereby resulting in lower ber digestion. In our study, ruminal pH in goats supplemented with inosine declined beyond 6.2, which is deemed as the minimum for optimal growth and functioning of cellulolytic rumen bacteria (Jetana et al., 2000). We postulated that high intakes of concentrate coupled with hydrolysis of the ribose sugar in the inosine molecule may have been responsible for the extended decline in ruminal pH in inosine supplemented goats. Obviously, rumen pH lower than 6.2 would impede growth, multiplication and fermentative activities of the ber degrading rumen microorganisms, therefore, explains the lower protozoa counts at 4 h after feeding, as well as a tendency for digestibility of NDF to be lower in this group. Many studies (Slyter et al., 1979; Erdman et al., 1986; Preston, 1995) have determined the minimum concentration of ammonia required for ruminal microbial growth and optimum rate and extent of degradation of feeds. According to Erdman et al. (1986), the concentration of ammonia required for ruminal microorganisms to achieve optimum degradation is dependent on the potential degradability of the feed material. Although 5 mmol/dl was determined to be the minimum required to support ruminal microbial growth (Slyter et al., 1979), the more degradable the feed material, the higher is the required ammonia concentration. In the present study, ruminal ammonia concentrations averaged 13.41 and 14.16 mmol/dl for inosine and urea groups, respectively, which were much higher than the minimum concentration level of 5 mmol/dl required to support ruminal microbial growth. Thus, adequate amounts of ammonia-N were available to the ruminal microorganisms in both groups of goats. In fact, it may be justiable to anticipate ammonia toxicity, and gastrointestinal disturbances in the animals with these high ruminal ammonia concentrations. Ammonia, if produced in large amounts and not utilized for microbial protein production, is absorbed predominately through the rumen wall and is transported to the liver, where it is detoxied and converted to urea which is much less toxic than ammonia. This mechanism may elevate the level of BUN. However, at high ammonia concentrations, this is not achieved and some ammonia enters general circulation. This may result in ammonia toxicity and its associated clinical signs. In our study, however, ruminal ammonia concentrations averaged over time in both treatments were much lower than the toxic levels of 50 mmol/dl proposed by Fonnesbeck et al. (1975), and BUN level was within the normal range (BUN, 10 to 20 mg/dl) proposed by Kaneko (1989). Additionally, inclusion of high amounts of concentrates consisting largely of barley grains, the starch of which is highly fermentable in the rumen, met recommendations (Ensminger et al., 1990) for using urea in ruminants. Thus, inosine at this level of supplementation coupled with high amounts of concentrates appears to be safe and is unlikely to cause ammonia toxicity. Total VFA concentration in the rumen is a balance between degradation of ingested feed materials (carbohydrates and proteins) and VFA absorbed across the ruminal wall, as well as that which passes with liquid to the abomasum (Lpez et al., 2003). Additionally, the proportions of total organic acid concentrations in the rumen are dependent on the type of carbohydrates supplemented, and the amount consumed (Heldt et al., 1999). Because the amounts of concentrate consumed by the two groups of animals in the present study were similar, concentrations of ruminal acetate, propionate, valerate and iso-acids were also similar. However, butyrate concentration was higher in animals supplemented with inosine than with urea 4 h after the morning feeding. Increased proportion of butyrate in animals supplemented with high grain diets is not surprising or without precedence. Kunkle et al. (1996) and Hall (1998) reported an increased butyrate concentration after supplementation with molasses which contain 0.60 to 0.65 sugar (predominantly glucose, fructose, and sucrose). Heldt et al. (1999) suggested that increased intake of readily fermentable carbohydrates, such as the starch in cereal grains, tends to increase the concentration of lactate in the rumen uid, particularly during the rst 3 h as an adaptive way by rumen microorganisms to handle excess carbohydrates or low ruminal pH. Thus it was speculated that under high concentrate feeding, the lower ruminal pH in goats supplemented with inosine favored production of lactate, which is associated with the high butyrate proportion as suggested by Satter and Esdale (1968).

F. Kanyinji et al. / Animal Feed Science and Technology 152 (2009) 1220

19

Lack of statistical differences in N retained as a proportion of N intake or digested between treatments, suggests that supplementation of inosine did not greatly change the animals N balance. Moreover, blood plasma metabolites were consistent with normal levels (BUN, 10 to 20 mg/dl; NEFA, < 0.45 mEq/l; albumin, 2.7 to 3.9 g/dl; glucose, 50 to 85 mg/dl; total cholesterol, 70 to 130 mg/dl) proposed by Kaneko (1989). Since concentrations of blood plasma metabolites have been commonly used to assess the plane of nutrition in ruminants (Russell and Wright, 1983; Jurez-Reyes et al., 2004), consistency of the present results with normal ranges indicate that the supplements (i.e., inosine and urea) did not change the nutritional status of the animals. 5. Conclusion When high amounts of concentrate were included in timothy hay fed to goats supplemented with inosine, nutrient intake, digestibility, ruminal fermentation pattern and N balance did not differ from those that received urea. Thus, though inosine has potential to be a valuable NPN source for ruminants, supplementing it with high amounts of concentrates may not have any advantages over urea in improving nutrient intake, digestibility, ruminal fermentation and N balance. Acknowledgements Authors are grateful to Ajinomoto Co., Inc., Tokyo, Japan, for supporting this study, and to Dr. Takeo Obitsu for helping in the analysis of data. References
AOAC. 1990. Ofcial methods of analysis, 15th edition. Association of Ofcial analytical chemists, Washington, DC, USA. Caton, J.S., Dhuyvetter, D.V., 1997. Inuence of energy supplementation on grazing ruminants: Requirements and responses. J. Anim. Sci. 75, 533542. Cheng, K.J., Stewart, C.S., Dinsdale, D., Costerton, J.W., 1984. Electron microscopy of bacteria involved in the digestion of plant cell walls. Anim. Feed Sci. Technol. 10, 93120. Conway, E.J., OMalley, E., 1942. Microdiffusion methods: Ammonia and urea using buffered absorbents (revised methods for ranges greater than 10 g N). Biochem. J. 36, 655661. Erdman, R.A., Proctor, G.H., Vandersall, J.H., 1986. Effects of rumen ammonia concentrations on in situ rate and extent of digestion of feed stuffs. J. Dairy Sci. 69, 23122320. Ensminger, M.E., Oldeld, J.E., Heinemann, W.W., 1990. Feeds and Nutrition. The Ensminger Publishing Co., Clovis, CA, USA, pp. 417423. Fonnesbeck, P.V., Kearl, L.C., Harris, L.E., 1975. Feed grade biuret as a protein replacement for ruminants. J. Anim. Sci. 40, 11501184. Galo, E., Emanuele, S.M., Sniffen, C.J., White, J.H., Knapp, J.R., 2003. Effects of a polymer-coated urea product on nitrogen metabolism in lactating Holstein dairy cattle. J. Dairy Sci. 86, 21542162. Golombeski, G.L., Kalscheur, K.F., Hippen, A.R., Schingoethe, D.J., 2006. Slow-release urea and highly fermentable sugars in diets fed to lactating dairy cows. J. Dairy Sci. 89, 43954403. Hall, M.B., 1998. Making nutritional sense of nonstructural carbohydrates. In: Proceedings of 9th Annual Florida Ruminant Nutrition Symposium, Gainesville, FL, USA, pp. 108121. Heldt, J.S., Cochran, R.C., Mathis, C.P., Woods, B.C., Olson, K.C., Titgemeyer, E.C., Nagaraja, T.G., Vanzant, E.S., Johnson, D.E., 1999. Effects of level and source of carbohydrates and level of degradable intake protein on intake and digestion of low-quality tallgrass-prairie hay by beef steers. J. Anim. Sci. 77, 28462854. Henning, P.H., Steyn, D.G., Meissner, H.H., 1993. Effect of synchronization of energy and nitrogen supply on rumen characteristics and microbial growth. J. Anim. Sci. 71, 25162528. Herrera-Saldana, R., Gomez-Alarcon, R., Torabi, M., Huber, J.T., 1990. Inuence of synchronizing protein and starch degradation in the rumen on nutrient utilization and microbial protein synthesis. J. Dairy Sci. 73, 142148. Jetana, J., Abdullah, N., Halim, R.A., Jalaludin, S., Ho, Y.W., 2000. Effects of energy and protein supplementation on microbial-N synthesis and allantoin excretion in sheep fed guinea grass. Anim. Feed Sci. Technol. 84, 167181. Jurez-Reyes, A.S., Cerrillo-Soto, M.A., Meza-Herrera, C.A., Nevrez-Carrasco, G., 2004. Diet composition, intake, plasma metabolites, reproductive and metabolic hormones during pregnancy in goats under semi-arid grazing conditions. J. Agric. Sci. 142, 697704. Kaneko, J.J., 1989. Clinical Biochemistry of Domestic Animals, 4th Ed. Academic Press, New York, NY, USA, pp. 886891. Kunkle, W.E., Moore, J.E., Balbuena, O., 1996. Self-fed molasses-based products to alter plane of nutrition. In: Proceedings of Grazing livestock nutrition conference, Custer, SD, USA, pp. 104117. Lpez, S., Hovell, F.D.D., Dijkstra, J., France, J., 2003. Effects of volatile fatty acid supply on their absorption and water kinetics in the rumen of sheep sustained by intragastric infusions. J. Anim. Sci. 81, 26092616. Lest, C.A., Titgermeyer, E.C., Drouillard, J.S., Lambert, B.D., Trater, A.M., 2001. Urea and biuret as non-protein nitrogen sources in cooked molasses blocks for steers fed praire hay. Anim. Feed Sci. Technol. 94, 115126.

20

F. Kanyinji et al. / Animal Feed Science and Technology 152 (2009) 1220

Matras, J., Bartle, S.J., Preston, R.L., 1991. Nitrogen utilization in growing lambs: effects of grain (starch) and protein sources with various rates of ruminal degradation. J. Anim. Sci. 69, 339347. McAllan, A.B., 1982. The fate of nucleic acids in ruminants. Proc. Nutr. Soc. 41, 309317. Preston, T.R. 1995. Tropical animal feeding. A manual for research workers. FAO animal production and health paper No. 126. Pub. FAO, Rome, Italy. pp 124. Russell, A.J.F., Wright, I.A., 1983. The use of blood metabolites in the determination of energy status in beef cows. J. Anim. Prod. 34, 335343. SAS Inc., 2002. SAS Users Guide: Statistics, Version 9.1 Edition. SAS Inc., Cary, NC, USA. Satter, L.D., Esdale, W.J., 1968. In vitro lactate metabolism by ruminal ingesta. Appl. Microbiol. 16, 680688. Slyter, L.L., Satter, L.D., Dinius, D.A., 1979. Effect of ruminal ammonia concentration on nitrogen utilization by steers. J. Anim Sci. 48, 906912. Van Soest, P.J., Robertson, J.B., Lewis, B.A., 1991. Methods for dietary ber, neutral detergent bre, and nonstarch polysaccharides in relation to animal nutrition. J. Dairy Sci. 74, 35833597.