Effect of Feeding Ammoniated Wheat Straw Treated with Hydrochloric Acid on Blood Biochemical Profile in Growing Male Buffalo (Bubalus bubalis) Calves

An experiment was conducted to study the effect of feeding ammoniated wheat straw treated with HCl on blood biochemical profiles in growing male buffalo (Bubalus bubalis) calves. Twenty-four growing male buffalo calves (one year of age, 88.54±3.81 kg average body weight) were divided into three groups in a completely randomized design on the basis of their body weight. Animals in all the three groups were fed on concentrate mixture. In addition, they were offered wheat straw, ammoniated wheat straw (4% urea at 50% moisture level) and HCI treated ammoniated wheat straw (4% urea at 50% moisture level and HCl added to trap 30% of the NH3 evolved) in groups I, II and III, respectively for a period of 180 days, as per Kearl (1982) for body weight gain of 500 g/d. In all diets, concentrate:roughage ratio was fixed at 50:50 and were made isonitrogenous by adjusting CP levels of conc. mixtures. Blood was collected from jugular vein of each buffalo calf at the beginning and subsequently at two months interval of experimental feeding. Due to urea-ammoniation, the CP content of wheat straw increased from 2.90 to 6.96% and addition of HCl along with urea further increased the CP content to 10.09%. In all the three groups, the mean values of plasma glucose (mg %) and serum globulin (g %), showed a decreasing trend, while the mean value of serum TP (g %), serum A:G ratio, serum urea (mg %), serum creatinine (mg %), serum ALP (KA units), SGOT (units/ml.), SGPT (units/ml), serum T3 and T4 (ng/ml) showed an increasing trend with the advancement of feeding period. The cumulative period mean values of serum TP (6.15 to 6.20 g %), serum albumin (3.07 to 3.18, g %), serum globulin (2.98 to 3.09, g %), serum A:G ratio (1.03 to 1.10), serum ALP (23.15 to 23.63, KA units), serum T3 (1.20 to 1.23 ng/ml) and serum T4 (21.33 to 21.88 ng/ml) were comparable among the groups. The cumulative period mean plasma glucose (mg %) in group III (57.28) was similar to groups I (55.31) and II (59.41), however, the cumulative period mean plasma glucose in group II was significantly (p<0.01) higher than group I. The cumulative period mean serum urea (mg %) in group III (47.34) was significantly (p<0.001) higher than group I (38.38) and II (42.24), which were statistically alike. However, the cumulative period mean serum creatinine values (mg %) in groups II (1.43) and III (1.52) were similar and were significantly (p<0.01) higher than group I (1.24). The cumulative period mean SGOT (units/ml) in groups I, II and III was 91.71, 96.04 and 96.64, respectively. Similarly the cumulative period mean SGPT (units/ml) was 19.00, 19.93 and 20.01 in groups I, II and III, respectively. The cumulative period mean values of SGOT (p<0.05) and SGPT (p<0.001) in groups II and III were similar and were significantly higher than group I. The cumulative period mean serum T3 and T4 values in groups I (1.21 and 21.81), II (1.23 and 21.42) and III (1.20 and 21.33) were comparable. From the present study it may be concluded that feeding of AWS treated with and without HCI to growing male buffalo calves for 180 days had no significant adverse effect on blood biochemical profile. (Asian-Aust. J. Anim. Sci. 2005. Vol 18, No. 2: 185-191)


INTRODUCTION
The main hindrance in utilisation of crop-residues are their low nutritive value and poor palatability.Out of the several methods tried in India and abroad to enhance the nutritive value of crop-residues, urea-ammoniation has been found to be the most promising, practicable and users friendly (Mehra et al., 1989;Khan et al., 1999;Fadal Elseed et al., 2003;Sarwar et al., 2003).But the losses of ammonia-nitrogen during urea-ammoniation of straw has been reported to be as high as 60-66% (MandaI et al., 1995;Dass et al., 2000).Various acids have been used to trap this excess ammonia (Borhami et al., 1982;Dass et al., 2001;Mehra et al., 2001) with different degree of success.But there is very little information on the effect of feeding ammoniated wheat straw treated with hydrochloric acid on the health status of the animals.Since the health status of the animals is reflected in the blood biochemical profile, an experiment was conducted to study the effect of feeding ammoniated wheat straw treated with hydrochloric acid on the blood biochemical profile of growing male buffalo (Bubalus bubalis) calves.

Preparation of concentrate mixture
To make the diet isonitrogenous three different types of concentrate mixtures namely CM 1 , CM 2 and CM 3 (Table 1) were prepared.The CP content of CM 1 , CM 2 and CM 3 was 22.07, 18.97 and 17.05%, respectively.Vitablend was added @ 25 g/100 kg concentrate mixture to meet out the vitamin A and D 3 requirement.

Ammoniation of wheat straw
Ammoniated wheat straw without HCl (type A) and with HCl (type B) were prepared.In type 'A', wheat straw was treated with 4% fertilizer grade urea at 50% moisture level and in type 'B', wheat straw was treated simultaneously with 4% fertilizer grade urea and 3.5 litres hydrochloric acid (specific gravity 1.18 and purity 35%) to trap 30% of the free ammonia evolved.In both the cases, treated wheat straw was covered with polythene sheet and was kept air tight at room temperature for 21 days, as described by Dass et al. (1984).

Animals management and feeding
Twenty four growing male buffalo (Bubalus bubalis) calves (one year age, 88.54±3.81kg average body weight) were randomly divided into three groups on the basis of their body weight.During the experiment, the animals were kept in well ventilated shed with individual feeding and watering arrangements.The animals were offered concentrate mixtures (CM 1 CM 2 and CM 3 ) along with wheat straw, ammoniated wheat straw and HCI treated ammoniated wheat straw in groups I, II and III, respectively for a period of 180 days as per Kearl (1982) to meet the nutrients requirement for 500 g gain/day.In all diets, the concentrate: roughage ratio was fixed at 50:50 and were made isonitrogenous.Roughage part was fed ad libitum to the experimental animals only after complete consumption of respective concentrate mixtures.Clean and fresh drinking water was provided ad libitum to all the experimental animals twice daily at 10 am and 3.30 pm.

Collection and processing of blood samples
Before feeding and watering of the experimental animals, about 10 ml, blood was collected from the jugular vein of each buffalo calf at the beginning and subsequently at two months interval of the experimental feeding.
Out of the 10 ml collected blood, 6 ml was taken in clean, dry test tube and was centrifuged at 3,000 rpm for 10 min for separation of serum.The remaining 4 ml blood was pipette into clean dry test tube containing a pinch of anticoagulant (mixtures of EDTA and Na 2 F) and was shaken gently for proper mixing of whole blood with the anticoagulant and was centrifuged at 3,000 rpm for 10 min for separation of plasma.The respective serum and plasma samples were collected in plastic vials and were preserved in deep freezer (-20°C) till the completion of biochemical analysis.During the entire period of collection and processing, precautions were taken to avoid haemolysis of the blood samples.

Analytical techniques
Plasma glucose was estimated by the glucose oxidase (GOD) and peroxidase (POD) method, as described by Henry (1963).Serum total protein and albumin were determined by the modified Biuret and Dumas method as described by Dumas et al. (1971).Serum globulin was calculated by subtracting serum albumin from serum total protein.The serum A:G ratio was calculated by dividing the values of serum albumin by serum globulin.Diacetyl mono oxime (DAM) method as described by Wybenga et al. (1971) was followed to estimate the concentration of serum urea.The concentration of creatinine in serum was determined by alkaline picrate method as described by Bonses and Tausskay (1945).The serum alkaline phosphatase activity was estimated as per the method of Kind and King (1954).SGOT and SGPT were determined by the method suggested by Reitman and Frankel (1957).The concentrations of T 3 and T 4 were estimated by following the method of Bhandarkar and Pillai (1982).

Statistical analysis
Data were subjected to the statistical analysis by following the standard procedures (Snedecor and Cochran, 1980) by statistical software package (SPSS) for the test of significance.

RESULTS AND DISCUSSION
The physical and chemical composition of the various feeds are presented in Tables 1 and 2, respectively.Due to urea-ammoniation, the CP content of wheat straw increased from 2.90 to 6.96%.This might be due to the binding of ammonia released from the hydrolysis of urea inside the intermolecular spaces of wheat straw (Dass et al., 1984;Reddy et al., 1989).However, addition of HCl along with urea during urea-ammoniation further increased the CP content to 10.09%.This might be due to the trapping of the excess ammonia by forming ammonium chloride (Dass et al., 2001;Nair et al., 2002).It was evident from Table 2 that there was some increase in cellulose content in ureaammoniated wheat straw, which further increased due to combined effect of HCl and urea during urea-ammoniation.The hemicellulose and soluble residues contents decreased due to urea ammoniation and addition of HCI during urea ammoniation resulted in further reduction.At the start of the experiment, the mean body weight (kg) of buffalo calves in groups I, II and III was 88.3, 88.9 and 88.5, respectively and were comparable among the groups.At the end of the experimental period of 180 days, the mean body weight (kg) was 179.1, 197.8 and 172.0 in groups I, II and III, respectively and were also comparable among the groups.The gain in body weight (kg) during the period of 180 days was 90.9, 109.0 and 83.5 in groups I, II and III, respectively.The corresponding values for ADG (g) was 504.86, 605.56 and 463.89 in groups I, II and III, respectively.The mean values of BW gain (kg) and ADG (g) in group III were significantly (p<0.05)lower than group II and the mean values in both groups were simi1ar to group I.The fortnightly body weight changes in buffalo calves in all three groups are presented in Figure 1.In all the three groups, there was an increase in BW with the advancement of feeding period and age.
The results of the blood biochemical profile in buffalo calves are presented in Table 3 and depicted in Figure 2.There was a lowering trend in the level of plasma glucose with advancement of age.These observations are depiction of higher plasma glucose in young animals which is tending to achieve its normal adult stage level.Similar period wise decreasing trend was also reported by earlier workers in buffalo calves (Puri et al., 1983;Helal et al., 1999) and crossbred calves (Sikka et al., 1994;Pattanaik, 1997).In group II, the values at 6 months post feeding (53.58 mg %) and the cumulative period mean value (59.41 mg %) remained significantly (p<0.01)higher than group I (45.13 and 55.13 mg %).This might be due to the higher OM intake in group II than group I. (96.45 g/kg W 0.75 vs. 89.99g/kg W 0.75 ).However, the similar cumulative period mean plasma glucose in group III (57.28 mg %) with group II (59.41 mg %) ascribed no impact of addition of HCl during ammoniatoin.The mean serum TP values observed in this experiment are higher than values reported by Sil (1992) and slightly lower than values (7.75 g %) reported by Singh (1991) in buffalo calves fed on AWS based ration.However, the values obtained in this experiment were with in normal range of 5.7 to 8.1 g/dl and were comparable to those values observed by Nawaz and Siddiqui (1974).The gradual increasing trend in value of serum TP in all three groups might be due to the effect on their growth.This is also supported by the findings of Afzal and Anjum (1982) in buffalo calves.The comparable serum TP at 0, 2, 4 and 6 months post feeding and the comparable cumulative period mean values (6.16, 6.15 and 86.20 g %) among the groups revealed that the protein available from AWS and HCl-AWS for absorption at intestinal level were similar to control diet.Similarly, no effect of sources of proteins on serum TP was recorded by earlier workers (Giri and Dass, 1993;Tiwari et al., 2001).The mean values of serum albumin were parallel with earlier findings (Verma et al., 1975).The significant (p<0.001)gradual increase in serum albumin values in all three groups might be due to their body weight gain.The mean values of serum globulin are similar to the values reported earlier (Sahoo, 1994;Pattanaik, 1997).The significant decreasing trend of serum globulin level in group II at 4 months post feeding and onwards might be due to the cyto-toxic effect of urea in AWS on lymphoid organs.However, the cumulative period mean serum globulin values (3.09, 2.98 and 3.03 g %) among the groups were with in normal range.The significant (p<0.001)increasing trend of serum A:G ratio at  4 months post feeding and onwards in group II could be attributed to the increasing and decreasing trend of serum albumin and globulin, respectively, and may be due to the cyto-toxic effect of urea of AWS on lymphoid organs.But the simultaneous addition of HCl during urea ammoniation of WS did not show any significant effect on the cyto-toxic property of AWS, as has been reflected by significant (p<0.001)increase in A:G ratio at 4 months post feeding and onwards in group III.However, the comparable cumu1ative period mean A:G ratio value among the groups were with in normal range and are supported by the findings of Ahuja et al. (1977).There was a significant (p<0.001)rising trend in mean serum urea values in all three groups.Similar trend was also noticed by Giri and Dass (1993) in buffalo calves.The serum urea level at 4 months post feeding and onwards was significantly (p<0.05)higher in group III than other two groups.The cumulative period mean serum urea (mg %) in group III was significantly (p<0.01)more than group I.This upswing, may presumably be either due to the more amount of NPN of the HCI treated AWS or the changes in vital organs like liver and kidney.Similarly, on supplementation of NPN compounds to the basal diet, high level of BUN was reported by earlier workers (Nolan and Leng, 1972;Dass et al., 1996;Bakshi et al., 1997).
Values of blood enzymes and hormones like T 3 and T 4 are given in Table 4 and depicted in Figure 3.The gradual increasing trend in mean values of serum ALP with advancement of age might be due to the normal physiological effect of their growth in all three groups.The cumulative period mean serum ALP values were comparable among three groups and were within normal range.Ahuja et al. (1977) also did not find any change in scrum ALP concentration in buffalo bulls fed on urea for a prolonged period.The serum level of SGOT and SGPT were well with in normal range.But when the values were compared on the basis of the effect of ammoniation, then the mean values of SGOT in groups II (p<0.05) and III (p<0.01) and the mean values of SGPT in groups I and II (p<0.001)showed a significant increasing trend with advancement of feeding.Similarly, the cumulative period mean values of SGOT (p<0.05) and SGPT (p<0.001) were significantly higher in groups II and III than group I.These higher values might be due to some pathological alterations in vital organs.The serum creatinine level at 4 months post feeding (p<0.05) and the cumulative period mean serum creatinine (mg %) values (p<0.001) in groups II (1.43) and III (1.52) were comparable and were significantly higher than group I (1.24).The changes might be due to the renal and muscular damage.However, the values found in the present study were well within normal range of 1.0 to 2.7 mg/dl, reported for the domestic animals (Kaneko, 1980).The mean values of serum T 3 and T 4 were well with in the normal physiological range.There was an increasing trend in both serum T 3 and T 4 values in all three groups.The cumulative period mean values of serum T 3 (1.21,1.23 and 1.20 ng/ml) and T 4 (21.81,21.42 and 21.33 ng/ml) in groups I, II and III were similar in all three groups, indicating no adverse effect of AWS and HCI-AWS on basal metabolic rate of buffalo calves.

CONCLUSION
From the study, it may be concluded that feeding of ammoniated wheat straw treated with and without HCl to growing male buffalo calves for 180 days had no significant adverse effect on any blood biochemical constituents.

Figure 2 .
Figure 2. Effect of feeding ammoniated and ammoniated plus hydrochloric acid treated wheat straw on blood biochemical constituents.

Figure 3 .
Figure 3.Effect of feeding ammoniated and hydrochloric acid treated wheat straw on some blood enzymes, T 3 and T 4 concentration in buffalo calves.

Table 3 .
Blood biochemical profile in buffalo calves a, b, c, d Means with different superscripts in a row differ significantly.x,y, z Means with different superscripts in a column differ significantly.

Table 4 .
Blood enzyme and hormone concentration in buffalo calves a, b, c, d Means with different superscripts in a row differ significantly.x,y, z Means with different superscripts in a column differ significantly.