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Effects of isoacids, urea and sulfur on ruminal fermentation in sheep fed pineapple tops
Small Ruminant Research, 6 ( 1991 ) 49-54
49
Elsevier Science Publishers B.V., Amsterdam
Effects of isoacids, urea and sulfur on ruminal fermentation in sheep fed pineapple tops M.E. Quispe a, H. Barradas b and R.M. Cooka'l aDepartment of Animal Science, Michigan State University, East Lansing, M148824 USA bNational Institute for Forestry, Agriculture and Livestock Research, Paso del Toro, Veracruz, Mexico (Accepted 9 January 1991 )
ABSTRACT Quispe, M.E., Barradas, H. and Cook, R.M., 1991. Effects of isoacids, urea and sulfur on ruminal fermentation in sheep fed pineapple tops. Small Rumin. Res., 6: 49-54. Eight Tabasco rams were used in a 2 3 factorial crossover experiment conducted in two 4 × 4 quasiLatin squares to study effects of supplementary isoacids, urea and sulfur on ruminal fermentation of pineapple tops. 'Isoacids' (ISO) refers to an equal-weight mixture of isobutyrate, 2-methylbutyrate, isovalerate and valerate. Treatments in g kg BW- l d a y - ~were: ISO, 0.07 and 0.14; urea, 0 and 0.43; sulfur (S), 0 and 0.086. After each weekly experimental period, the rate of ruminal acetate production was taken as a measure of ruminal fermentation. There was significant interaction only between ISO and S. Acetate production ranged from 0.17 to 0.44 tool h - i per sheep for the eight treatment combinations. Acetate was increased ( P < 0.05 ) by 0.17 mol h - t per sheep, or 81%, when ISO were fed at the high level in diets supplemented with S. When S was added to diets containing a high level of ISO, acetate production increased ( P < 0.01 ) by 0.14 mol h - ~per sheep, or 56%. When urea was added to high ISO and S, acetate production was 33% higher (0.33 vs. 0.44 tool h - ~per sheep ). Optimum N: S ratio was 5: 1, and this apparently was more important than absolute concentration of rumen ammonia. Ruminal fermentation of pineapple tops appears to be improved by supplementing the diet with 0.14 g ISO, 0.43 g urea and 0.086 g S per kg BW of sheep.
INTRODUCTION
Crop residues are an important source of feed for ruminants, especially in developing countries (Muller, 1978 ). For example, in Mexico 350 000 metric tons of pineapple fruit were processed in six states in 1985 (FAO, 1987). World production was 9.5 million metric tons in 1985. Processing plants utilize only 15 to 25% of the fruit, the rest is waste. Pineapple tops are 17% of the residue from the pineapple canning industry and frequently used fodder for livestock. They are low in crude protein (CP, 7% ) and high in fiber ( 25% ) (Muller, 1978). ~To w h o m correspondence should be addressed.
0921-4488/91/$03.50
© 1991 Elsevier Science Publishers B.V. All rights reserved.
50
M.E. QUISPE ET AL.
Isoacids (ISO) are either essential or stimulatory for growth of several rumen cellulolytic bacteria (Allison, 1969). Ammonia is the main form of nitrogen used by rumen bacteria (Bryant, 1973). Sulfur (S) is required for growth of rumen bacteria (Bray and Till, 1975), but its interaction with ISO and urea has not been studied. The objective of the work reported herein was to determine the effects of various combinations of ISO, urea and S on the ruminal fermentation of a high-fiber crop residue. Pineapple tops were chosen because they are high in fiber and are an important crop residue. Ruminal acetate production was taken as an indicator of fermentation parameters. Utilization of other by-products and crop residues may benefit from this information. MATERIALS AND METHODS
The experiment was conducted at the La Posta Experiment Station, Paso del Toro, Veracruz, Mexico. Eight yearling Tabasco sheep were sorted by weight into two groups of four animals. Each sheep in group I weighed 25 kg and each sheep in group 2 weighed 35 kg. Sheep in groups I and 2 were each fed 1215 and 1700 g/day of pineapple tops and 20 and 27 g/day of minerals, respectively. Pineapple tops were 6.6 and 0.13% CP and S, respectively. All sheep had ruminal fistulae and were kept in individual metabolism cages. Pineapple tops were chopped in a silage cutter, air-dried to 20% moisture and finally ground for formulating rations. Isoacids (equal weights of isobutyrate, 2-methylbutyrate, isovalerate and valerate) were mixed into rations at two levels to provide 0.07 and 0.14 g kg BW-1 day-1. In order to achieve ruminal NH3-N levels of approximately 5 and 15 mg/dl, pineapple tops were fed without or with urea at 0.43 g kg B W - 1 d a y - 1. Similarly, either 0 or 0.086 g S kg B W - 1 d a y - 1 was provided by adding elemental sulfur. The statistical design was a 3-factor (23) crossover in two 4 × 4 quasi-Latin squares (Gill, 1978; A X B × C interaction confounded with squares). Double blocking criteria were animals and time (non-random repeated measurements were obtained from each subject assigned to a sequence of treatment combinations). The animals were randomly assigned to rows of one square, corresponding to a predetermined sequence of treatment combinations (Gill, 1978). The three factors were isoacids, urea and sulfur, each at two levels. Overall treatment differences were determined by ANOVA (Gill, 1978 ) and specific differences among treatment means within each interaction by Bonferroni t-tests (Gill, 1978 ). Experimental periods were 1 week. Acetate production was measured on the last day of each period. Ruminal acetate production rates were determined using the single injection method described by Cook (1966). Each animal received a tracer dose of 100/zCi Na-acetate-1~4C and 10 g of polyethylene glycol (m.w. 4000) 30 min after feeding. Serial rumen fluid samples were taken every 15 rain for 3 h. Ruminal fluid volume
RUMINAL FERMENTATION IN SHEEP
51
was determined according to Smith (1959). Ruminal volatile fatty acids (VFA) were analyzed by gas chromatography according to Felix et al. (1980). For measuring the specific radioactivity of acetate, 3 ml of strained ruminal fluid was centrifuged at 39 000 g for 20 min. Two ml of supernatant was deproteinized with Ba (OH)2-ZnSO4. The filtrate was made alkaline with KOH and freeze-dried. Samples were dissolved in 1% H3PO4 and adjusted to pH 2 with 50% H2SO 4. For isolation of [ ~4C] acetate a 50-#1 aliquot was subjected to high-pressure liquid chromatography using a C8 (4.6 m m × 2 5 cm) LiChromosorb, 10/lm Hibar II analytical column (E.M. Reagent # A 0 0 / 0 4 ) . A 4 . 6 × 7 - m m perisorb RP-8 guard column (E.M. Reagent #910436-94) was fitted in front of the analytical column. The mobile phase was 1% H3PO 4 at a flow rate of 2.0 m l / m i n requiring 1 500 to 2 000 PSI. After each sample, the guard column was washed with 10 ml 65% aqueous acetonitrile, 10 ml deionized water and 10 ml 1% ( v / v ) phosphoric acid. Acetate in the eluate was determined by measuring light absorption at 214 nm and standard acetate solutions with a chart recorder. Fractions (0.8 ml) from the column were collected in 7 ml scintillation vials, 5 ml scintillation cocktail was added and radioactivity in the vials measured with a liquid scintillation spectrometer. Ruminal H2S was determined with a sensing electrode (Lazar, model GS-136) and digital (Lazar) potentiometer. Ruminal NH3-N was determined with an Orion (model 95-10 ) ammonia-sensing electrode and potentiometer. RESULTS AND DISCUSSION
Two different ruminal levels of H2S and N H a - N were achieved by supplementing pineapple tops with elemental sulfur and urea (Table 1 ). Ruminal H2S averaged 7 to 9/tl/ml when S was added compared to 0.7 to 4/tg/ml with no S. Similarly, ruminal NH3-N was 13 to 16 mg/1 when urea was added compared to 5 to 7 mg/dl without urea. Ruminal concentrations of ISO were not increased with high levels of ISO. This may be due to rapid utilization in the rumen. The ruminal N: S ratio (Table 1 ) reflected the dietary N : S ratio with the exception of the treatment At.BLCL. For this treatment the wide ruminal ratio was due to unusually low H2S concentrations in two sheep, for which there was no apparent explanation. For acetate production, interactions were only significant between ISO and S. Therefore, the Bonferroni t-test (Gill, 1978) was used to test differences among treatment means within each level of ISO or S. These results show that acetate production was increased ( P < 0.05) with S supplementation. Similarly, acetate production was higher when adding S and high ISO ( P < 0.01 ). Acetate production was higher with a dietary N: S ratio of 5 : 1 relative to 3: I, 8: 1 or 12: 1 (Fig. 1 ). This ratio is narrower than the ratio usually considered adequate for sheep (Bray and Till, 1975 ). The dietary N: S ratio of 5: 1
0.7 6.7 90 8 0.21
(ALBLCL)
Treatmentsi
4.0 14.8 36 12 0.22 c
(AHBHCL) 7.8 7.4 9 3 0.33 a
(AHBLCH) 7.3 12.8 18 5 0.25 b
(ALBHCt-I) 7.0 7.0 10 3 0.17 b
(ALBLCH)
2.9 15.9 54 12 0.27
(ALBHCL)
3.5 4.8 14 8 0.28 c
(ArIBLCL)
9.1 13.9 16 5 0.44 a
(AnB,C~)
'A, B, C are levels ofisoacids, urea and sulfur, respectively. L, H are low and high levels. ZSE for H2S and NH3-N is 0.6 and 1.2, respectively. ab~l'wo pooled treatment contrasts were different: a vs b ( P < 0.05), positive effect of isoacids when sulfur was present, and a vs c ( P < 0.1 ), positive effect of sulfur when isoacids were high. All other treatment comparisons were not different ( P > 0. l ).
H2S (/*g/ml) 2 NH3-N ( m g / d l ) 2 Rumen ( NH3-N: H2S ) Ration ( N : S ) Acetate (mol h - 1 per sheep)
Item
Effects of interaction of isoacids, urea and sulfur, each at two levels, on ruminal acetate production, H2S and N H 3 - N in sheep fed pineapple tops
TABLE 1
> t-"
m
.g
RUMINALFERMENTATIONIN SHEEP
53
0.4 Optimum N:S rotio
"|
0.3
L o
0.2
~"
0.1
0.0 1
2
3
4
5
6
7
8
9
10
11
12
N:S RATIO Fig. 1. Effects of N: S ratios in the ration on mean rumen acetate production in sheep (SE=0.05).
resulted in ruminal N H 3 - N to H2S ratio of 17: 1, which is close to the N:S ratios for ruminal bacteria of approximately 13 : 1 proposed by Bray and Till (1975). Acetate production was more correlated to N:S ratios (r2= 14.6%) than to ruminal N H 3 - N concentration (r2= 3.2%). There was a trend towards more acetate production as ruminal N H 3 - N increased, but differences were not significant. In general, acetate production was 42% greater when higher levels of ISO and S were added to the feed ration. Adding urea to diets containing high levels of ISO and S increased acetate production by another 33%. It is concluded that 0.14 g of isoacids, 0.43 g urea and 0.086 g S kg BW -~ gave the maximum fermentation of pineapple tops. These treatments gave a dietary N:S ratio of 5: 1. ACKNOWLEDGEMENT
Research was funded under NSF Grant INT77-0636, CONACYT Grant 1396 and by the Michigan Agricultural Experiment Station.
REFERENCES
Allison, M.J., 1969. Biosynthesis of amino acids by ruminal micro-organisms. J. Anim. Sci., 29: 797-807. Bray, A.C. and Till, A.R., 1975. Metabolism of sulfur in the gastro-intestinal tract. In: I.W.
54
M.E.QUISPEETAL.
McDonald and A.I.C. Warner (Editors), Digestion and Metabolism in the Ruminant. University of New England Publishing Unit, Armidale, N.S.W. 2351, Australia, p. 243-260. Bryant, M.P., 1973. Nutritional requirements of ceUulolyticbacteria. Fed. Proc., 32:1809-1813. Cook, R.M., 1966. Use of t4C to study utilization of substrates in ruminants. J. Dairy Sci., 49: 1018-1023. FAO, 1987. 1948-1985 World Crop and Livestock Statistics. Area, Yield and Production of Crops; Production of Livestock Products. FAO Processed Statistics Series, Vol. 1. Food and Agriculture Organization of the United Nations, Rome, p. 565. Felix, A., Cook, R.M. and Huber, J., 1980. Isoacids and urea as a protein supplement for lactating cows fed corn silage. J. Dairy Sci., 63:1098-1103. Gill, J.L., 1978. Design and Analysis of Experiments in the Animal and Medical Sciences, Vols. 1, 2, 3. Iowa State University Press, Ames, Iowa. Muller, Z.O., 1978. Feeding potential of pineapple waste for cattle. World Anim. Rev., 25: 2536. Smith, R.H., 1959. The development and function of the rumen in milk-fed calves. J. Agric. Sci., 52: 72-78.
49
Elsevier Science Publishers B.V., Amsterdam
Effects of isoacids, urea and sulfur on ruminal fermentation in sheep fed pineapple tops M.E. Quispe a, H. Barradas b and R.M. Cooka'l aDepartment of Animal Science, Michigan State University, East Lansing, M148824 USA bNational Institute for Forestry, Agriculture and Livestock Research, Paso del Toro, Veracruz, Mexico (Accepted 9 January 1991 )
ABSTRACT Quispe, M.E., Barradas, H. and Cook, R.M., 1991. Effects of isoacids, urea and sulfur on ruminal fermentation in sheep fed pineapple tops. Small Rumin. Res., 6: 49-54. Eight Tabasco rams were used in a 2 3 factorial crossover experiment conducted in two 4 × 4 quasiLatin squares to study effects of supplementary isoacids, urea and sulfur on ruminal fermentation of pineapple tops. 'Isoacids' (ISO) refers to an equal-weight mixture of isobutyrate, 2-methylbutyrate, isovalerate and valerate. Treatments in g kg BW- l d a y - ~were: ISO, 0.07 and 0.14; urea, 0 and 0.43; sulfur (S), 0 and 0.086. After each weekly experimental period, the rate of ruminal acetate production was taken as a measure of ruminal fermentation. There was significant interaction only between ISO and S. Acetate production ranged from 0.17 to 0.44 tool h - i per sheep for the eight treatment combinations. Acetate was increased ( P < 0.05 ) by 0.17 mol h - t per sheep, or 81%, when ISO were fed at the high level in diets supplemented with S. When S was added to diets containing a high level of ISO, acetate production increased ( P < 0.01 ) by 0.14 mol h - ~per sheep, or 56%. When urea was added to high ISO and S, acetate production was 33% higher (0.33 vs. 0.44 tool h - ~per sheep ). Optimum N: S ratio was 5: 1, and this apparently was more important than absolute concentration of rumen ammonia. Ruminal fermentation of pineapple tops appears to be improved by supplementing the diet with 0.14 g ISO, 0.43 g urea and 0.086 g S per kg BW of sheep.
INTRODUCTION
Crop residues are an important source of feed for ruminants, especially in developing countries (Muller, 1978 ). For example, in Mexico 350 000 metric tons of pineapple fruit were processed in six states in 1985 (FAO, 1987). World production was 9.5 million metric tons in 1985. Processing plants utilize only 15 to 25% of the fruit, the rest is waste. Pineapple tops are 17% of the residue from the pineapple canning industry and frequently used fodder for livestock. They are low in crude protein (CP, 7% ) and high in fiber ( 25% ) (Muller, 1978). ~To w h o m correspondence should be addressed.
0921-4488/91/$03.50
© 1991 Elsevier Science Publishers B.V. All rights reserved.
50
M.E. QUISPE ET AL.
Isoacids (ISO) are either essential or stimulatory for growth of several rumen cellulolytic bacteria (Allison, 1969). Ammonia is the main form of nitrogen used by rumen bacteria (Bryant, 1973). Sulfur (S) is required for growth of rumen bacteria (Bray and Till, 1975), but its interaction with ISO and urea has not been studied. The objective of the work reported herein was to determine the effects of various combinations of ISO, urea and S on the ruminal fermentation of a high-fiber crop residue. Pineapple tops were chosen because they are high in fiber and are an important crop residue. Ruminal acetate production was taken as an indicator of fermentation parameters. Utilization of other by-products and crop residues may benefit from this information. MATERIALS AND METHODS
The experiment was conducted at the La Posta Experiment Station, Paso del Toro, Veracruz, Mexico. Eight yearling Tabasco sheep were sorted by weight into two groups of four animals. Each sheep in group I weighed 25 kg and each sheep in group 2 weighed 35 kg. Sheep in groups I and 2 were each fed 1215 and 1700 g/day of pineapple tops and 20 and 27 g/day of minerals, respectively. Pineapple tops were 6.6 and 0.13% CP and S, respectively. All sheep had ruminal fistulae and were kept in individual metabolism cages. Pineapple tops were chopped in a silage cutter, air-dried to 20% moisture and finally ground for formulating rations. Isoacids (equal weights of isobutyrate, 2-methylbutyrate, isovalerate and valerate) were mixed into rations at two levels to provide 0.07 and 0.14 g kg BW-1 day-1. In order to achieve ruminal NH3-N levels of approximately 5 and 15 mg/dl, pineapple tops were fed without or with urea at 0.43 g kg B W - 1 d a y - 1. Similarly, either 0 or 0.086 g S kg B W - 1 d a y - 1 was provided by adding elemental sulfur. The statistical design was a 3-factor (23) crossover in two 4 × 4 quasi-Latin squares (Gill, 1978; A X B × C interaction confounded with squares). Double blocking criteria were animals and time (non-random repeated measurements were obtained from each subject assigned to a sequence of treatment combinations). The animals were randomly assigned to rows of one square, corresponding to a predetermined sequence of treatment combinations (Gill, 1978). The three factors were isoacids, urea and sulfur, each at two levels. Overall treatment differences were determined by ANOVA (Gill, 1978 ) and specific differences among treatment means within each interaction by Bonferroni t-tests (Gill, 1978 ). Experimental periods were 1 week. Acetate production was measured on the last day of each period. Ruminal acetate production rates were determined using the single injection method described by Cook (1966). Each animal received a tracer dose of 100/zCi Na-acetate-1~4C and 10 g of polyethylene glycol (m.w. 4000) 30 min after feeding. Serial rumen fluid samples were taken every 15 rain for 3 h. Ruminal fluid volume
RUMINAL FERMENTATION IN SHEEP
51
was determined according to Smith (1959). Ruminal volatile fatty acids (VFA) were analyzed by gas chromatography according to Felix et al. (1980). For measuring the specific radioactivity of acetate, 3 ml of strained ruminal fluid was centrifuged at 39 000 g for 20 min. Two ml of supernatant was deproteinized with Ba (OH)2-ZnSO4. The filtrate was made alkaline with KOH and freeze-dried. Samples were dissolved in 1% H3PO4 and adjusted to pH 2 with 50% H2SO 4. For isolation of [ ~4C] acetate a 50-#1 aliquot was subjected to high-pressure liquid chromatography using a C8 (4.6 m m × 2 5 cm) LiChromosorb, 10/lm Hibar II analytical column (E.M. Reagent # A 0 0 / 0 4 ) . A 4 . 6 × 7 - m m perisorb RP-8 guard column (E.M. Reagent #910436-94) was fitted in front of the analytical column. The mobile phase was 1% H3PO 4 at a flow rate of 2.0 m l / m i n requiring 1 500 to 2 000 PSI. After each sample, the guard column was washed with 10 ml 65% aqueous acetonitrile, 10 ml deionized water and 10 ml 1% ( v / v ) phosphoric acid. Acetate in the eluate was determined by measuring light absorption at 214 nm and standard acetate solutions with a chart recorder. Fractions (0.8 ml) from the column were collected in 7 ml scintillation vials, 5 ml scintillation cocktail was added and radioactivity in the vials measured with a liquid scintillation spectrometer. Ruminal H2S was determined with a sensing electrode (Lazar, model GS-136) and digital (Lazar) potentiometer. Ruminal NH3-N was determined with an Orion (model 95-10 ) ammonia-sensing electrode and potentiometer. RESULTS AND DISCUSSION
Two different ruminal levels of H2S and N H a - N were achieved by supplementing pineapple tops with elemental sulfur and urea (Table 1 ). Ruminal H2S averaged 7 to 9/tl/ml when S was added compared to 0.7 to 4/tg/ml with no S. Similarly, ruminal NH3-N was 13 to 16 mg/1 when urea was added compared to 5 to 7 mg/dl without urea. Ruminal concentrations of ISO were not increased with high levels of ISO. This may be due to rapid utilization in the rumen. The ruminal N: S ratio (Table 1 ) reflected the dietary N : S ratio with the exception of the treatment At.BLCL. For this treatment the wide ruminal ratio was due to unusually low H2S concentrations in two sheep, for which there was no apparent explanation. For acetate production, interactions were only significant between ISO and S. Therefore, the Bonferroni t-test (Gill, 1978) was used to test differences among treatment means within each level of ISO or S. These results show that acetate production was increased ( P < 0.05) with S supplementation. Similarly, acetate production was higher when adding S and high ISO ( P < 0.01 ). Acetate production was higher with a dietary N: S ratio of 5 : 1 relative to 3: I, 8: 1 or 12: 1 (Fig. 1 ). This ratio is narrower than the ratio usually considered adequate for sheep (Bray and Till, 1975 ). The dietary N: S ratio of 5: 1
0.7 6.7 90 8 0.21
(ALBLCL)
Treatmentsi
4.0 14.8 36 12 0.22 c
(AHBHCL) 7.8 7.4 9 3 0.33 a
(AHBLCH) 7.3 12.8 18 5 0.25 b
(ALBHCt-I) 7.0 7.0 10 3 0.17 b
(ALBLCH)
2.9 15.9 54 12 0.27
(ALBHCL)
3.5 4.8 14 8 0.28 c
(ArIBLCL)
9.1 13.9 16 5 0.44 a
(AnB,C~)
'A, B, C are levels ofisoacids, urea and sulfur, respectively. L, H are low and high levels. ZSE for H2S and NH3-N is 0.6 and 1.2, respectively. ab~l'wo pooled treatment contrasts were different: a vs b ( P < 0.05), positive effect of isoacids when sulfur was present, and a vs c ( P < 0.1 ), positive effect of sulfur when isoacids were high. All other treatment comparisons were not different ( P > 0. l ).
H2S (/*g/ml) 2 NH3-N ( m g / d l ) 2 Rumen ( NH3-N: H2S ) Ration ( N : S ) Acetate (mol h - 1 per sheep)
Item
Effects of interaction of isoacids, urea and sulfur, each at two levels, on ruminal acetate production, H2S and N H 3 - N in sheep fed pineapple tops
TABLE 1
> t-"
m
.g
RUMINALFERMENTATIONIN SHEEP
53
0.4 Optimum N:S rotio
"|
0.3
L o
0.2
~"
0.1
0.0 1
2
3
4
5
6
7
8
9
10
11
12
N:S RATIO Fig. 1. Effects of N: S ratios in the ration on mean rumen acetate production in sheep (SE=0.05).
resulted in ruminal N H 3 - N to H2S ratio of 17: 1, which is close to the N:S ratios for ruminal bacteria of approximately 13 : 1 proposed by Bray and Till (1975). Acetate production was more correlated to N:S ratios (r2= 14.6%) than to ruminal N H 3 - N concentration (r2= 3.2%). There was a trend towards more acetate production as ruminal N H 3 - N increased, but differences were not significant. In general, acetate production was 42% greater when higher levels of ISO and S were added to the feed ration. Adding urea to diets containing high levels of ISO and S increased acetate production by another 33%. It is concluded that 0.14 g of isoacids, 0.43 g urea and 0.086 g S kg BW -~ gave the maximum fermentation of pineapple tops. These treatments gave a dietary N:S ratio of 5: 1. ACKNOWLEDGEMENT
Research was funded under NSF Grant INT77-0636, CONACYT Grant 1396 and by the Michigan Agricultural Experiment Station.
REFERENCES
Allison, M.J., 1969. Biosynthesis of amino acids by ruminal micro-organisms. J. Anim. Sci., 29: 797-807. Bray, A.C. and Till, A.R., 1975. Metabolism of sulfur in the gastro-intestinal tract. In: I.W.
54
M.E.QUISPEETAL.
McDonald and A.I.C. Warner (Editors), Digestion and Metabolism in the Ruminant. University of New England Publishing Unit, Armidale, N.S.W. 2351, Australia, p. 243-260. Bryant, M.P., 1973. Nutritional requirements of ceUulolyticbacteria. Fed. Proc., 32:1809-1813. Cook, R.M., 1966. Use of t4C to study utilization of substrates in ruminants. J. Dairy Sci., 49: 1018-1023. FAO, 1987. 1948-1985 World Crop and Livestock Statistics. Area, Yield and Production of Crops; Production of Livestock Products. FAO Processed Statistics Series, Vol. 1. Food and Agriculture Organization of the United Nations, Rome, p. 565. Felix, A., Cook, R.M. and Huber, J., 1980. Isoacids and urea as a protein supplement for lactating cows fed corn silage. J. Dairy Sci., 63:1098-1103. Gill, J.L., 1978. Design and Analysis of Experiments in the Animal and Medical Sciences, Vols. 1, 2, 3. Iowa State University Press, Ames, Iowa. Muller, Z.O., 1978. Feeding potential of pineapple waste for cattle. World Anim. Rev., 25: 2536. Smith, R.H., 1959. The development and function of the rumen in milk-fed calves. J. Agric. Sci., 52: 72-78.