- Email: [email protected]
Effect of Dietary Acetic Acid Levels on Protein and Energy Utilization in Chicks1
Effect of Dietary Acetic Acid Levels on Protein and Energy Utilization in Chicks MITSUHIRO FURUSE and JUN-ICHI OKUMURA Laboratory of Animal Nutrition, School of Agriculture, Nagoya University, Chikusa-ku, Nagoya 464-01, Japan (Received for publication February 24, 1988) ABSTRACT Chicks were given diets containing varying amounts of dietary acetic acid (0,12.7,25.4,38.1, 50.8, and 63.5 g/kg diet) under ad libitum feeding in Experiment 1 and under equalized feeding (average feed intake of Experiment 1) conditions in Experiment 2. In Experiment 1, values for body weight gain (g/10 days), feed intake (g/10 days), fat retention (g/10 days), and energy retention (kJ/10 days) were linearly lower as dietary acetic acid was increased. In Experiment 2, values for body weight gain, protein retention, and fat retention (grams/10 days), and for energy retention (kiloJoules/10 days) were changed curvilinearly. These values tended to increase until diets contained 25.4 g acetic acid/kg diet and then decreased significantly when diets contained 50.8 g acetic acid/kg diet. All chicks given the 63.5-g acetic acid/kg diet died during Experiment 2. Dietary acetic acid had a detrimental rather than a beneficial effect on chick performance, particularly at the high level. (Key words: acetic acid, growth, protein utilization, energy utilization, chick) 1989 Poultry Science 68:795-798 INTRODUCTION
Volatile fatty acids (VFA), mainly acetic acid, propionic acid, and butyric acid, are major end products of the microbial digestion of carbohydrates in the alimentary canal. These acids are major energy sources in ruminants and are rapidly produced and absorbed in the rumen. In the chicken, VFA are produced mainly in the hind gut, with acetic acid predominating (Annison et ai, 1968; Moore et al., 1969). Organic acids have been used in pig diets and have improved growth rate and feed utilization (Kirchgessner and Roth, 1982; Falkowski and Aherne, 1984; Giesting and Easter, 1985). In chicks, Cave (1984) reported that feed intake was reduced by the supplement of organic acids. There is, however, little information concerning the effects of dietary organic acids on protein and energy deposition. This report describes experiments designed to assess chick responses to diets containing graded levels of acetic acid. MATERIALS AND METHODS
In two experiments, 1-day-old Single Comb White Leghorn male chicks were given a commercial chick mash (CP, 21.5%; ME, 12.1
'1 kJ = .239 kcal.
kJ/g,1 Marubeni Siryou Ltd., Tokyo, Jpn) for 4 days. On Day 4, the chicks were individually weighed after starving for 3 h and were then selected and distributed into seven groups of five chicks each, so that mean body weights were as uniform as possible, being 52 g in Experiment 1 and 58 g in Experiment 2. At this stage, chicks were housed individually in stainless-steel metabolism cages. In both experiments, six groups of five chicks each were given experimental diets from Day 4 to Day 14. On Day 4, five chicks in a seventh group were killed by cervical dislocation and used to determine the initial body composition. In Experiment 2, diets were force-fed at the level of 138 g/bird per 10 days, which corresponds to the average feed intake in Experiment 1. Feed input was equalized but energy input was not. The daily quantities were increased as follows: 5, 7, 9, 11, 13, 15, 17, 19, 21, and 21 g/bird. All chicks were force-fed twice a day. Diets were blended with water in the ratio of 10 g of feed to 9 g of water. A 20-mL syringe with a plastic tube, 5 mm diam and 60 mm long, was used to deliver the slurry through the esophagus into the crop. Table 1 shows the composition of the basal diet. Powdered distilled vinegar (acetic acid 15.0%) used in the present study was made by mixing with dextrin as 12.7% acetic acid and 87.3% dextrin, donated by Nakano Vinegar Company Ltd., Handa-shi, Aichi-ken, Jpn.
795
796
FURUSE AND OKUMURA TABLE 1. Composition of basal diet
Ingredient
Amount
Isolated soybean protein Corn oil Mineral mixture1 Vitamin mixture2 Choline chloride (95%) Inositol L-Methionine Glycine L-Threonine Cellulose Dextrin
(g/kg) 226 55 58.5 2 1.5 1 2.9 4.2 1.2 100 547.7
'Contained 20.7 g CaHPCy2H20, 14.8 g CaC03, 10 g KHjP04, 3 g KCl, 6 g NaCl, 3 g MgSO„, .5 g FeSO^HjO, .35 g MnSCy5HjO, 2.6 mg KI, 40 mg CuSCy5H20, 62 mg ZnO, 1.7 mg CoCl2-6HjO, 8.3 mg NajMoO^HjO, and .4 mg NajSeCv Untamed 15 mg calcium pantothenate, 6 mg riboflavin, 4 mg pyridoxine hydrochloride, 40 mg nicotinic acid, 1.5 mg folic acid, 200 ug biotin, 20 |Xg cyanocobalamine, 3 mg thiamine hydrochloride, 200 ICU vitamin D3, .5 mg vitamin K3, and 1.930 g glucose. The DL-a-tocopheryl acetate (10IU) andretinylacetate (1,700 IU) were dissolved in the com oil.
Powdered distilled vinegar was supplemented to the basal diet at levels of 12.7, 25.4, 38.1, 50.8, and 63.5 g acetic acid/kg diet at the expense of dextrin. Dietary pH was decreased by increasing acetic acid levels, being 5.94, 4.91, 4.64, 4.42, 4.31, and 4.21, respectively. These values were obtained using a glass electrode and pH meter to measure the pH of a blend of 10 g diet and 9 g water. Experimental diets were mixed separately for the two experiments. Diets were prepared before starting an experiment (within a week) and were kept in nylon bags at room temperature (29 ± 1 C) during an experiment. At Day 14, chicks were killed by cervical dislocation and frozen at -20 C. The frozen carcasses were minced in a meat grinder. The mince was frozen again with liquid nitrogen, minced for a second time and dried at 55 C for 48 h. Nitrogen and protein (N x 6.25) in the diets and carcasses were determined by the Kjeldahl procedure. Fat content in carcasses was extracted overnight (about 16 h) with diethyl ether using a Soxhlet apparatus and determined gravimetrically. Gains in protein, fat, and energy over the experimental period were determined by subtracting the initial from the final values for body composition. Energy content of the chick was calculated using the values of 9.35 and 5.66 kcal/g (39.12 and
23.68 kJ/g) for fat and protein in the body, respectively (Fraps, 1946). Data were subjected to analysis of variance, and significance of difference between means was determined by the Duncan's multiple range test using a commercially available statistical package (SAS, 1985). Regression equations were also fitted to the data. RESULTS AND DISCUSSION
The 10 chicks when 4 days of age at the start of the experiments contained 150 g protein/kg body weight and 73 g fat/kg body weight in Experiment 1 and 154 g protein and 75 g fat in Experiment 2. Within 6 days after commencement of the experiment, in Experiment 2, all chicks given diets containing the highest level (63.5 g/kg diet) of acetic acid and one chick given the 50.8-g acetic acid/kg diet were dead with vomiting, hematemesis, and corrosion of the proventriculus. Under ad libitum feeding (Experiment 1), some acetic acid might have been vaporized from the feed trough, but loss of this acid would be negligible under a force-feeding condition (Experiment 2). Acetic acid might combine with the other compounds in the diet, although after ingestion this acid would be dissociated in the gizzard. With the increase of dietary acetic acid in Experiment 1, body weight gain decreased in a linear manner (Table 2). There was also a linear relationship between dietary acetic acid and feed consumption. The value for feed efficiency was curvilinear with dietary acetic acid. Cave (1984) reported that feed intake was reduced with the increase of dietary propionic acid. Lee and Blair (1972) also discussed the possibility that dietary citrate affected the acidity or the palatability or both acidity and palatability of the diet. Dietary acetic acid can also have an effect similar to that of dietary citrate, because the pH of diets was decreased by the supplement of acetic acid and the acetic acid has a repulsive smell, which can cause a depression in feed consumption. In Experiment 2, chicks were fed by the regimen of equalized feeding to remove the influence of variation in feed consumption, but energy inputs were not equal. Values for body weight gain and feed efficiency showed curvilinear relationships (Table 3). These values tended to increase until diets contained 25.4 g acetic acid/kg diet and then decreased significantly from diets con-
63.5
50.83
1 kJ = .239 kcal.
One missing value due to death.
3
4
9.14 .58x 702 - 2.72x
y = 77.8 - .215x y = 145.3 - .217x y = .511 + .002245x - .000047x2
Regression equation
3.0 .022 .61 .284 20
Pooled SEM y y y y y
= = = = =
Stan of i
75.2 + .557x - .017x2 2.7 .546 + .004034X - .000124x2 .0 2 14.1 + .079x - .0029x .5 10.2 + .068x - .0018x2 .3 2 730 + 4.61x - .140x 19
Regression equation
Feed consumption was 138 g for 10 days. The daily quantities (grams per bird) were increased as follows: 5, 7, 9, 11, 13, 15, 17, 19
Number of observation was five birds per treatment.
•^Means having different superscript letters are significantly different (P<.05).
79"b 81" 70b 61' 76*b .507" .571* .586" .438' .548,b 14.5' 14.4' 12.7" 10.7b 14.1' b b 10.2 10.2 10.4*" 11.2" 8.9' 742 ,b 780" 698b 603' 739"b
38.1
Body weight gain, g/10 days Feed efficiency, gain:feed Protein retention, g/10 days Fat retention, g/10 days Energy retention, kJ/10 days4
25.4
Acetic acid level (g/kg)
12.7
0
2
2.9 4.9 .021 .50 .46 28
Pooled SEM
TABLE 3. Body weight gain, feed efficiency, and protein, fat, and energy retention of chicks given diets containing under an equalized-feeding condition (Experiment 2) 2
Measurement
1 kJ = .239 kcal.
3
'One missing value due to an outlier.
Number of observation was five birds per treatment.
Means having different superscript letters are significantly different (P<.05).
50.8
76* 76" 72" 70" 71" 61° 151 139 139 131 137 133 .510 .541 .522 .532 .515 .459 14.2 14.3 14.4 14.0 14.5 12.6 b b 1 9.2' 8.3" 7.5 6.8' 7.3 * 4.8d ,ab 694' 665 ,b 631" 595b 627*b 484'
38.1
Body weighl gain, g/]0 days Feed consumption, g/10 days Feed efficiency, gain:feed Protein retention, g/10 days Fat retention, g/10 days Energy retention, kJ/10 days3
25.4
Acetic acid level (g/kg diet)
0'
Measurement
12.7
TABLE 2. Body weight gain, feed consumption, feed efficiency, and protein, fat, and energy retention of chicks giv dietary acetic acid under an ad libitum -feeding condition (Experiment 1)
798
FURUSE AND OKUMURA
taining 50.8 g acetic acid/kg diet. In Experiment 1, there were no significant differences between protein retention rates of the two treatments (Table 2). Fat retention and energy retention followed patterns similar to those for body weight gain, and showed a linear decrease with the increase of dietary acetic acid. The lower fat and energy retention from feeding diets with added acetic acid was very likely due to the lower feed consumption. In contrast, Yoshida et al. (1970) showed low availability of energy in fatty acids with less than a 6-carbon chain. Bolton and Dewar (1965) also suggested that the bird can obtain only a small amount of energy from acetate. Thus, the reported low availability of energy in acetic acid may account for part of the reduced energy retention in the high acetic acid diets. In Experiment 2, the values for protein retention, fat retention, and energy retention showed curvilinear relationships (Table 3). However, only small, but nonsignificant, increases in these values occurred until diets contained 25.4 g acetic acid/kg diet, and additional acetic acid produced significant detrimental effects on performance. It was concluded that dietary acetic acid had a detrimental effect rather than a beneficial effect on chick performance, particularly at high dietary concentrations.
REFERENCES Annison, E. F., K. J. Hill, and R. Kenworthy, 1968. Volatile fatty acids in the digestive tract of the fowl. Br. J. Nutr. 22:207-216. Bolton, W., and W. A. Dewar, 1965. The digestibility of acetic, propionic and butyric acids by the fowl. Br. Poult. Sci. 6:103-105. Cave, N.A.G., 1984. Effect of dietary propionic and lactic acids on feed intake by chicks. Poultry Sci. 63: 131-134. Falkowski, J. F„ and F. X. Aherne, 1984. Fumaric and citric acid as feed additives in starter pig nutrition. J. Anim. Sci. 58:935-938. Fraps, G. S., 1946. Composition and productive energy of poultry feeds and rations. Tex. Agric. Exp. Stn. Bull. 678:5-38. Giesting, D. W., and R. A. Easter, 1985. Response of starter pigs to supplementation of corn-soybean meal diets with organic acids. J. Anim. Sci. 60:1288-1294. Kirchgessner, M., and F. X. Roth, 1982. Fumaric acid as a feed additive in pig nutrition. Pig News Inform. 3: 259-264. Lee, D.J.W., and R. Blair, 1972. Effects of chick growth of adding various non-protein nitrogen sources or dried autoclaved poultry manure to diets containing crystalline essential amino acids. Br. Poult. Sci. 13:243-249. Moore, W.E.C., E. P. Cato, and L. V. Holdeman, 1969. Anaerobic bacteria of the gastrointestinal flora and their occurrence in clinical infections. J. Infect. Dis. 119:641-649. SAS, 1985. SAS User's Guide: Statistics. SAS Inst. Inc., Cary, NC. Yoshida, M., H. Morimoto, and R. Oda, 1970. Availability of energy in aliphatic carboxylic acids by growing chicks. Agric. Biol. Chem. 34:1301-1307.
Volatile fatty acids (VFA), mainly acetic acid, propionic acid, and butyric acid, are major end products of the microbial digestion of carbohydrates in the alimentary canal. These acids are major energy sources in ruminants and are rapidly produced and absorbed in the rumen. In the chicken, VFA are produced mainly in the hind gut, with acetic acid predominating (Annison et ai, 1968; Moore et al., 1969). Organic acids have been used in pig diets and have improved growth rate and feed utilization (Kirchgessner and Roth, 1982; Falkowski and Aherne, 1984; Giesting and Easter, 1985). In chicks, Cave (1984) reported that feed intake was reduced by the supplement of organic acids. There is, however, little information concerning the effects of dietary organic acids on protein and energy deposition. This report describes experiments designed to assess chick responses to diets containing graded levels of acetic acid. MATERIALS AND METHODS
In two experiments, 1-day-old Single Comb White Leghorn male chicks were given a commercial chick mash (CP, 21.5%; ME, 12.1
'1 kJ = .239 kcal.
kJ/g,1 Marubeni Siryou Ltd., Tokyo, Jpn) for 4 days. On Day 4, the chicks were individually weighed after starving for 3 h and were then selected and distributed into seven groups of five chicks each, so that mean body weights were as uniform as possible, being 52 g in Experiment 1 and 58 g in Experiment 2. At this stage, chicks were housed individually in stainless-steel metabolism cages. In both experiments, six groups of five chicks each were given experimental diets from Day 4 to Day 14. On Day 4, five chicks in a seventh group were killed by cervical dislocation and used to determine the initial body composition. In Experiment 2, diets were force-fed at the level of 138 g/bird per 10 days, which corresponds to the average feed intake in Experiment 1. Feed input was equalized but energy input was not. The daily quantities were increased as follows: 5, 7, 9, 11, 13, 15, 17, 19, 21, and 21 g/bird. All chicks were force-fed twice a day. Diets were blended with water in the ratio of 10 g of feed to 9 g of water. A 20-mL syringe with a plastic tube, 5 mm diam and 60 mm long, was used to deliver the slurry through the esophagus into the crop. Table 1 shows the composition of the basal diet. Powdered distilled vinegar (acetic acid 15.0%) used in the present study was made by mixing with dextrin as 12.7% acetic acid and 87.3% dextrin, donated by Nakano Vinegar Company Ltd., Handa-shi, Aichi-ken, Jpn.
795
796
FURUSE AND OKUMURA TABLE 1. Composition of basal diet
Ingredient
Amount
Isolated soybean protein Corn oil Mineral mixture1 Vitamin mixture2 Choline chloride (95%) Inositol L-Methionine Glycine L-Threonine Cellulose Dextrin
(g/kg) 226 55 58.5 2 1.5 1 2.9 4.2 1.2 100 547.7
'Contained 20.7 g CaHPCy2H20, 14.8 g CaC03, 10 g KHjP04, 3 g KCl, 6 g NaCl, 3 g MgSO„, .5 g FeSO^HjO, .35 g MnSCy5HjO, 2.6 mg KI, 40 mg CuSCy5H20, 62 mg ZnO, 1.7 mg CoCl2-6HjO, 8.3 mg NajMoO^HjO, and .4 mg NajSeCv Untamed 15 mg calcium pantothenate, 6 mg riboflavin, 4 mg pyridoxine hydrochloride, 40 mg nicotinic acid, 1.5 mg folic acid, 200 ug biotin, 20 |Xg cyanocobalamine, 3 mg thiamine hydrochloride, 200 ICU vitamin D3, .5 mg vitamin K3, and 1.930 g glucose. The DL-a-tocopheryl acetate (10IU) andretinylacetate (1,700 IU) were dissolved in the com oil.
Powdered distilled vinegar was supplemented to the basal diet at levels of 12.7, 25.4, 38.1, 50.8, and 63.5 g acetic acid/kg diet at the expense of dextrin. Dietary pH was decreased by increasing acetic acid levels, being 5.94, 4.91, 4.64, 4.42, 4.31, and 4.21, respectively. These values were obtained using a glass electrode and pH meter to measure the pH of a blend of 10 g diet and 9 g water. Experimental diets were mixed separately for the two experiments. Diets were prepared before starting an experiment (within a week) and were kept in nylon bags at room temperature (29 ± 1 C) during an experiment. At Day 14, chicks were killed by cervical dislocation and frozen at -20 C. The frozen carcasses were minced in a meat grinder. The mince was frozen again with liquid nitrogen, minced for a second time and dried at 55 C for 48 h. Nitrogen and protein (N x 6.25) in the diets and carcasses were determined by the Kjeldahl procedure. Fat content in carcasses was extracted overnight (about 16 h) with diethyl ether using a Soxhlet apparatus and determined gravimetrically. Gains in protein, fat, and energy over the experimental period were determined by subtracting the initial from the final values for body composition. Energy content of the chick was calculated using the values of 9.35 and 5.66 kcal/g (39.12 and
23.68 kJ/g) for fat and protein in the body, respectively (Fraps, 1946). Data were subjected to analysis of variance, and significance of difference between means was determined by the Duncan's multiple range test using a commercially available statistical package (SAS, 1985). Regression equations were also fitted to the data. RESULTS AND DISCUSSION
The 10 chicks when 4 days of age at the start of the experiments contained 150 g protein/kg body weight and 73 g fat/kg body weight in Experiment 1 and 154 g protein and 75 g fat in Experiment 2. Within 6 days after commencement of the experiment, in Experiment 2, all chicks given diets containing the highest level (63.5 g/kg diet) of acetic acid and one chick given the 50.8-g acetic acid/kg diet were dead with vomiting, hematemesis, and corrosion of the proventriculus. Under ad libitum feeding (Experiment 1), some acetic acid might have been vaporized from the feed trough, but loss of this acid would be negligible under a force-feeding condition (Experiment 2). Acetic acid might combine with the other compounds in the diet, although after ingestion this acid would be dissociated in the gizzard. With the increase of dietary acetic acid in Experiment 1, body weight gain decreased in a linear manner (Table 2). There was also a linear relationship between dietary acetic acid and feed consumption. The value for feed efficiency was curvilinear with dietary acetic acid. Cave (1984) reported that feed intake was reduced with the increase of dietary propionic acid. Lee and Blair (1972) also discussed the possibility that dietary citrate affected the acidity or the palatability or both acidity and palatability of the diet. Dietary acetic acid can also have an effect similar to that of dietary citrate, because the pH of diets was decreased by the supplement of acetic acid and the acetic acid has a repulsive smell, which can cause a depression in feed consumption. In Experiment 2, chicks were fed by the regimen of equalized feeding to remove the influence of variation in feed consumption, but energy inputs were not equal. Values for body weight gain and feed efficiency showed curvilinear relationships (Table 3). These values tended to increase until diets contained 25.4 g acetic acid/kg diet and then decreased significantly from diets con-
63.5
50.83
1 kJ = .239 kcal.
One missing value due to death.
3
4
9.14 .58x 702 - 2.72x
y = 77.8 - .215x y = 145.3 - .217x y = .511 + .002245x - .000047x2
Regression equation
3.0 .022 .61 .284 20
Pooled SEM y y y y y
= = = = =
Stan of i
75.2 + .557x - .017x2 2.7 .546 + .004034X - .000124x2 .0 2 14.1 + .079x - .0029x .5 10.2 + .068x - .0018x2 .3 2 730 + 4.61x - .140x 19
Regression equation
Feed consumption was 138 g for 10 days. The daily quantities (grams per bird) were increased as follows: 5, 7, 9, 11, 13, 15, 17, 19
Number of observation was five birds per treatment.
•^Means having different superscript letters are significantly different (P<.05).
79"b 81" 70b 61' 76*b .507" .571* .586" .438' .548,b 14.5' 14.4' 12.7" 10.7b 14.1' b b 10.2 10.2 10.4*" 11.2" 8.9' 742 ,b 780" 698b 603' 739"b
38.1
Body weight gain, g/10 days Feed efficiency, gain:feed Protein retention, g/10 days Fat retention, g/10 days Energy retention, kJ/10 days4
25.4
Acetic acid level (g/kg)
12.7
0
2
2.9 4.9 .021 .50 .46 28
Pooled SEM
TABLE 3. Body weight gain, feed efficiency, and protein, fat, and energy retention of chicks given diets containing under an equalized-feeding condition (Experiment 2) 2
Measurement
1 kJ = .239 kcal.
3
'One missing value due to an outlier.
Number of observation was five birds per treatment.
Means having different superscript letters are significantly different (P<.05).
50.8
76* 76" 72" 70" 71" 61° 151 139 139 131 137 133 .510 .541 .522 .532 .515 .459 14.2 14.3 14.4 14.0 14.5 12.6 b b 1 9.2' 8.3" 7.5 6.8' 7.3 * 4.8d ,ab 694' 665 ,b 631" 595b 627*b 484'
38.1
Body weighl gain, g/]0 days Feed consumption, g/10 days Feed efficiency, gain:feed Protein retention, g/10 days Fat retention, g/10 days Energy retention, kJ/10 days3
25.4
Acetic acid level (g/kg diet)
0'
Measurement
12.7
TABLE 2. Body weight gain, feed consumption, feed efficiency, and protein, fat, and energy retention of chicks giv dietary acetic acid under an ad libitum -feeding condition (Experiment 1)
798
FURUSE AND OKUMURA
taining 50.8 g acetic acid/kg diet. In Experiment 1, there were no significant differences between protein retention rates of the two treatments (Table 2). Fat retention and energy retention followed patterns similar to those for body weight gain, and showed a linear decrease with the increase of dietary acetic acid. The lower fat and energy retention from feeding diets with added acetic acid was very likely due to the lower feed consumption. In contrast, Yoshida et al. (1970) showed low availability of energy in fatty acids with less than a 6-carbon chain. Bolton and Dewar (1965) also suggested that the bird can obtain only a small amount of energy from acetate. Thus, the reported low availability of energy in acetic acid may account for part of the reduced energy retention in the high acetic acid diets. In Experiment 2, the values for protein retention, fat retention, and energy retention showed curvilinear relationships (Table 3). However, only small, but nonsignificant, increases in these values occurred until diets contained 25.4 g acetic acid/kg diet, and additional acetic acid produced significant detrimental effects on performance. It was concluded that dietary acetic acid had a detrimental effect rather than a beneficial effect on chick performance, particularly at high dietary concentrations.
REFERENCES Annison, E. F., K. J. Hill, and R. Kenworthy, 1968. Volatile fatty acids in the digestive tract of the fowl. Br. J. Nutr. 22:207-216. Bolton, W., and W. A. Dewar, 1965. The digestibility of acetic, propionic and butyric acids by the fowl. Br. Poult. Sci. 6:103-105. Cave, N.A.G., 1984. Effect of dietary propionic and lactic acids on feed intake by chicks. Poultry Sci. 63: 131-134. Falkowski, J. F„ and F. X. Aherne, 1984. Fumaric and citric acid as feed additives in starter pig nutrition. J. Anim. Sci. 58:935-938. Fraps, G. S., 1946. Composition and productive energy of poultry feeds and rations. Tex. Agric. Exp. Stn. Bull. 678:5-38. Giesting, D. W., and R. A. Easter, 1985. Response of starter pigs to supplementation of corn-soybean meal diets with organic acids. J. Anim. Sci. 60:1288-1294. Kirchgessner, M., and F. X. Roth, 1982. Fumaric acid as a feed additive in pig nutrition. Pig News Inform. 3: 259-264. Lee, D.J.W., and R. Blair, 1972. Effects of chick growth of adding various non-protein nitrogen sources or dried autoclaved poultry manure to diets containing crystalline essential amino acids. Br. Poult. Sci. 13:243-249. Moore, W.E.C., E. P. Cato, and L. V. Holdeman, 1969. Anaerobic bacteria of the gastrointestinal flora and their occurrence in clinical infections. J. Infect. Dis. 119:641-649. SAS, 1985. SAS User's Guide: Statistics. SAS Inst. Inc., Cary, NC. Yoshida, M., H. Morimoto, and R. Oda, 1970. Availability of energy in aliphatic carboxylic acids by growing chicks. Agric. Biol. Chem. 34:1301-1307.