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Effect of Dietary Sorbose on Lipid Metabolism in Male and Female Broilers
Effect of Dietary Sorbose on Lipid Metabolism in Male and Female Broilers MTTSUHIRO FURUSE Laboratory of Animal Nutrition, School of Agriculture, Nagoya University, Nagoya 464-01, Japan TOSHIYA ISHH, SYUICHI MIYAGAWA, and JIRO NAKAGAWA Toyohashi Feed Mills Co. Ltd., Toyohashi 440, Japan TOSHIO SHTMIZU Food Research Group, Food Division, Asahi Chemical Industry Co. Ltd., Fuji 416, Japan TOHRU WATANABE Laboratory of Animal Anatomy, School of Agriculture, Nagoya University, Nagoya 464-01, Japan JUN-ICHI OKUMURA Laboratory of Animal Nutrition, School of Agriculture, Nagoya University, Nagoya 464-01, Japan (Received for publication April 26, 1990) ABSTRACT Male and female broilers were given diets (6 males and 6 females per diet) containing varying percentages of sorbose (0,3,6, and 9%) and fed for ad libitum access from 28 to 56 days of age. Body weight gain and feed intake were decreased with increasing dietary sorbose, particularly in male birds fed diets containing 9% sorbose, although feed efficiency and N retention rate were not influenced by dietary treatments. Absolute and relative abdominal fat weights were higher in females than in males and decreased with the increasing levels of dietary sorbose in both sexes. Fat content in the pectoral muscle also decreased as dietary sorbose increased. Dietary sorbose did not have significant effects on serum glucose, triglyceride, total cholesterol, low density lipoprotein, very low density lipoprotein, and chylomicron levels in either male or female birds. The ME values of diets decreased as dietary sorbose increased. Palmitic acid content of abdominal fat was significantly lower in birds fed the 9% sorbose diet than in birds fed the control diet. The reverse was true for linoleic acid content. It was concluded that dietary sorbose can be used as a potential regulator of lipid deposition in broilers. (Key words: sorbose, lipid metabolism, abdominal fat, metabolizable energy, fatty acid composition) 1991 Poultry Science 70:95-102
content, and energy utilization in growing rats (Furuse et al, 1989). In laying hens, Furuse et al. (1990a) reported that dietary sorbose decreased liver fat and abdominal fat contents. Serum triglyceride, total cholesterol, low density lipoprotein (LDL), very low density lipoprotein (VLDL), and chylomicron levels were also found to be lower when 10% sorbose was included in the diet. Accordingly, it was suggested that dietary sorbose may be able to regulate lipid accumulation of growing broilers, hi the present study, the effect of dietary sorbose on lipid metabolism of male and female broilers was investigated.
INTRODUCTION
Both laying-type and meat-type chickens have problems with excess fat deposition. For example, broiler chickens fed a high quality diet mainly deposit protein in muscle. However, this feeding regimen also results in considerable fat deposition, and the excess adipose tissue represents a useless energy deposit in the body. Consumers are concerned about decreasing their animal fat consumption. Thus, lean meat is desired to keep a healthy life in a modern society. Dietary sorbose has been shown to lower, in a dose-dependent manner, feed intake, body fat 95
96
FURUSE ET AL. MATERIALS AND METHODS
Male and female broilers (Chunky, 28 dayold) were allocated to four groups. Groups contained six male and six female birds per diet except for five male birds in the 3% sorbose group and five females in the 6% sorbose group, so that mean body weights were as uniform as possible. Birds were housed individually in cages with a wire mesh bottom under continuous lighting. Table 1 shows the composition of the control diet. Sorbose was supplemented to the control diet at 3, 6, or 9% at the expense of cornstarch. Birds were given the experimental diets and water for ad libitum access for 4 wk. On the final experimental day, blood samples were obtained from the wing vein. Determinations of serum glucose, cholesterol, triglyceride, LDL, VLDL, and chylomicron were described elsewhere (Furuse et al., 1990a). After blood sampling, birds were killed by decapitation. Liver and a pair of pectoral muscles (M. pectoralis profundus) were weighed, and protein and fat contents were determined. The Kjeldahl method was used to determine N content, and protein content was obtained ?.
Mizuho Rika, Nagoya, Japan. Shimadzu CA-4, Shimadzu Co., Kyoto, Japan. SP-2300, Gasukuro Kogyo, Inc., Japan.
2 3
TABLE 1. Composition of the control diet Ingredient
(g/kg)
Ground yellow corn Milo Soybean meal Rape seed meal Fish meal Meat and bone meal Rice bran Tallow Calcium carbonate Sodium chloride Vitamin and mineral mixture1 Choline chloride DL-methionine Lysine Virgiruamycin Colistin sulfate2 Salinomycin Na 2 Cornstarch Crude protein, %
447.34 130 165 25 40 40 20 31 6 1.7 1 .4 1.6 .2 .01 .25 .5 90 18.2
Provided the following per kilogram of mixture: retinoL, 8,000,000 IU; cholecalciferol, 1,800,000 IU; DLa-tocopherol, 8 g; menadione, 2 g; thiamin, 1.22 g; riboflavin, 5 g; pyridoxine, 500 mg; cyanocobalamin, 10 mg; nicotinic acid amide, 30 g; pantothenic acid, 10 g; folic acid, 500 mg; biotV 40 mg; MnC0 3 , 60 g; ZnC0 3 , 45 g; Q1SO4, 20.1 g; CoS04> 263 mg; FeS0 4> 136 g; and Ca(I03)2, 537.6 mg. 2 Removed from the diets 7 days before the end of the experiment.
pentobarbital and then perfused through the common carotid artery with 4% paraformaldehyde in .1 M sodium phosphate buffer (pH 7.3). Liver was dissected out and immersed in the same perfusate, containing 30% sucrose, overnight. Ten-um thick frozen sections were cut in a cryostat, mounted on glass slides, and dried. In order to find the neutral fat and lipids, sections were stained for 1 h in a saturated solution of Sudan black B in 60% alcohol, mounted in glycerol, and observed under a light microscope. Two-way analysis of variance was used to analyze the data, and for comparison of the individual treatment differences, Duncan's multiple range test was applied. Regression lines were estimated according to the methods of Snedecor and Cochran (1980). Statistical analysis was done using a commercially available statistical package (SAS Institute, 1985). Differences between sexes at each sorbose level were compared only when a significant interaction between sex and diet was detected.
SORBOSE AND LIPID METABOLISM IN BROILERS
97
TABLE 2. Body weight gain and feed intake of broilers fed diets containing graded levels of sorbose Sorbose Parameters
Sex
0%
3%
6%
9%
662 Male (M) 681 671 691 Female (F]1 605 588 581 579 X 643 618 633 635 Final body weight, g M 2,982 2,658 3,137 3,128 F 2,452 2,346 2,425 2,499 M - F 782* 483* 685* 233 X 2,795a 2,701 ab 2,762* 2,541 b Body weight gain, g/day M 87.7 88.1 82.5 70.3 F 65.9 63.0 68.2 65.9 M- F 4.4 21.8* 25.1* 14.3* a a 74.4 X 76.8 76.0* 68.1 b Feed intake, g/day M 168 161 140 169 F 140 133 143 138 M- F 2 28* 36* 18* X 154* 150* 153* 139b .521 Feed efficiency, g/g M 322 .514 .498 F .472 .473 .478 .480 X .497 .495 .498 .489 Initial body weight, g
Residual mean square
X
677 588*
3,155
2,970 2,427*
43,569
81.9 65.7*
159 138*
.513 .476*
43.7
134
.00096
a,b
Means within a row with no common superscripts are significantly different (P<05). 'Experiment was conducted from 28 to 56 days of age. •Significantly different (P<05).
RESULTS
Table 2 shows the values for initial body weight, final body weight, body weight gain, feed intake, and feed efficiency in male and female broilers fed diets containing graded levels of sorbose. Initial body weights were not significantly different among the dietary treatments, although male chicks had significantly heavier initial body weights than female chicks. Final body weight, body weight gain, and feed intake were significantly reduced in birds fed the 9% sorbose diet and in female birds compared with the other diet-sex subgroups. Significant interactions were detected in these three parameters, implying that these parameters were drastically depressed due to 9% sorbose in the male but not in the female birds. There were no significant differences in feed efficiency due to dietary treatments, though the values for male birds were significantly higher than those for female counterparts. Absolute and relative weights of liver, pectoral muscle, and abdominal fat in male and female broilers are listed in Table 3. There were no significant differences in absolute liver weight among the dietary treatments, although liver weights of male birds were
significantly higher than those of female counterparts. Relative weights of liver significantly increased with the increase of dietary sorbose, but no significant differences were observed between sexes. Pectoral muscles were heavier in male than in female birds but were not significantly different among bird groups fed the different diets. There was a significant interaction between sex and sorbose level in absolute weight of pectoral muscle; the difference in weights between sexes disappeared when dietary sorbose levels increased. Relative weights of pectoral muscle were significantly higher in females than in males. A significant difference was not observed in relative weights of pectoral muscle among the dietary treatments. Absolute and relative weights of abdominal fat were significantly higher in female than in male birds. These two values decreased with increasing dietary sorbose levels. The values for protein and fat contents of liver and pectoral muscle in broilers fed diets containing graded levels of sorbose are shown in Table 4. Liver protein content was significantly lower in birds fed the 9% sorbose diet than in birds fed the other diets, whereas an effect of sex was not detected. Liver fat and
98
FURUSE ET AL.
TABLE 3. Absolute and relative weights of liver, pectoral muscle, and abdominal fat in 56-day-old broilers fed diets containing graded levels of sorbose Residual mean square
Sorbose Parameters
Sex
0%
Liver weight, g
Male (M) SS.8 Female (F)42.5 x 49.1 Relative liver weight, gflcg BW M 17.7 F 17.4 X 17.5C Pectoral muscle weight1, g M 93.0 F 75.8 M -F 17.2* 84.4 X Relative muscle weight, g/kg BW M 29.7 F 30.9 X 30.3 Abdominal fat weight, g M 58.1 F 69.6 X 63.9* Relative abdominal fat weight, g/ kg BW M 18.4 28.3 F 23.4* X
3%
6%
9%
X
51.8 44.0 47.6 16.6 18.8 17.8*°
60.5 46.2 54.0 20.2 18.5 19.4ab
55.8 44.9*
98.9 81.1 17.8* 89.2
90.7 87.2 3.5 89.1 30.5 34.8 32.5 34.0 53.2 42.7 b
54.3 47.2 50.8 20.5 19.6 20.0 s 79.2 82.2 -3.0 80.7 29.9 33.9 31.9 37.1 47.7 42.4 b
30.4 33.5*
31.7 34.4 33.2 55.0 55.8
17.6 23.8 21.0*
11.3 21.2 15.8°
14.1 19.6
I^S1*
18.8 18.5 90.1 81.3*
45.7 56.7*
15.3 23.3*
54.7
4.34
81.5
7.72
221
26.7
""""Means within a row with no common superscripts are significantly different (P<05). 'Weight of bilateral pectoral muscles. •Significantly different at (P<05).
pectoral muscle protein contents were not influenced by either dietary treatment or sex. Pectoral muscle fat content was lowered by the inclusion of dietary sorbose, but the difference between sexes was not significantly different.
The data in Table 5 indicate an effect of dietary sorbose on serum glucose, triglyceride, total cholesterol, LDL, VLDL, and chylomicron in male and female broilers. There were no significant differences in these
TABLE 4. Protein and fat contents of liver and pectoral muscle in 56-day-old broilers fed diets containing graded levels of sorbose Sorbose Parameters
Sex
0%
Protein in liver
Male (M) 21.6 Female (F) 21.3 x 21.5*
3%
6%
9%
X
Residual mean square
1.1
-(%) —
Fat in liver
Protein in pectoral muscle
Fat in pectoral muscle
21.1 21.3 21.2*
21.5 21.1 21.3*
20.0 20.6 20.3 b
21.1 21.1
M F x
3.1 3.5 3.3
3.4 3.6 3.5
3.6 3.5 3.6
3.3 4.4 3.9
3.4 3.8
.57
M F x
23.5 23.5 23.5
24.0 23.8 23.7
23.6 24.2 23.9
24.1 24.1 24.1
23.8 23.9
.37
M F x
.59 .47 .53"
.35 .27 .31 b
.40 .41 .40 ab
.28 .31 .30b
•bMeans within a row with no common superscripts are significantly different (P<05).
.41 .36
.039
99
SORBOSE AND LIPID METABOLISM IN BROILERS
TABLE 5. Effect of dietary sorbose on serum glucose, triglyceride, total cholesterol, low density lipoprotein (LDL), very low density lipoprotein (VLDL), and chylomicron in 56-day-old broilers Sorbose Parameters
Sex
3%
6%
9%
X
Glucose, mg/100 mL
Male (M) 224 Female (F) 218 M- F 6 X 221 M 35.5 F 36.5 X 36.0
208 215 -7 212
223 206 17 215 35.0 33.0 34.1
225 194 31* 209 45.0 34.7 39.8
220 208*
258
37.7 34.3
128
M F
113 106 110 153 119 134
118 92 106 137 157 146
109 95 102 130 161 146 46.5 16.7 31.6
115 105
333
150 151
2,943
Triglyceride, mg/100 mL
Total cholesterol, mg/100 mL
X
LDL, mg/100 mL
M F X
VLDL, mg/100 mL
M F X
Chylomicron, mg/100 mL
M F X
0%
120 123 122 181 169 175 47.0 20.0 33.5 33.8 30.2 32.0
34.8 32.7 33.6
26.6 17.8 21.8 36.2 46.2 41.6
27.8 16.8 22.8 34.8 35.4 35.1
43.3 24.5 33.9
mean square
37.4 17.9* 381 37.1 34.0
341
•Significantly different (P<05).
parameters among the treatments. Serum glucose and VLDL levels were significantly higher in male than in female birds. Effect of dietary sorbose on N intake, N excretion, N retention, N retention rate, and ME values in broilers are given in Table 6. The values for N intake and N retention were significantly higher in male than in female birds, and significant interactions were detected among diets and sexes. These interactions imply that the difference between sexes is small when dietary sorbose levels increase. Significant difference in N excretion was not observed due to dietary treatment and sex. The N retention rate was not influenced by dietary treatment, although the value was significantly higher in male birds than in female counterparts. The values for ME gradually decreased with the increase of dietary sorbose levels, although those were not influenced by sex. Table 7 shows the fatty acid composition of abdominal fat in male and female broilers fed 0 or 9% sorbose diets. Palmitic acid content of abdominal fat was significantly lower in birds fed the 9% sorbose diet than in birds fed the control diet. The reverse was true for linoleic acid content. These results showed a lower proportion of saturated fatty acid and higher proportion of unsaturated fatty acid in birds
fed diets containing sorbose. Consequently, the saturated:unsaturated ratio was significantly lower in birds fed the diet containing sorbose. Female birds had higher proportion of oleic acid compared with male counterparts, and this changed the proportion of saturated and unsaturated fatty acids. The proportions of myristic, stearic, and linolenic acids were not influenced by either dietary or sex treatments. There were many fat droplets that stained strongly with Sudan black B in the liver of female broilers fed both 0 or 9% sorbose diets. However, no differences in the distribution of fat droplets were found by light microscopy. Many large fat droplets were observed in the connective tissue around large or medium sized portal veins, but not around the hepatic artery. They were also seen just beneath epithelia of bile duct Very fine droplets were detected within epithelial cells of bile duct and parenchymal cells of the liver at high magnification. It is well-known that mere are fatstoring cells in the perisinusoidal space. However, strong aggregation of fat droplets was not observed in that space. DISCUSSION
Body weight gain of growing rats (Furuse et al, 1989) and body weights of laying hens
100
FURUSE ET AL. TABLE 6. Effect of dietary sorbose on N intake, N excretion, N retention, N retention rate, and ME values in 56-day-old broilers Sorbose
Parameters
Sex
3%
6%
9%
X
Residual mean square
N intake, g/3 days
Male (M) 18.5 Female (F)14.1 M - F 4.4* x 16.3* M 8.4 F 7.9 X 8.1 M 10.2 6.2 F 4.0* M - F 8.2* X
17.6 14.5 3.1* 15.9* 8.7 7.9 8.3 8.8 6.6 2.2* 7.6b
19.0 14.7 4.3* 17.1" 9.0 8.2 8.6 10.0 6.6 3.4* 8.4 a
15.6 15.3 .3 15.4b
17.7 14.6*
2.58
7.5 8.2 7.8 8.1 7.1 1.0* 7.6b
8.4 8.0
1.60
M F
.507 .455 .479 12.8 13.0 12.9*
.531 .446 .493 12.5 12.6 12.6b
.524 .463 .493 12.7 12.5 12.6b
N excretion, g/3 days
N retention, g/3 days
N retention rate (N retained/N intake)
X
ME value, kj/g1
M F X
0%
.550 .442 .496 12.8 13.2 13.0"
9.3 6.6*
.529 .452* 12.7 12.9
.639
.00182
.089
a,b
Means within a row with no common superscripts are significantly different (P<05). UkJ = .239 kcal. •Significantly different (P<05).
(Furuse et al., 1990a) decreased with the supplementation of sorbose in diets. Male broilers in the present study showed the tendency observed in other species, though body weight gain of female broilers was not influenced by dietary sorbose. The response to sorbose was more sensitive in male than in female counterparts. The reason for this might be explained by hormonal status, but it was not investigated in the present study. Feed intake of growing rats (Furuse et al., 1989) and laying hens (Furuse et al., 1990a) was lowered, in a dose-dependent fashion, with increasing levels of sorbose. This was also confirmed in the present study where inclusion of sorbose at the 9% level significantly decreased feed intake. However, this result was mostly due to the value for the male birds fed the 9% sorbose diet, because feed intake of female birds was not influenced by dietary sorbose levels. The regulation mechanism of feed intake could be different for laying hens and female broilers. In any event, even if feed intake was altered, feed efficiency was not influenced by dietary sorbose. Abdominal fat weight and muscle fat content decreased with the increase of dietary
sorbose regardless of similar feed efficiency values. Even in female birds that showed no changes in body weight gain by dietary treatments, excessive fat was reduced by dietary sorbose. Dietary sorbose was useful for producing a lean carcass without decreasing feed utilization. Not only amount but also fatty acid composition of abdominal fat was changed by dietary sorbose. In birds fed the sorbose diet, saturated fatty acid decreased and unsaturated fatty acid increased; these changes were mainly due to the decrease of palmitic acid and the increase of linoleic acid. It is well known that insulin increases lipid synthesis by providing acetyl-Coenzyme A and p-nicotinamide adenine dinucleotide phosphate (NADPH). Furuse et al. (1989) reported that me serum insulin level was lowered due to dietary sorbose in rats. It might be possible that de novo palmitate synthesis was reduced by the supplementation of dietary sorbose in the present study. Nir et al. (1988) reported that linoleic acid concentration was higher in the chicken selected for low abdominal fat than in the chicken selected for high abdominal fat. Increase in linoleic acid composition might be important in producing the chicken with the low abdominal fat.
101
SORBOSE AND LIPID METABOLISM IN BROILERS
TABLE 7. Fatty acid composition of abdominal fat in male and female 56-day-old broilers fed diets containing sorbose at 0 or 9% Fatty acid
Sorbose level Sex
0%
9%
Male (M) Female (F)
.6 .5 .5 22.9 22.9 22.9 a 6.5 6.4 6.4 a 5.8 52 5.5 44.7 45.6 45.2 18.5 18.4 18.4b 1.0 1.1 1.1 29.3 28.5 28.9 a 70.7 71.5 71.1 b 41.4 40.0 40.7 a
.5 .5 .5 20.5 19.3 19.9b 4.5 52 4.8 b 6.9 6.0 6.5 43.7 46.8 45.3 22.8 20.8 21.8a 1.1 1.4 1.3 27.9 25.8 26.8b 72.1 74.2 73.2a 38.7 34.8 36.8b
Residual mean square
X
(%) Myristic (CI4 : Q)
X
Palmitic (Ci&o)
M F X
Palmitoleic (Cig : i)
M F X
Stearic (C 18 . 0 )
M F X
Oleic (Cig:1)
M F X
Linoleic (Ci8:2)
M F X
Linolenic (Cig.3)
M F X
Saturated fatty acid (S)
M F X
Unsaturated fatty acid (U)
M F X
S:U ratio, %
M F X
.5 .5
.003
21.7 21.1
1.01
5.5 5.8
.85
6.4 5.6
.60
44.2 46.3*
1.82
20.6 19.6
1.14
1.1 12
.05
28.6 27.2*
.90
71.4 72.8*
.90
40.1 37.4*
.03
^kMeans within a row with no common superscripts are significantly different (P<.05). •Significantly different (P<.05).
According to Furuse et al. (1990a), serum levels of triglyceride, total cholesterol, LDL, VLDL, and chylomicron in the laying hen were greatly reduced by the inclusion of 10% sorbose in the diet. It was also shown that serum total cholesterol level of growing rats (Tamura et al., 1991) and VLDL level of growing pigs were lowered by the supplementation of sorbose in diets (Furuse et al., 1990b). In the present study, however, even when sorbose was given in die diet at me 9% level, serum lipid contents were not influenced. The liver as well as the adipose tissue contribute to the synthesis of fatty acids in animals. In mammalian species, adipose tissue is an important site for the synthesis step, hi avian species, in contrast, the liver is the major site for fatty acid synthesis (Goodridge and Ball, 1967; Leveille, 1969). The liver of laying
hens exhibits greater lipid synthesis than the liver of broilers, because the hen synthesizes triglyceride and secretes VLDL in sufficient amounts to meet a high egg production rate. Indeed, decreased accumulation of liver fat in laying hens fed sorbose diets has been observed from the liver color. The hens fed control diets had a light and yellow liver, but livers of birds fed sorbose diets was less light and yellow (Furuse et al., 1990a). hi the present study, liver fat content among dietary treatments was unaltered; this supported die values for serum lipid contents. One of the reasons for differences between broilers and laying hens in the literature is that broilers may be younger Uian laying hens. Accordingly, amounts of a hormone such as estrogen, which regulates lipid metabolism, might be different in the two types of bird populations.
102
FURUSE ET AL.
Nitrogen retention was influenced by dietary sorbose levels. However, this was due to the difference in N intake, because N retention rate was not affected by dietary treatments. Furase et al. (1989) reported that digestible energy and ME values of diets and efficiency of energy utilization decreased with the supplement of dietary sorbose in growing rats when the ME value of sorbose itself was 12.4 kJ/g.4 In the present study, ME values of diets containing sorbose also decreased; the ME of sorbose for broilers was calculated as follows. First the equations relating metabolizability of gross energy to the proportion of sorbose in the diet were expressed. Then the equation relating ME (kJ/g) to the proportion of sorbose (X) was calculated by multiplying the gross energy of the control diet (17.1 kJ/g): ME = 13.0(SE = .07) - 5.3(SE = 1.3) X (R2 = .27, P<001). The value for the ME of sorbose was calculated using a published value, 15.3 kJ/g (Scott et al, 1982), for the ME for the cornstarch that was replaced by sorbose. The ME value of sorbose was 10.0 (15.3 - 5.3) kJ/ g, less than that observed in rats (12.4 kJ/g). The difference for ME values between two species could be explained by the difference in transit time and retention time of the diets. In birds, diets are in rapid transit through the gastro-intestinal tract. Even if sorbose had 10 kJ ME/g, its energy was less available for broilers, than as previously reported for rats (Furuse et al, 1989). It was clearly shown that a small reduction in ME value due to inclusion of sorbose could cause a large reduction in abdominal fat weight and muscle fat content. For example, although the difference in ME value between the 0 and 6% sorbose diets was .4 kJ/g, The inclusion of 6% sorbose reduced
1 kJ = .239 kcal.
abdominal fat weight by 21.2 g and pectoral muscle fat content by .13%. However, it was difficult to separate the effect of sorbose itself from effects due to lower dietary ME, because four diets used in the present study were not formulated to be isocaloric. It was concluded that dietary sorbose can be used as a potential regulator of lipid deposition in broilers without reducing feed efficiency. REFERENCES Furuse, M., S. Nakajima, J. Nakagawa, T. Shimizu, and J. Okumura, 1990a. Regulation of lipid metabolism by dietary sorbose in laying hens. Poultry Sci. 69: 1508-1512. Furuse, M, T. Tagishi, H, Narita, T. Shimizu, and J. Okumura, 1990b. Effect of dietary sorbose on lipid metabolism in growing pigs. Jpn. J. Zootech. Sci. 61: 371-374. Furuse, M, Y. Tamura, S. Matsuda, T. Shimizu, and J. Okumura, 1989. Lower fat deposition and energy utilization of growing rats fed diets containing sorbose. Comp. Biochem. Physiol. 94A:813-817. Goodridge, A. G., and E. G. Ball, 1967. Lipogenesis in the pigeon: in vivo studies. Am. J. Physiol. 213:245-249. Hill, F. W., and D. L. Anderson, 1958. Comparison of metabolizable energy and productive energy determinations with growing chicks. J. Nutr. 64:587-603. Leveille, G. A., 1969. In vitro hepatic lipogenesis in the hen and chick. Comp. Biochem. Physiol. 28: 431-435. Nir, I., Z. Nitsan, and S. Keren-Zvi, 1988. Fat deposition in birds. Pages 141-174 in: Leanness in Domestic Birds. Genetic, Metabolic and Hormonal Aspects. B. Leclercq and C. C. Whitehead, ed. Butterworths, London, England. SAS Institute, 1985. SAS® User's Guide: Statistics. SAS tost Inc., Cary, NC. Scott, M. L., M. C. Nesheim, and R. J. Young, 1982. Nutrition of the chicken. 3rd ed. M. L. Scott and Associates, Ithaca, NY. Snedecor, G. W., and W. G. Cochran, 1980. Pages 149-174 in: Statistical Methods. 7th ed. Iowa State University Press, Ames, IA. Tamura, Y„ M. Furuse, S. Matsuda, T. Shimizu, and J. Okumura, 1991. Energy utilization of sorbose in comparison with maltitol in growing rats. J. Agric. Food. Chem. (in press).
content, and energy utilization in growing rats (Furuse et al, 1989). In laying hens, Furuse et al. (1990a) reported that dietary sorbose decreased liver fat and abdominal fat contents. Serum triglyceride, total cholesterol, low density lipoprotein (LDL), very low density lipoprotein (VLDL), and chylomicron levels were also found to be lower when 10% sorbose was included in the diet. Accordingly, it was suggested that dietary sorbose may be able to regulate lipid accumulation of growing broilers, hi the present study, the effect of dietary sorbose on lipid metabolism of male and female broilers was investigated.
INTRODUCTION
Both laying-type and meat-type chickens have problems with excess fat deposition. For example, broiler chickens fed a high quality diet mainly deposit protein in muscle. However, this feeding regimen also results in considerable fat deposition, and the excess adipose tissue represents a useless energy deposit in the body. Consumers are concerned about decreasing their animal fat consumption. Thus, lean meat is desired to keep a healthy life in a modern society. Dietary sorbose has been shown to lower, in a dose-dependent manner, feed intake, body fat 95
96
FURUSE ET AL. MATERIALS AND METHODS
Male and female broilers (Chunky, 28 dayold) were allocated to four groups. Groups contained six male and six female birds per diet except for five male birds in the 3% sorbose group and five females in the 6% sorbose group, so that mean body weights were as uniform as possible. Birds were housed individually in cages with a wire mesh bottom under continuous lighting. Table 1 shows the composition of the control diet. Sorbose was supplemented to the control diet at 3, 6, or 9% at the expense of cornstarch. Birds were given the experimental diets and water for ad libitum access for 4 wk. On the final experimental day, blood samples were obtained from the wing vein. Determinations of serum glucose, cholesterol, triglyceride, LDL, VLDL, and chylomicron were described elsewhere (Furuse et al., 1990a). After blood sampling, birds were killed by decapitation. Liver and a pair of pectoral muscles (M. pectoralis profundus) were weighed, and protein and fat contents were determined. The Kjeldahl method was used to determine N content, and protein content was obtained ?.
Mizuho Rika, Nagoya, Japan. Shimadzu CA-4, Shimadzu Co., Kyoto, Japan. SP-2300, Gasukuro Kogyo, Inc., Japan.
2 3
TABLE 1. Composition of the control diet Ingredient
(g/kg)
Ground yellow corn Milo Soybean meal Rape seed meal Fish meal Meat and bone meal Rice bran Tallow Calcium carbonate Sodium chloride Vitamin and mineral mixture1 Choline chloride DL-methionine Lysine Virgiruamycin Colistin sulfate2 Salinomycin Na 2 Cornstarch Crude protein, %
447.34 130 165 25 40 40 20 31 6 1.7 1 .4 1.6 .2 .01 .25 .5 90 18.2
Provided the following per kilogram of mixture: retinoL, 8,000,000 IU; cholecalciferol, 1,800,000 IU; DLa-tocopherol, 8 g; menadione, 2 g; thiamin, 1.22 g; riboflavin, 5 g; pyridoxine, 500 mg; cyanocobalamin, 10 mg; nicotinic acid amide, 30 g; pantothenic acid, 10 g; folic acid, 500 mg; biotV 40 mg; MnC0 3 , 60 g; ZnC0 3 , 45 g; Q1SO4, 20.1 g; CoS04> 263 mg; FeS0 4> 136 g; and Ca(I03)2, 537.6 mg. 2 Removed from the diets 7 days before the end of the experiment.
pentobarbital and then perfused through the common carotid artery with 4% paraformaldehyde in .1 M sodium phosphate buffer (pH 7.3). Liver was dissected out and immersed in the same perfusate, containing 30% sucrose, overnight. Ten-um thick frozen sections were cut in a cryostat, mounted on glass slides, and dried. In order to find the neutral fat and lipids, sections were stained for 1 h in a saturated solution of Sudan black B in 60% alcohol, mounted in glycerol, and observed under a light microscope. Two-way analysis of variance was used to analyze the data, and for comparison of the individual treatment differences, Duncan's multiple range test was applied. Regression lines were estimated according to the methods of Snedecor and Cochran (1980). Statistical analysis was done using a commercially available statistical package (SAS Institute, 1985). Differences between sexes at each sorbose level were compared only when a significant interaction between sex and diet was detected.
SORBOSE AND LIPID METABOLISM IN BROILERS
97
TABLE 2. Body weight gain and feed intake of broilers fed diets containing graded levels of sorbose Sorbose Parameters
Sex
0%
3%
6%
9%
662 Male (M) 681 671 691 Female (F]1 605 588 581 579 X 643 618 633 635 Final body weight, g M 2,982 2,658 3,137 3,128 F 2,452 2,346 2,425 2,499 M - F 782* 483* 685* 233 X 2,795a 2,701 ab 2,762* 2,541 b Body weight gain, g/day M 87.7 88.1 82.5 70.3 F 65.9 63.0 68.2 65.9 M- F 4.4 21.8* 25.1* 14.3* a a 74.4 X 76.8 76.0* 68.1 b Feed intake, g/day M 168 161 140 169 F 140 133 143 138 M- F 2 28* 36* 18* X 154* 150* 153* 139b .521 Feed efficiency, g/g M 322 .514 .498 F .472 .473 .478 .480 X .497 .495 .498 .489 Initial body weight, g
Residual mean square
X
677 588*
3,155
2,970 2,427*
43,569
81.9 65.7*
159 138*
.513 .476*
43.7
134
.00096
a,b
Means within a row with no common superscripts are significantly different (P<05). 'Experiment was conducted from 28 to 56 days of age. •Significantly different (P<05).
RESULTS
Table 2 shows the values for initial body weight, final body weight, body weight gain, feed intake, and feed efficiency in male and female broilers fed diets containing graded levels of sorbose. Initial body weights were not significantly different among the dietary treatments, although male chicks had significantly heavier initial body weights than female chicks. Final body weight, body weight gain, and feed intake were significantly reduced in birds fed the 9% sorbose diet and in female birds compared with the other diet-sex subgroups. Significant interactions were detected in these three parameters, implying that these parameters were drastically depressed due to 9% sorbose in the male but not in the female birds. There were no significant differences in feed efficiency due to dietary treatments, though the values for male birds were significantly higher than those for female counterparts. Absolute and relative weights of liver, pectoral muscle, and abdominal fat in male and female broilers are listed in Table 3. There were no significant differences in absolute liver weight among the dietary treatments, although liver weights of male birds were
significantly higher than those of female counterparts. Relative weights of liver significantly increased with the increase of dietary sorbose, but no significant differences were observed between sexes. Pectoral muscles were heavier in male than in female birds but were not significantly different among bird groups fed the different diets. There was a significant interaction between sex and sorbose level in absolute weight of pectoral muscle; the difference in weights between sexes disappeared when dietary sorbose levels increased. Relative weights of pectoral muscle were significantly higher in females than in males. A significant difference was not observed in relative weights of pectoral muscle among the dietary treatments. Absolute and relative weights of abdominal fat were significantly higher in female than in male birds. These two values decreased with increasing dietary sorbose levels. The values for protein and fat contents of liver and pectoral muscle in broilers fed diets containing graded levels of sorbose are shown in Table 4. Liver protein content was significantly lower in birds fed the 9% sorbose diet than in birds fed the other diets, whereas an effect of sex was not detected. Liver fat and
98
FURUSE ET AL.
TABLE 3. Absolute and relative weights of liver, pectoral muscle, and abdominal fat in 56-day-old broilers fed diets containing graded levels of sorbose Residual mean square
Sorbose Parameters
Sex
0%
Liver weight, g
Male (M) SS.8 Female (F)42.5 x 49.1 Relative liver weight, gflcg BW M 17.7 F 17.4 X 17.5C Pectoral muscle weight1, g M 93.0 F 75.8 M -F 17.2* 84.4 X Relative muscle weight, g/kg BW M 29.7 F 30.9 X 30.3 Abdominal fat weight, g M 58.1 F 69.6 X 63.9* Relative abdominal fat weight, g/ kg BW M 18.4 28.3 F 23.4* X
3%
6%
9%
X
51.8 44.0 47.6 16.6 18.8 17.8*°
60.5 46.2 54.0 20.2 18.5 19.4ab
55.8 44.9*
98.9 81.1 17.8* 89.2
90.7 87.2 3.5 89.1 30.5 34.8 32.5 34.0 53.2 42.7 b
54.3 47.2 50.8 20.5 19.6 20.0 s 79.2 82.2 -3.0 80.7 29.9 33.9 31.9 37.1 47.7 42.4 b
30.4 33.5*
31.7 34.4 33.2 55.0 55.8
17.6 23.8 21.0*
11.3 21.2 15.8°
14.1 19.6
I^S1*
18.8 18.5 90.1 81.3*
45.7 56.7*
15.3 23.3*
54.7
4.34
81.5
7.72
221
26.7
""""Means within a row with no common superscripts are significantly different (P<05). 'Weight of bilateral pectoral muscles. •Significantly different at (P<05).
pectoral muscle protein contents were not influenced by either dietary treatment or sex. Pectoral muscle fat content was lowered by the inclusion of dietary sorbose, but the difference between sexes was not significantly different.
The data in Table 5 indicate an effect of dietary sorbose on serum glucose, triglyceride, total cholesterol, LDL, VLDL, and chylomicron in male and female broilers. There were no significant differences in these
TABLE 4. Protein and fat contents of liver and pectoral muscle in 56-day-old broilers fed diets containing graded levels of sorbose Sorbose Parameters
Sex
0%
Protein in liver
Male (M) 21.6 Female (F) 21.3 x 21.5*
3%
6%
9%
X
Residual mean square
1.1
-(%) —
Fat in liver
Protein in pectoral muscle
Fat in pectoral muscle
21.1 21.3 21.2*
21.5 21.1 21.3*
20.0 20.6 20.3 b
21.1 21.1
M F x
3.1 3.5 3.3
3.4 3.6 3.5
3.6 3.5 3.6
3.3 4.4 3.9
3.4 3.8
.57
M F x
23.5 23.5 23.5
24.0 23.8 23.7
23.6 24.2 23.9
24.1 24.1 24.1
23.8 23.9
.37
M F x
.59 .47 .53"
.35 .27 .31 b
.40 .41 .40 ab
.28 .31 .30b
•bMeans within a row with no common superscripts are significantly different (P<05).
.41 .36
.039
99
SORBOSE AND LIPID METABOLISM IN BROILERS
TABLE 5. Effect of dietary sorbose on serum glucose, triglyceride, total cholesterol, low density lipoprotein (LDL), very low density lipoprotein (VLDL), and chylomicron in 56-day-old broilers Sorbose Parameters
Sex
3%
6%
9%
X
Glucose, mg/100 mL
Male (M) 224 Female (F) 218 M- F 6 X 221 M 35.5 F 36.5 X 36.0
208 215 -7 212
223 206 17 215 35.0 33.0 34.1
225 194 31* 209 45.0 34.7 39.8
220 208*
258
37.7 34.3
128
M F
113 106 110 153 119 134
118 92 106 137 157 146
109 95 102 130 161 146 46.5 16.7 31.6
115 105
333
150 151
2,943
Triglyceride, mg/100 mL
Total cholesterol, mg/100 mL
X
LDL, mg/100 mL
M F X
VLDL, mg/100 mL
M F X
Chylomicron, mg/100 mL
M F X
0%
120 123 122 181 169 175 47.0 20.0 33.5 33.8 30.2 32.0
34.8 32.7 33.6
26.6 17.8 21.8 36.2 46.2 41.6
27.8 16.8 22.8 34.8 35.4 35.1
43.3 24.5 33.9
mean square
37.4 17.9* 381 37.1 34.0
341
•Significantly different (P<05).
parameters among the treatments. Serum glucose and VLDL levels were significantly higher in male than in female birds. Effect of dietary sorbose on N intake, N excretion, N retention, N retention rate, and ME values in broilers are given in Table 6. The values for N intake and N retention were significantly higher in male than in female birds, and significant interactions were detected among diets and sexes. These interactions imply that the difference between sexes is small when dietary sorbose levels increase. Significant difference in N excretion was not observed due to dietary treatment and sex. The N retention rate was not influenced by dietary treatment, although the value was significantly higher in male birds than in female counterparts. The values for ME gradually decreased with the increase of dietary sorbose levels, although those were not influenced by sex. Table 7 shows the fatty acid composition of abdominal fat in male and female broilers fed 0 or 9% sorbose diets. Palmitic acid content of abdominal fat was significantly lower in birds fed the 9% sorbose diet than in birds fed the control diet. The reverse was true for linoleic acid content. These results showed a lower proportion of saturated fatty acid and higher proportion of unsaturated fatty acid in birds
fed diets containing sorbose. Consequently, the saturated:unsaturated ratio was significantly lower in birds fed the diet containing sorbose. Female birds had higher proportion of oleic acid compared with male counterparts, and this changed the proportion of saturated and unsaturated fatty acids. The proportions of myristic, stearic, and linolenic acids were not influenced by either dietary or sex treatments. There were many fat droplets that stained strongly with Sudan black B in the liver of female broilers fed both 0 or 9% sorbose diets. However, no differences in the distribution of fat droplets were found by light microscopy. Many large fat droplets were observed in the connective tissue around large or medium sized portal veins, but not around the hepatic artery. They were also seen just beneath epithelia of bile duct Very fine droplets were detected within epithelial cells of bile duct and parenchymal cells of the liver at high magnification. It is well-known that mere are fatstoring cells in the perisinusoidal space. However, strong aggregation of fat droplets was not observed in that space. DISCUSSION
Body weight gain of growing rats (Furuse et al, 1989) and body weights of laying hens
100
FURUSE ET AL. TABLE 6. Effect of dietary sorbose on N intake, N excretion, N retention, N retention rate, and ME values in 56-day-old broilers Sorbose
Parameters
Sex
3%
6%
9%
X
Residual mean square
N intake, g/3 days
Male (M) 18.5 Female (F)14.1 M - F 4.4* x 16.3* M 8.4 F 7.9 X 8.1 M 10.2 6.2 F 4.0* M - F 8.2* X
17.6 14.5 3.1* 15.9* 8.7 7.9 8.3 8.8 6.6 2.2* 7.6b
19.0 14.7 4.3* 17.1" 9.0 8.2 8.6 10.0 6.6 3.4* 8.4 a
15.6 15.3 .3 15.4b
17.7 14.6*
2.58
7.5 8.2 7.8 8.1 7.1 1.0* 7.6b
8.4 8.0
1.60
M F
.507 .455 .479 12.8 13.0 12.9*
.531 .446 .493 12.5 12.6 12.6b
.524 .463 .493 12.7 12.5 12.6b
N excretion, g/3 days
N retention, g/3 days
N retention rate (N retained/N intake)
X
ME value, kj/g1
M F X
0%
.550 .442 .496 12.8 13.2 13.0"
9.3 6.6*
.529 .452* 12.7 12.9
.639
.00182
.089
a,b
Means within a row with no common superscripts are significantly different (P<05). UkJ = .239 kcal. •Significantly different (P<05).
(Furuse et al., 1990a) decreased with the supplementation of sorbose in diets. Male broilers in the present study showed the tendency observed in other species, though body weight gain of female broilers was not influenced by dietary sorbose. The response to sorbose was more sensitive in male than in female counterparts. The reason for this might be explained by hormonal status, but it was not investigated in the present study. Feed intake of growing rats (Furuse et al., 1989) and laying hens (Furuse et al., 1990a) was lowered, in a dose-dependent fashion, with increasing levels of sorbose. This was also confirmed in the present study where inclusion of sorbose at the 9% level significantly decreased feed intake. However, this result was mostly due to the value for the male birds fed the 9% sorbose diet, because feed intake of female birds was not influenced by dietary sorbose levels. The regulation mechanism of feed intake could be different for laying hens and female broilers. In any event, even if feed intake was altered, feed efficiency was not influenced by dietary sorbose. Abdominal fat weight and muscle fat content decreased with the increase of dietary
sorbose regardless of similar feed efficiency values. Even in female birds that showed no changes in body weight gain by dietary treatments, excessive fat was reduced by dietary sorbose. Dietary sorbose was useful for producing a lean carcass without decreasing feed utilization. Not only amount but also fatty acid composition of abdominal fat was changed by dietary sorbose. In birds fed the sorbose diet, saturated fatty acid decreased and unsaturated fatty acid increased; these changes were mainly due to the decrease of palmitic acid and the increase of linoleic acid. It is well known that insulin increases lipid synthesis by providing acetyl-Coenzyme A and p-nicotinamide adenine dinucleotide phosphate (NADPH). Furuse et al. (1989) reported that me serum insulin level was lowered due to dietary sorbose in rats. It might be possible that de novo palmitate synthesis was reduced by the supplementation of dietary sorbose in the present study. Nir et al. (1988) reported that linoleic acid concentration was higher in the chicken selected for low abdominal fat than in the chicken selected for high abdominal fat. Increase in linoleic acid composition might be important in producing the chicken with the low abdominal fat.
101
SORBOSE AND LIPID METABOLISM IN BROILERS
TABLE 7. Fatty acid composition of abdominal fat in male and female 56-day-old broilers fed diets containing sorbose at 0 or 9% Fatty acid
Sorbose level Sex
0%
9%
Male (M) Female (F)
.6 .5 .5 22.9 22.9 22.9 a 6.5 6.4 6.4 a 5.8 52 5.5 44.7 45.6 45.2 18.5 18.4 18.4b 1.0 1.1 1.1 29.3 28.5 28.9 a 70.7 71.5 71.1 b 41.4 40.0 40.7 a
.5 .5 .5 20.5 19.3 19.9b 4.5 52 4.8 b 6.9 6.0 6.5 43.7 46.8 45.3 22.8 20.8 21.8a 1.1 1.4 1.3 27.9 25.8 26.8b 72.1 74.2 73.2a 38.7 34.8 36.8b
Residual mean square
X
(%) Myristic (CI4 : Q)
X
Palmitic (Ci&o)
M F X
Palmitoleic (Cig : i)
M F X
Stearic (C 18 . 0 )
M F X
Oleic (Cig:1)
M F X
Linoleic (Ci8:2)
M F X
Linolenic (Cig.3)
M F X
Saturated fatty acid (S)
M F X
Unsaturated fatty acid (U)
M F X
S:U ratio, %
M F X
.5 .5
.003
21.7 21.1
1.01
5.5 5.8
.85
6.4 5.6
.60
44.2 46.3*
1.82
20.6 19.6
1.14
1.1 12
.05
28.6 27.2*
.90
71.4 72.8*
.90
40.1 37.4*
.03
^kMeans within a row with no common superscripts are significantly different (P<.05). •Significantly different (P<.05).
According to Furuse et al. (1990a), serum levels of triglyceride, total cholesterol, LDL, VLDL, and chylomicron in the laying hen were greatly reduced by the inclusion of 10% sorbose in the diet. It was also shown that serum total cholesterol level of growing rats (Tamura et al., 1991) and VLDL level of growing pigs were lowered by the supplementation of sorbose in diets (Furuse et al., 1990b). In the present study, however, even when sorbose was given in die diet at me 9% level, serum lipid contents were not influenced. The liver as well as the adipose tissue contribute to the synthesis of fatty acids in animals. In mammalian species, adipose tissue is an important site for the synthesis step, hi avian species, in contrast, the liver is the major site for fatty acid synthesis (Goodridge and Ball, 1967; Leveille, 1969). The liver of laying
hens exhibits greater lipid synthesis than the liver of broilers, because the hen synthesizes triglyceride and secretes VLDL in sufficient amounts to meet a high egg production rate. Indeed, decreased accumulation of liver fat in laying hens fed sorbose diets has been observed from the liver color. The hens fed control diets had a light and yellow liver, but livers of birds fed sorbose diets was less light and yellow (Furuse et al., 1990a). hi the present study, liver fat content among dietary treatments was unaltered; this supported die values for serum lipid contents. One of the reasons for differences between broilers and laying hens in the literature is that broilers may be younger Uian laying hens. Accordingly, amounts of a hormone such as estrogen, which regulates lipid metabolism, might be different in the two types of bird populations.
102
FURUSE ET AL.
Nitrogen retention was influenced by dietary sorbose levels. However, this was due to the difference in N intake, because N retention rate was not affected by dietary treatments. Furase et al. (1989) reported that digestible energy and ME values of diets and efficiency of energy utilization decreased with the supplement of dietary sorbose in growing rats when the ME value of sorbose itself was 12.4 kJ/g.4 In the present study, ME values of diets containing sorbose also decreased; the ME of sorbose for broilers was calculated as follows. First the equations relating metabolizability of gross energy to the proportion of sorbose in the diet were expressed. Then the equation relating ME (kJ/g) to the proportion of sorbose (X) was calculated by multiplying the gross energy of the control diet (17.1 kJ/g): ME = 13.0(SE = .07) - 5.3(SE = 1.3) X (R2 = .27, P<001). The value for the ME of sorbose was calculated using a published value, 15.3 kJ/g (Scott et al, 1982), for the ME for the cornstarch that was replaced by sorbose. The ME value of sorbose was 10.0 (15.3 - 5.3) kJ/ g, less than that observed in rats (12.4 kJ/g). The difference for ME values between two species could be explained by the difference in transit time and retention time of the diets. In birds, diets are in rapid transit through the gastro-intestinal tract. Even if sorbose had 10 kJ ME/g, its energy was less available for broilers, than as previously reported for rats (Furuse et al, 1989). It was clearly shown that a small reduction in ME value due to inclusion of sorbose could cause a large reduction in abdominal fat weight and muscle fat content. For example, although the difference in ME value between the 0 and 6% sorbose diets was .4 kJ/g, The inclusion of 6% sorbose reduced
1 kJ = .239 kcal.
abdominal fat weight by 21.2 g and pectoral muscle fat content by .13%. However, it was difficult to separate the effect of sorbose itself from effects due to lower dietary ME, because four diets used in the present study were not formulated to be isocaloric. It was concluded that dietary sorbose can be used as a potential regulator of lipid deposition in broilers without reducing feed efficiency. REFERENCES Furuse, M., S. Nakajima, J. Nakagawa, T. Shimizu, and J. Okumura, 1990a. Regulation of lipid metabolism by dietary sorbose in laying hens. Poultry Sci. 69: 1508-1512. Furuse, M, T. Tagishi, H, Narita, T. Shimizu, and J. Okumura, 1990b. Effect of dietary sorbose on lipid metabolism in growing pigs. Jpn. J. Zootech. Sci. 61: 371-374. Furuse, M, Y. Tamura, S. Matsuda, T. Shimizu, and J. Okumura, 1989. Lower fat deposition and energy utilization of growing rats fed diets containing sorbose. Comp. Biochem. Physiol. 94A:813-817. Goodridge, A. G., and E. G. Ball, 1967. Lipogenesis in the pigeon: in vivo studies. Am. J. Physiol. 213:245-249. Hill, F. W., and D. L. Anderson, 1958. Comparison of metabolizable energy and productive energy determinations with growing chicks. J. Nutr. 64:587-603. Leveille, G. A., 1969. In vitro hepatic lipogenesis in the hen and chick. Comp. Biochem. Physiol. 28: 431-435. Nir, I., Z. Nitsan, and S. Keren-Zvi, 1988. Fat deposition in birds. Pages 141-174 in: Leanness in Domestic Birds. Genetic, Metabolic and Hormonal Aspects. B. Leclercq and C. C. Whitehead, ed. Butterworths, London, England. SAS Institute, 1985. SAS® User's Guide: Statistics. SAS tost Inc., Cary, NC. Scott, M. L., M. C. Nesheim, and R. J. Young, 1982. Nutrition of the chicken. 3rd ed. M. L. Scott and Associates, Ithaca, NY. Snedecor, G. W., and W. G. Cochran, 1980. Pages 149-174 in: Statistical Methods. 7th ed. Iowa State University Press, Ames, IA. Tamura, Y„ M. Furuse, S. Matsuda, T. Shimizu, and J. Okumura, 1991. Energy utilization of sorbose in comparison with maltitol in growing rats. J. Agric. Food. Chem. (in press).