Adipose Cellularity in Nonselected and Selected Broiler Stocks: Measurements at Equal Weights and Ages1

PHYSIOLOGY AND REPRODUCTION Adipose Cellularity in Nonselected and Selected Broiler Stocks: Measurements at Equal Weights and Ages1 A. L. CARTWRIGHT United States Department of Agriculture, Agriculture Research Service, Poultry Research Laboratory, Georgetown, Delaware 19947 H. L. MARKS Southeast Poultry Research Laboratory, Athens, Georgia 30613 D. R. CAMPION Richard B. Russell Agricultural Research Center, Athens, Georgia 30613

ABSTRACT A population of nonselected broilers (AC) and a stock of commercial broilers (C) differ in growth characteristics. The two stocks of broilers were examined for differences in adipose tissue at equal ages at 28 and 54 days of age and at equal body weights. Body composition and abdominal adipose tissue were measured. Total adipocyte number, adipocyte size distributions, and DNA content of the abdominal fat pad were determined. Partial correlations of abdominal fat, expressed as a percentage of body weight, with mean adipocyte volume and abdominal adipocyte number were calculated. Body weights of C birds were greater than body weights of AC birds at 28 days of age (903 ± 21 and 355 ± 12 g; X ± SE) and at 54 days of age (2,410 ± 45 and 892 ± 31 g). Abdominal fat pads were heavier in C birds than in AC birds on an absolute basis (P<.01) and as a proportion of body weight (P<,01). The C birds had more and larger adipocytes than AC birds at equal weights (P<.05) and equal ages (P<.01). Age did not significantly affect adipocyte size in AC birds, but adipocytes were larger in C birds at 28 days of age (117.3 vs. 78.8 pL, P<.01) and further increased in size at 54 days (177.3 vs. 79.1 pL, P<.01) when compared with those in AC birds at the same age. Hypertrophy and not hyperplasia of adipocytes was associated with development and maintenance of excessive abdominal fat deposits in the C stock at 28 and 54 days of age. The increased adiposity of C broilers was not attributed to differences in maturation rates. Adiposity was concluded to be a product of innate differences in adipocyte size regulatory systems. {Key words: adipose, cellularity, growth, broilers, chickens) 1988 Poultry Science 67:1338-1344 INTRODUCTION

Genetic selection readily yields birds that differ in growth characteristics and body composition (Lilburnef al, 1982; Hood and Pym, 1982). Selection for growth characteristics desirable for commercial development results in increased deposition of fat (Proudman et al, 1970; Lin, 1981; Chambers et al, 1981). Indeed, Becker et al. (1979) describes a positive correlation between fat and body weight.

'Mention of a trade name, proprietary product, or specific equipment does not constitute a guarantee or warranty by the United States Department of Agriculture and does not imply its approval to the exclusion of other products that may be suitable.

Adipose tissue cellularity of the chicken is under scientific scrutiny (Pfaff and Austic, 1976; March and Hansen, 1977; March et al, 1982; Allain and Simon, 1982; Hood, 1982; Hood and Pym, 1982; Cherry et al, 1984; March et al, 1984; Cartwright et al., 1986b). Adipose mass is dependent upon the number and size of individual adipocytes. Development of adipose cellularity is a physiologically controlled process that is affected by hormonal factors (Nixon and Green, 1984; Zezulak and Green, 1986). Recent findings suggest that adipose cellularity in the growing broiler may be relatively inelastic (Cartwright, 1986; 1987a) when compared with the cellularity of mammals (Lemonnier, 1972; Allen et al, 1976; Faust et al., 1978; Schneider etal., 1981; Smith etal, 1983). Neither lipectomy (Cartwright, 1986) nor overfeeding alters

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(Received for publication September 3, 1987)

ADIPOSE CELLULARITY IN BROILERS

adipocyte hyperplasia, nor permanently affects adipocyte hypertrophy of the abdominal fat pad (Cartwright, 1986; 1987a). Dietary restriction affects adipose cellularity in the growing broiler, but this effect may be temporary (Cartwright et al, 1986c). This study was designed to compare cellularity characteristics of adipose tissue at equal weight and equal age in chickens that differ in propensity to develop body size and body fat. As tissue development accounts for body size and composition differences, these observations may suggest mechanisms by which body size and composition are controlled. MATERIALS AND METHODS

Giles and Myers, 1965; Munro and Fleck, 1966). Percentage data were transformed to arc sine for the analysis of variance. Analyses were performed by using the GLM and ANOVA procedures (SAS, 1982). Stock X age interaction was detected with GLM by following the model: Yijk = ti + Ai + Bj + AB y + eijk where Yijk denoted the k* observation in the 1th broiler stock (A), j * age (B), |JL was the overall mean, and e is the random error. Means separation was accomplished with Duncan's multiple range test. Partial correlations were calculated by regression analysis of variance, with percentage abdominal fat as the dependent variable and mean adipocyte volume and total adipocyte number as the independent variables. RESULTS AND DISCUSSION

Birds of the C stock had more and larger abdominal adipocytes than AC birds, controlling for age and body weight. Body weight and abdominal fat weight at similar ages, and body composition data and abdominal fat weights at similar body weights, showed that the C stock was heavier bodied and fatter than the AC stock. Furthermore, the data suggest that differences in regulation of adipose cellularity and not differences in maturation rate produce the observed differences in adiposity. The significant interactions between stock and age detected for body weight and abdominal fat size were attributed to the obvious body size differences between the AC and C stocks. Adipose tissue of the C stock was hypertrophic compared with that of AC adipose tissue (Table 1). Total volume of abdominal adipocytes, distributed by diameter (Figure 1), illustrates how adipocytes of various size ranges contributed to abdominal adipose mass in each stock-age subclass. The areas under the distribution graphs are representative of the abdominal fat pad weights that were observed in each subclass (Table 1). The effects of stock and age on adipocyte size distribution are more clearly presented when the volume distribution is expressed as a proportion of the total volume of adipocytes from the fat pad, as shown in Figure 2. Adipocyte volume distributions from AC tissue at 28 and 54 days of age were not significantly different. However, a greater proportion of adipocytes was distributed among larger diameters in C birds than in AC birds. Thus, mean adipocyte volume for 28-day C birds was larger (P<.05)

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Athens-Canadian (AC) and Cobb (C) chickens were examined. The AC birds, described by Hess (1962), are small-bodied chickens representative of the 1950's meat-type bird. The C birds are a large-bodied stock maintained in our laboratory representative of current commercial broiler stock. Male chickens of each stock were identified with wingbands at 1 day of age and randomly assigned to battery brooding pens. Chicks were fed the University of Georgia broiler starter diet (23% protein, 3,120 kcal/kg ME) ad libitum and weighed periodically. At both 28 days of age and at 54 days of age, when data indicated that body weight of the remaining AC birds were the same as the body weight of C birds at 28 days of age, 16 birds from each stock were killed and processed. Sixteen birds of each stock and age were weighed and killed by cervical dislocation. Eight birds of each age and stock were defeathered and prepared for body composition analysis. Gastrointestinal contents were removed, and the digestive tract was returned to the carcass. Carcasses were ground, and homogeneous samples of the ground carcasses were divided and analyzed for moisture, Kjeldahl nitrogen, ether extract, and ash. Abdominal fat samples were removed from the remaining eight birds of each subclass immediately after death and fixed with osmium tetroxide for electronic quantification of adipocyte number and size by a modification (Cartwright, 1987b) of the procedures described by Hirsch and Gallian (1968) and Etherton et al. (1977). The remainder of the fat pad was excised and weighed. Gastrointestinal contents were removed and the body weight was recorded. The DNA content of the abdominal fat pad was determined chemically (Burton, 1956;

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than the mean volumes of 28-day and 54-day AC birds (Table 1). Adipocyte mean volume increased from 117 pL at 28 days of age to 177 pL at 54 days of age in C birds. Mean cell volume did not increase significantly between 28 and 54 days of age in the AC stock, which accounted for a significant stock X age interaction. Mean adipocyte cell volume at both 28 and 54 days for AC birds was less than the mean volume of adipocytes from C birds at 28 days of age. The relative hypertrophy of adipocytes from C birds was manifest by 28 days of age, persisted even when comparisons were made between C and AC birds at equal weights, and continued to increase with age. Total adipocyte number increased with age and weight in both stocks, but adipose tissue of the C stock was hyperplastic compared with AC adipose tissue (Table 1). Total abdominal adipocyte number increased from 28 to 54 days of age in both AC (27.95 x 106 and 80.07 X 106 adipocytes) and C birds (120.95 x 106 and 233.34 X 106 adipocytes). Fat pad DNA values were approximately proportional to measurable adipocyte number.

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number of maturing adipocytes might be underestimated for the AC birds, this difference probably did not significantly affect the results of this study. The peak of the distribution of total adipocyte number (Figure 3) was substantially less in AC birds than in C birds of the same body weight and age. Partial correlations between abdominal fat weight expressed as a percentage of body weight and mean abdominal adipocyte volume and total abdominal adipocyte number are shown in Table 2. Partial correlations from AC birds at both ages and from C birds at 54 days of age indicate that mean adipocyte volume had greater effect than adipocyte cell number on percentage abdominal fat in the broilers. The high correlation of total adipocyte number with percentage abdominal fat weight in the 28-day-old C birds might have resulted from a precocious filling of preadipocytes, which increased the number of adipocytes detected. The early filling was concurrent with relatively early hypertrophy of adipocytes in C birds when compared with AC birds. The C birds were heavier and fatter than AC birds at the same age. Body weights of the AC birds at 54 days and body weight of the C birds at 28 days were not significantly different (Table 1). At equal ages the C stock was more than twice the weight of the AC stock. Birds within a stock were heavier at 54 days than at 28 days. Similarly, fat pad weights increased with age within each stock. Abdominal fat pads from C birds were heavier than fat pads from AC birds in all age comparisons. The proportion of body weight that was abdominal fat (percentage fat pad) did not significantly increase from 28 to 54 days in either AC or C birds. However, the abdominal fat pad as a proportion of body weight

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The C birds had 50% more detectable adipocytes (P<.01) at 28 days of age than AC birds of equal weight at 54 days of age (Figure 3, Table 1). However, part of the difference in cell number might be attributed to the interaction among the methodology used to determine adipose cellularity and the line difference in adipocyte size distribution. The frequency distribution of adipocyte number by diameter (Figure 4) demonstrates that the C distribution, when compared with the AC distribution, was skewed to the right. As the limit of detection of the cell counting and sizing technique was set at approximately 25 n-m, it is assumed that a proportionally greater number of adipocytes in AC birds was not detected. This phenomenon might account for the observed stock x age interaction. Comparison of Figures 1 and 3 illustrates that, as cell volume increases as a cubed function of cell diameter, small cells contribute a smaller proportion of total adipose tissue volume than their numbers would suggest. Although the

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CARTWRIGHT ET AL.

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that AC birds would deposit a proportion of fat similar to that deposited by C birds as they matured. The differences in adiposity of the two stocks of birds were innate and not a consequence of differences in maturation. Genetic selection dramatically affects adipose cellularity. Hood and Pym (1982) examined cellularity of abdominal adipose tissue in three selected lines of chickens and a control line at 9 wk of age. Eight generations of selection for increased body weight gain (Line W), increased food consumption (Line F), and decreased food conversion ratio (Line E) yielded birds with body weights significantly higher than body weight of a randombred control line (Line C). Abdominal fat and body fat were also affected by selection. Mean abdominal adipocyte volume significantly decreased in Lines E and W in comparison with that of Lines C and F. Abdominal adipocyte number increased as body weight increased. However, the authors suggested that line differences in abdominal fat are more reflective of the effects of mean adipocyte volume than of total adipocyte number. Increased fat cell number is a major contributor to abdominal adipose tissue growth in broiler chickens from 2 wk to more than 12 wk of age (Hood, 1982; Cherry et al, 1984). The observed increase in adipocyte number with age in this report was consistent with that reported by Hood (1982) and Cherry et al. (1984). A previous study (Cartwright et al., 1986b) showed that excessive fat deposition in chickens was not a result of hyperplasia but rather was the result of hypertrophy of fat cells. Likewise, in this study, both AC and C birds increased in body weight by approximately 2.5 times from 28 to 54 days of age. Mean adipocyte volume

TABLE 3. Mean (± SE) body composition by stock-age subclass1 Stock 2

Age

Water

Dry m a t t e r

Crude protein

Ether extract

Ash

(days) AC

28 54

a 69.1 ± .4 67.5 ± . 6 b

30.9 ± . 4 b 32.5 ± . 6 *

18.4 + 2abc 18.8 ± . 2 a

8.4 ± . 5 b 9.2 ± . 7 b

C

28 54

66.6 ± ^ 66.2 + . 5 b

33.4 + . 6 a 33.8 ± . 5 a

17.6 ± . 3 C 18.1 ± 2 b c

12.3 ± . 7 a 11.4 ± , 6 a

Values within a column with no common superscripts are significantly different (P-C05). 1

Differences between stocks were significant (P<.01) for all variables.

2

AC = Athens-Canadian stock; C = Cobb stock.

3.0 + 2 ab 3.3 ± . l a 2.8 ± . l b 2.9 ± , l b

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in C birds was twice the proportion found in AC birds at both ages. Body compositions for the two stocks were significantly different (Table 3). The body component that was most notably affected by stock was the ether extract value. At 28 and 54 days, respectively, C birds had a 46 and 24% higher proportion of ether extract than AC birds. At ages when C and AC birds were not significantly different in body weight, C birds had 33% higher ether extract values. The percentage dry matter was lower in the 28-day AC birds, but the dry matter content of 28-day C stock was not significantly different from those of the older birds from both stocks. The percentage protein from 28-day C birds was significantly lower than the percentage protein from 54-day AC birds. The change in percentage of the ether extract accounted for much of the aforementioned body composition differences. The C stock was fatter than the AC stock. The commercial broiler stock, C, was larger and fatter than was the unselected stock, AC. These observations confirm the results of an earlier study (Cartwright et al., 1986b), which used the same bird populations. Genetic selection, which has been used to improve commercial chickens, has yielded larger birds with higher percentages of body fat (Chambers et al., 1981). The commercial broiler stock already was depositing excessive amounts of adipose tissue when compared with the AC stock by 28 days of age. This early onset of accelerated fat accretion might be observed more predictably in small body weight breeds because of early maturation (Hood, 1983). At the termination of the experiment at 54 days of age, there was no indication

ADIPOSE CELLULARITY IN BROILERS

REFERENCES Allain, X., and J. Simon, 1982. Ineffiacite d'une suralimentation imposee des la naissance pour modifier le comportement alimentaire, la croissance et la composition corporelle ulterieurs du poulet. Reprod. Nutr. Dev. 22:27^0. Allen, C. E., D. C. Beitz, D. A. Cramer, and R. G. Kauffman, 1976. Biology of fat in meat animals. North Central Regional Research Publication No. 234. Research Division, College of Agriculture and Life Sciences, University of Wisconsin, Madison, WI. Becker, W. A., J. V. Spencer, L. W. Mirosh, and J. A. Verstrate, 1979. Prediction of fat and fat free live weight in broiler chickens using backskin fat, abdominal fat, and live body weight. Poultry Sci. 58:835842. Burton, K., 1956. A study of the conditions and mechanism of the diphenylamine reaction for the colorimetric estimation of deoxyribonucleic acid. Biochem. J. 62:315-323. Cartwright, A. L., 1986. Effects of hyperalimentation and lipectomy on abdominal adipose cellularity and feed intake in growing broilers. Poultry Sci. 65(Suppl. 1):21. (Abstr.)

Cartwright, A. L., 1987a. Adipocyte hypertrophy and voluntary feed intake in growing broilers. Poultry Sci. 66(Suppl. 1):77. (Abstr.) Cartwright, A. L., 1987b. Determination of adipose tissue cellularity. Pages 229-254 in: Biology of the Adipocyte: Research Approaches. G. J. Hausman and R. J. Martin, ed. Van Nostrand Reinhold Company, Inc., New York, NY. Cartwright, A. L., J. M. Leatherwood, and E. J. Eisen, 1986a. Insulin responsiveness of diaphragm tissue and adipose cellularity in mice selected for rapid growth. Growth 50:155-168. Cartwright, A. L., H. L. Marks, andD. R. Campion, 1986b. Adipose tissue cellularity and growth characteristics of unselected and selected broilers: Implications for the development of body fat. Poultry Sci. 65:10211027. Cartwright, A. L., J. P. McMurtry, and I. Plavnik, 1986c. Effect of early feed restriction on adipose cellularity of broilers. Poultry Sci. 65(Suppl. 1):21. (Abstr.) Chambers, J. R., J. S. Gavora,andA. Fortin, 1981. Genetic changes in meat-type chickens in the last twenty years. Can. J. Anim. Sci. 61:555-563. Cherry, J. A., W. J. Swartworth, and P. B. Siegel, 1984. Adipose cellularity in commercial broiler chicks. Poultry Sci. 63:97-108. Etherton, T. D., E. H. Thompson, and C. E. Allen, 1977. Improved techniques for studies of adipocyte cellularity and metabolism. J. Lipid Res. 18:552-557. Faust, I. M., P. R. Johnson, J. S. Stern, and J. Hirsch, 1978. Diet-induced adipocyte number increase in adult rats: A new model of obesity. Am. J. Physiol. 235:E279-E286. Giles, K. W., and A. Myers, 1965. An improved diphenylamine method for the estimation of deoxyribonucleic acid. Nature 206:93. Hess, C. W., 1962. Randombred populations of the Southern Regional Poultry Breeding Project. World's Poult. Sci. J. 18:147-152. Hirsch, J., and E. Gallian, 1968. Methods for the determination of adipose cell size in man and animals. J. Lipid Res. 9:110-119. Hood, R. L., 1982. The cellular basis for growth of the abdominal fat pad in broiler-type chickens. Poultry Sci. 61:117-121. Hood, R. L., 1983. Changes in fatty acid synthesis associated with growth and fattening. Proc. Nutr. Soc. 42:303-313. Hood, R. L., andR.A.E. Pym, 1982. Correlated responses for lipogenesis and adipose tissue cellularity in chickens selected for body weight gain, food consumption, and food conversion efficiency. Poultry Sci. 61:122127. Lemonnier, D., 1972. Effect of age, sex and site on the cellularity of the adipose tissue in mice and rats rendered obese by a high fat diet. J. Clin. Invest. 51:29072915. Lilburn, M. S., R. M. Leach, Jr., E. G. Buss, and R. J. Martin, 1982. The developmental characteristics of two strains of chickens selected for differences in mature abdominal fat pad size. Growth 46:171-181. Lin, C. Y., 1981. Relationship between increased body weight and fat deposition in broilers. World's Poult. Sci. J. 37:106-110. March, B. E., and G. Hansen, 1977. Lipid accumulation and cell multiplication in adipose bodies in White Leghorn and broiler type chicks. Poultry Sci. 56:886894.

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increased considerably in C birds whereas mean volume of AC adipocytes was static. If the observations from this study with AC and C birds are generally representative of phenomena in broilers, the increased adiposity of genetically selected meat-type broilers is more correctly attributed to adipocyte hypertrophy than to hyperplasia. The observed increase of adipocyte number as well as adipocyte volume might be ascribed to increases in lean body mass rather than age. In a previous study in which C and AC chickens were compared, a relationship between genetic selection for large lean body mass and the occurrence of large adipocytes was observed (Cartwright et al., 1986b). Suggestion of the same relationship was observed in a report that examined selected lines of mice differing in lean body mass and body composition (Cartwright et al., 1986a). Pond and Mattacks (1985) surveyed adipose tissue of 91 wild and captive birds. Total number of adipocytes was proportional to (body mass) 68 ; this requires that adipocytes in larger birds must also be larger to maintain similar body compositions. Collectively, these studies suggest a natural tendency for the physiological coupling of development of lean body mass and control of adipocyte proliferation and development and maintenance of adipocyte volume. The work of Hood and Pym (1982) show that this tendency can be overcome. The nature of this coupling process and its effect on genetic selection of broilers remains to be elucidated.

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March, B. E., C. MacMillan, and S. Chu, 1984. Characteristics of adipose tissue growth inhroiler-typechickens to 22 weeks of age and the effects of dietary protein and lipid. Poultry Sci. 63:2207-2216. March, B. E., C. Stanley, and C. MacMillan, 1982. The effect of feed intake on adipocytes in the abdominal fat pad of mature broiler-type chickens. Poultry Sci. 61:1137-1146. Munro, H. N., and A. Fleck, 1966. The determination of nucleic acids. Methods Biochem. Anal. 14:133-142. Nixon, T., and H. Green, 1984. Contribution of growth hormone to the adipogenic activity of serum. Endocrinology 114:527-532. Pfaff, F. E., and R. E. Austic, 1976. Influence of diet on development of the abdominal fat pad in the pullet. J. Nutr. 106:443-450. Pond, C , and C. A. Mattacks, 1985. Cellular structure of

adipose tissue in birds. J. Morphol. 185:195-202. Proudman, J. A., W. J. Mellen, and D. L. Anderson, 1970. Utilization of feed in fast and slow growing lines of chickens. Poultry Sci. 49:961-972. SAS, 1982. SAS User's Guide: Statistics. SAS Inst. Inc., Raleigh, NC. Schneider, B. S., I. M. Faust, R. Hemmes, and J. Hirsch, 1981. Effects of altered adipose tissue morphology on plasma insulin levels in the rat. Am. J. Physiol. 240:E358-E362. Smith, K. J., J. M. Leatherwood, and E. J. Eisen, 1983. Effects of preweaning and postweaning feed restriction on the development of polygenic obese mice. Growth 47:35-52. Zezulak, K. M., and H. Green, 1986. The generation of insulin-like growth factor-1 sensitive cells by growth hormone action. Science 233:551-553.

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