COMPARATIVE STUDY OF PANCREATIC ENZYME ACTIVITY AND IT S HISTOLOGY IN NATIVE AND BROILER CHICKS (Studi Perbandingan Aktivitas Enzim dan Histologi dari Pankreas Ayam Kampung dan Broiler) HANNY I. WAHYUNI 1 and N.P. ROXAS 2 1 Faculty of Animal Agriculture Diponegoro University Jl. Drh. Soejono Koesoemowardojo, Tembalang Campus, Semarang 2 Institute of Animal Science College of Agriculture, University of the Philippines Los Baños, Laguna ABSTRAK Penelitian ini bertujuan untuk membandingkan kapasitas pancreas pada 44 ekor ayam kampung dan 44 ekor broiler dalam kaitannya dengan aktivitas relatif enzim amilase dan lipase serta histologinya pada umur 1, 7, 14 dan 21 setelah menetas. Aktivitas relatif amilase dan lipase dari ayam kampung dan broiler naik dengan konsisten dan mencapai puncak pada umur 21 hari. Amylase dan lipase pada broiler menunjukkan relatif aktifitas yang lebih tinggi dibanding pada ayam kampung. Hal ini sesuai dengan hasil observasi histologinya. Nampak bahwa ayam kampung mempunyai pankreas dengan kepadatan acini yang lebih sedikit dibandingkan pada broiler. Kesimpulan yang dapat diberikan adalah bahwa kepadatan acini yang rendah menunjukkan rendahnya kapasitas pancreas pada ayam kampung yang mempengaruhi rendahnya aktivitas relatif dari amylase dan lipase. Kata Kunci: Ayam Kampung, Broiler, Pancreas, Enzym pencernaan, Histologi ABSTRACT The purpose of this study is to compare the capacity of pancreas in 44 day-old native birds and 44 day-old broiler chicks in term of the relative activity of amylase and lipase as well as its histology at 1, 7, 14 and 21 days after hatching. The relative activities of pancreatic amylase and lipase increased rapidly to its maximum at day 21, both in native and broiler chicks. Both amylase and lipase of broiler showed higher relative activity than in native chicks. This finding was supported by the pancreatic histological figures. It was noted that native chicks have less dense acini and larger area of connective tissue compared to those found in broiler chicks. It is concluded that less dense of acini showed the lower capacity of native chicks pancreas that affected relatively low activity of amylase and lipase. Key Words: Native Chicks, Broiler, Pancreas, Digestive Enzyme, Histology INTRODUCTION Among all digestive organs, pancreas has a large capacity in producing several digestive enzymes that plays an important role in digestion processes. The influence of age has been clearly demonstrated in digestive enzyme activity. One study reported that amylase activity may increase following the age of birds (RODEHEAVER and WYATT, 1986). A separate study noted that pancreatic amylase, trypsin and lipase secreted into the duodenum were low at day 4 and increased by 100, 50 and 20 folds at age 21 days in broiler chicks, respectively (NOY and SKLAN, 1995). NITZAN et al. (1991) found that the relative activity of lipase in chicks increased until eight days of age, plateaued through day 17, then slowly decreased. Breeds may also contribute to the differences in digestive enzyme activity. Pancreatic digestive enzyme activities were similar between broiler and egg-type of chickens. However, the activities in the small intestinal contents were lower in meat-type chickens at the same age (NIR et al., 1993). UNI et al. (1995) showed that enzyme secretion 678
to the duodenum was higher per gram of feed intake in heavy-strain birds on four days after hatching but thereafter, no differences were apparent. Native chicken seen today is a product of long and continuous processes of natural crossing and selection between imported purebreds and the native stock. This indigenous chicken is used for both meat and eggs. Compared to the commercial breeds and other meat type stocks, native chickens are generally poor egg producers and the growth rate is slow. Nevertheless, native chickens are raised because of their ability to survive in scavenging conditions under minimal feeding, care and housing. On the other hand native chickens possess some special genetic merits, such good meat and egg quality as well as, high resistance to diseases and heat stress. Their eggs contain the desired intense yellow pigmentation, while their meat is widely preferred because of taste, leanness, and suitability for special dishes. Furthermore, native chickens are good breeders, foragers and are efficient mothers. This type of chicken is noted for their hardiness and resistance. They are able to survive and reproduce even under the very harsh environmental conditions. Hence, this genetic resource has to be conserved. Other basic potentials of the native chicken also need to be discovered in order to learn more about this species. Comparative study related to pancreatic histology, amylase and lipase activity in native and broiler chickens are non-existent. It is commonly known that native chickens are slow growers and have lower egg production, even when they are fed with high quality diet. Unlike in broiler, expensive dietary formulations should be avoided for native chickens because they may prove uneconomically. The last invariably points to a physiological explanation. MATERIALS AND METHODS Two groups consisting of 44 day-old broilers and 44 heads of day-old native chick were used in this experiment. Chicks were fed with commercial broiler booster ration from day-old to day 7, then shifted to broiler starter feed from day 8 up to the end of the experiment. Feed and water were made available at all times during the experimental period. Similar management practices were carried out in all the birds used. At days 1, 7, 14 and 21, eleven chicks from each group were weighed individually and sacrificed by cervical dislocation. The abdominal cavity was immediately opened and the whole viscera were removed. The pancreas was detached from intact intestine. Each pancreas from 9 chicks was homogenized using a mortar and pestle, while the left pancreas was used for histological preparation. Tissue sample of pancreas were immediately placed in a 10% formaldehyde solution, processed for histology preparations using the Humason Technique and stained with hematoxylin-eosin (HUMASON, 1979). Ten ml of phosphate buffer (ph 7.4) was added during the homogenization process of pancreas and when ready was poured into the test tubes following centrifugation at 10,000 rpm for 30 minutes. The supernatant was used for enzyme assay. The amylase activity was measured by the ability to reduce 3,5 dinitrosalicylic acid as explained by PUBOLS (1991) and the lipase activity based on the liberation of free fatty acid from coconut oil with specified amount of enzyme (CASTILLO, 1997). To compute the specific activity of the enzyme the protein content of pancreas should be measured. The protein content of the pancreas was measured using Bradford method. The relative activity of the enzyme was computed as follow: Specific activity x pancreas weight ------------------------------------------- x 100 Body weight The experiment was conducted in Completely Randomized Design. Significant means among various ages were judged using Duncan s Multiple Range Test (DMRT). Student s t-test was used to determine the differences between strains at the same age. RESULTS AND DISCUSSION The relative weight of pancreas and relative activity of pancreatic amylase and lipase were presented in Table 1. Considering the relative weight of pancreas, the two strains of chicks 679
except at day 14 was not significantly different. The data showed that the age was significantly affecting the relative weight of pancreas. The relative weights of pancreas in broilers were found increasing up to seven days of age and stabilized up to day 21. In native chicks, its maximum relative weight was attained at 14 days of age and declined thereafter. According to O'SULLIVAN et al. (1992a), the relative weight of pancreas declined from day 8 to 21 days of age. The pancreas of heavy- and lightstrain chicks matured together at seven days after hatching (DROR et al., 1977). Statistical analysis of the relative activity of pancreatic amylase and lipase in both native and broiler chicks were influenced by age. Comparing to the relative activities of amylase and lipase at the same age between the two strains of chicks revealed significant differences in all ages. As it is shown in Table 1, broiler had significantly higher relative activity of amylase and lipase at 1, 7, 14 and 21 days of age compared to those found in native chicks. According to O SULLIVAN et al. (1992b), selection for a higher body weight in broiler chicks would influenced their feeding behavior. They became hyperphagic. Observation was made on the relative weight of duodenal chyme in these two strains of chicks. The relative weight of broilers duodenal chyme was 0.39 g/100 g BW heavier than that of the native chicks (1.27 vs 0.88). The high digesta in small intestine would stimulate digestive enzyme syntheses in pancreas. As the age increases, feed consumption also increases resulting in the increase of intestinal chyme. The intestinal chyme serves as a mechanical stimulus. Mechanical dilation of the intestinal wall stimulates the digestive enzyme synthesis (BRANNON, 1990). On the other hand, according to O SULLIVAN et al. (1992b) that selection for high body weight affected the feeding behavior of chicken. As the result, broiler chicks became hyperphagic in selected group but hypophagic in unselected group, i.e. the native chicks. Due to hypophagic behavior, less stimulation may have occurred in native chicks, leading to reducing in synthesis and secretion of digestive enzyme. Table 1. Pancreas relative weight and relative activities of pancreatic amylase and lipase in broiler and native chicks at different ages Parametres Age strains (days) Broiler Native T-test Pancreas relative weight (g/100 g body weight) Amylase relative activity (unit/100 g body weight) Lipase relative activity (unit/100 g body weight) 1 0.21 B ± 0.05 0.23 C ± 0.03 NS 7 0.52 A ± 0.07 0.58 B ± 0.11 NS 14 0.57 A ± 0.07 0.68 A ± 0.06 ** 21 0.55 A ± 0.11 0.59 B ± 0.05 NS 1 421.88 D ± 114.45 55.92 D ± 11.05 ** 7 1193.20 C ± 301.24 135.17 C ± 26.13 ** 14 3661.61 B ± 531.72 341.05 B ± 68.40 ** 21 5635.77 A ± 1067.03 1163.14 A ± 209.45 ** 1 454.42 D ± 160.39 107.24 D ± 19.60 ** 7 2899.1 C ± 414.83 336.41 C ± 80.99 ** 14 5742.2 B ± 954.11 1007.72 B ± 121.19 ** 21 7080.50 A ± 1486.20 1886.04 A ± 311.33 ** Means in each parameter at the same column with similar letters are not significantly different at 1% level; ** Significantly different at 1 % level; NS=non significant 680
Pancreatic histological figures in native and broiler chicks at days 1, 7, 14 and 21 were shown in Plates 1 and 2. The pancreas is regarding as an exocrine and endocrine glands. The exocrine tissue of the pancreas represents at least 99% of the total fresh chicken pancreas, while remainder is the endocrine portion (HAZELWOOD, 1976). According to HILL (1971) and TELFORD and BRIDGMAN (1995), the exocrine or the glandular acini is composed of six to eight low columnar or irregular pyramidal serous cells surrounding a small lumen (Plate 2d). The acinar cells of native chicks pancreas at day-old were smaller as compared to that of the broilers at the same age (Plates 1b vs. 1a). However, after seven days, they have almost similar size of acinar cells (Plates 1d vs. 1c). The acini of native chicks pancreas at day 7 were already developed which is more or less similar to that of the acini of the day-old broilers (Plates 1d vs. 1a). As the chicks of both strains grow older, the acinar developed bigger as seen in Plate 2c for broiler chicks and Plate 2d in native chicks. At the same magnification, more connective tissues in the pancreas at day-old chicks are present and could be seen widely spread (Plates 1a and 1b) as compared to that in the older chicks (Plates 2c and 2d). Thus, the pancreas of day-old chicks have smaller and less number acini than those at 21 days. This feature implies that in young chick, less enzymes are produced. It is also expected that in young chicks, there is lower enzyme activity compared to the older chicks. These findings support the results obtained in the measurement of relative activities of pancreatic amylase and lipase; the activities of these two enzymes increase as the age increased (Table 1). Plate 1. Cross section of chicks pancreas at days 1 and 7 Longitudinal section in day-old broiler chick (800x) A = acinar gland; CT = connective tissue Longitudinal section in day-old native chicks (800x). A = acinar gland; CT = connective tissue. Transverse section in 7 day-old broiler chick (800x) Transverse section in 7 day-old native chick (800x) A = acinar gland; CT = connective tissue; L = lumen 681
Plate 2. Cross section of chicks pancreas at days 14 and 21 Longitudinal section in 14 day-old broiler chick (800x) showing the height of the acinar gland (A); Longitudinal section in 14 day-old native chick (800x) showing the height of the acinar gland (A); CT=Connective tissue; Transverse section in 21 day-old broiler chick (800x) showing the width cell of the acinar gland (A) and the lumen (L); Transverse section in 21 day-old native chick (800x) showing the individual cell of the acinar gland (1 to 8) and the lumen (L) Comparing to the two strains of chicks, the native chicks have less dense acini and larger area of connective tissue compared to those found in the commercial broiler chicks (Plates 1a vs. 1b; Plates 1c vs. 1d; Plates 2a vs. 2b and Plates 2c vs. 2d). The pancreas weight and growth in these two strains of chicks also support these findings. It was noted that the native chicks pancreas have the same relative weight to broiler chicks (Table 1), hence it could be said that both chicks pancreas have the same growth rate. On the other hand, at 800 x magnification level, the histological features showed that native chicks have wider connective tissue but similar size of acini. This suggests that the connective tissue in the native chicks pancreas would occupy a bigger space rather than the acinar cells. Consequently, the pancreas of native chicks would produce less enzymes. This result agrees with the findings in enzyme activity measurement. Pancreatic amylase and lipase activities measured in native chicks were lower than those found in the commercial broiler chicks (Table 1). CONCLUSIONS It could be concluded that the less dense of acini showed the lower capacity of native chicks pancreas in the production of amylase and lipase as compare to those found in broiler. This was showed by the low relative activity of these 2 enzymes in native chicks within three weeks post hatch. REFERENCES BRANON, P.M. 1990. Adaptation of the exocrine pancreas to diet. Annu. Rev. Nutr. 10: 88 105. 682
CASTILLO, E.M. 1997. Application of biochemical techniques in the purification of biologically active compounds. Hand out in Chem 263 (Analytical Biochemistry). Institute of Chemistry, University of the Philippines Los Baños, College, Laguna. DROR, Y., I. NIR and Z. NITZAN. 1977. The relative growth of intestinal organs in light and heavy breeds. British Poult. Sci. 18: 493 496. HAZELWOOD, R.L. 1976. The pancreas. In: Avian Physiology. 3 rd Edition. STURKIE, P.D. (Ed.) Springer-Verlag, New York. pp. 383 388. HILL, K.J. 1971. The structure of the alimentary tract. In: Physiology and Biochemistry of Domestic Fowl. BELL, D.J. and B.M. FREEMAN (Ed.). Academic Press. London. 1: 1 23. HUMASON, G.I. 1979. Animal Tissue Techniques. 4 th Edition. San Francisco:Witt. Freeman and Co. pp. 78. NIR, I., Z. NITZAN and M. MAHAGNA. 1993. Comparative growth and development of the digestive organs and of some enzymes in broiler and egg type chicks after hatching. Poult. Sci. 34: 523 532. NITZAN, Z., G. BEN-AVRAHAM, ZIPORA ZOREF and I. NIR. 1991. Growth and development of the digestive organs and some enzymes in broiler chicks after hatching. Poult. Sci. 32: 515 523. NOY, Y. and D. SKLAN. 1995. Digestion and absorption in the young chick. Poult. Sci. 74: 366 373. O SULLIVAN, N.P., E.A. DUNNINGTON, A.S. LARSEN and P.B. SIEGEL. 1992a. Correlated responses in lines of chickens divergently selected for fifty-six-day body weight. 2. Organ growth, deoxyribonucleic acid, ribonucleic acid, and protein content. Poult. Sci. 71: 598 609. O SULLIVAN, N.P., E.A. DUNNINGTON, A.S. LARSEN and P.B. SIEGEL. 1992b. Correlated responses in lines of chickens divergently selected for fifty-six-day body weight. 3. Digestive enzyme. Poult. Sci. 71: 610 617. PUBOLS, M.H. 1991. Ratio of digestive enzymes in chick pancreas. Poult. Sci. 70: 337 342. RODEHEAVER, D.P. and R.D. WYATT. 1986. Distribution of α amylase activity in selected broiler tissue. Poult. Sci. 65: 325 329. TELFORD, I.R. and C.F. BRIDGMAN. 1995. Introduction To Functional Histology. 2 nd Ed. Harper Collins College Publisher. pp. 316 336. UNI, Z., Y. NOY and D. SKLAN. 1995. Posthatch changes in morphology and function of the small intestine in heavy and light-strain chicks. Poult. Sci. 74: 1622 1629. 683