SCANNING electron - microscopy has

Characteristics of the Absorptive Surface of the Small Intestine of the Chicken from 1 Day to 14 Weeks of Age 1 R. C. BAYER, C. B. CHAWAN, F. H. BIRD AND S. D. MUSGRAVE Department of Animal and Veterinary Sciences, University of Maine, Orono, Maine 04473 (Received for publication April 15, 1974) ABSTRACT The epithelial surface of the intestine of chicks 1 day to 14 weeks of age was examined with both scanning and transmission electron microscopes. Most of the intestinal villi were seen to be plate-like structures. During the first week post-hatch, goblet cell orifices were easily seen. Goblet cell pores were not readily apparent in the older birds. During this stage of early development discontinuity was seen among some epithelial cells. The villi of birds one week or older showed a more convoluted mucosal surface than the younger birds. Areas of cell extrusion were observed at the tips of some villi. The three dimensional cylindrical structure of the microvilli was shown with the scanning electron microscope. SCANNING electron - microscopy has added a new dimension to the study of gastrointestinal morphology. Grey (1972) studied the developmental aspects of the chick duodenal epithelium from 1 day's incubation to 1 week past hatch with the scanning electron microscope (SEM). Grey observed that at 14 to 16 days of incubation the previllus ridges began development forming the pattern for later villus development. Numerous mammalian species have been studied with the SEM. Studies of the pig intestine with the SEM reveal finger-like villi (Mouwen, 1971; Waxier, 1972). Studies on the rat jejunum revealed a plate-like structure of the villi (Rao and Williams, 1972). Leafshaped villi have been observed in SEM studies of bovine intestine (Musgrave et al., 1973). Calhoun (1954) has described the histology of the intestine of the chick intestine and Humphrey and Turk (1970) have examined the ultrastructure of the intestine. The present study was designed to observe the absorptive surface of the intestine primarily with the 1. This work was funded by Hatch funds from the Maine Agricultural Experiment Station in the Department of Animal and Veterinary Sciences. POULTRY SCIENCE 54: 155-169, 1975 SEM but also with transmission electron microscope (TEM). PROCEDURE Twenty, day-old broiler strain chicks were fed a modified New England College Conference starter ration containing 24% protein and 3216 calories of metabolizable energy per kg. of feed. Animals were sacrificed periodically until they reached 14 weeks of age. Two chicks were sacrificed at one day, four days, one week, ten days, and at two, three, four, five, seven, ten and fourteen weeks of age. Specimens were prepared for SEM examination by the procedure of Bayer et al. (1974). Samples of duodenum, ileum and jejunum were removed from the birds as follows: The duodenal sample was taken at the mid point between the ventriculus and the cystic duct; the jejunum sample was taken mid point from the cystic duct and the yolk stalk; and the ileum sample was taken mid point between the yolk stalk and the caeca. Samples were immersed in 4% glutaraldehyde made up in 0.2M phosphate buffer (ph 7.2) (Sjostrand, 1967). Specimens were post-fixed in 1% osmium tetroxide. Samples were dehydrated in graded ethanol solutions (50%, 90%, 95%, 100%) and vacuum dried in a freeze-drying apparatus. The dried specimens were mount- 155

156 R. C. BAYER, C. B. CHAWAN, F. H. BIRD AND S. D. MUSGRAVE FIG. 1. Scanning electron micrograph of a duodenal villus from a day-old chick. The inset shows goblet cell pores pointed out by arrows. ed on aluminum stubs with electrically conducting cement. 2 Some specimens were also taken for transmission electron microscopy (TEM). TEM tissue preparation consisted of the same fixation procedure as was used for SEM. Tissues were dehydrated in acetone solutions (50%, 75%, 95%, 100%) and embedded in Spurr's embedding medium (Spurr, 1969). RESULTS AND DISCUSSION SEM examination of the duodenum from a day-old chick revealed plate-like villi (Fig. 1). At a higher magnification, (Fig. 1 inset), 2. Television Tube KoteG. C. Electronics Division of Hydrometals, Inc. Rockford, Illinois. goblet cell orifices are seen along with cell outlines toward the villus tip. Some of the villi examined from day-old chicks showed discontinuity of the epithelium (Fig. 2). The surface of the duodenum of the day-old chick (Fig. 1) is relatively smooth in comparison to the surface of the 7-day old chick duodenum, (Fig. 3). The villi surfaces of a 7-day old chick have the characteristic folds and recesses seen on villi of mature birds. This type of convoluted surface was observed in most birds one week or older (Figs. 3A, B, C and D). Observation of the jejunal villi from dayold chicks indicated two morphological types of villi, broad finger-shaped villi and more narrow plate-like villi (Fig. 4). Outlines of

SURFACE OF INTESTINE 157 FIG. 2. Scanning electron micrograph of an area of a duodenal villus from a day-old chick. Arrows point to discontinuity in the epithelium.

158 R. C. BAYER, C. B. CHAWAN, F. H. BIRD AND S. D. MUSGRAVE FIG. 3. Scanning electron micrographs of duodenal villi showing characteristic folds and recesses. Figure 1A and B were taken from 1-week old birds; Figure 1C was taken from a 2-week old bird; and Figure ID was taken from a 12-week old bird. individual cells were often observed (Fig. 5). Disruptions were seen in the epithelium (Fig. 6). Sub-epithelial crevices were observed in some of the jejunal villi from day-old chicks (Figs. 4 and 6A). The jejunal villi from birds one week of age and older (Fig. 7) were indistinguishable in morphology from the villi of the duodenum. Shape variation in the villi from the ileum of day-old chicks was similar to that seen in the jejunum. There were areas which showed epithelial disruptions on the villi (Fig. 8). Goblet cell pores were observed in some of the villi (Fig. 9). At one week of age or older numerous folds and recesses were easily viewed (Fig. 10).

SURFACE OF INTESTINE 159 ^» m my ^ > > 25 *" gfcrfc $ # FIG. 4. Scanning electron micrograph of jejunal villi from a day-old chick.

160 R. C. BAYER, C. B. CHAWAN, F. H. BIRD AND S. D. MUSGRAVE FIG. 5. Scanning electron micrograph of a jejunal villus. Arrows point to outlines of individual cells.

SURFACE OF INTESTINE 161 FIG. 6. Scanning electron micrographs showing disruption of epithelium in the jejunum of day-old chicks.

162 R. C. BAYER, C. B. CHAWAN, F. H. BIRD AND S. D. MUSGRAVE *IL -f* : : 50ja FIG. 7. Scanning electron micrographs of villi from 14-week old chickens. Note the numerous folds and recesses.

SURFACE OF INTESTINE 163 25 JU 3..y m FIG. 8. Scanning electron micrograph of villi in the ileum of a day-old chick. Arrows point to epithelial crevices. Inset shows an enlarged view of an epithelial crevice.

164 R. C. BAYER, C. B. CHAWAN, F. H. BIRD AND S. D. MUSGRAVE mat VM \ it FIG. 9. Scanning electron micrograph of ileum villi with arrows pointing to goblet cell pores. # 4

SURFACE OF INTESTINE 165 FIG. 10. Scanning electron micrograph showing the convoluted surface of the ileum villi from a one-week old chick. The inset shows similar appearing villi from a 14-week old bird.

166 R. C. BAYER, C. B. CHAWAN, F. H. BIRD AND S. D. MUSGRAVE FIG. 11. Scanning electron micrograph of a villus showing an extrusion area. The inset is a transmission electron micrograph showing the same phenomenon.

SURFACE OF INTESTINE 167 Hi fvss i,v fegra FIG. 12. Transmission electron micrograph showing the appearance of microvilli. The inset is a scanning electron micrograph showing the three-dimensional aspects of microvilli. Imondi and Bird (1966) demonstrated that epithelial cells of the chick intestine migrate from the crypts to the tips of the villi where extrusion takes place. The extrusion zone was readily seen in the SEM examination. Figure 11 shows several cells in an extrusion area separating from surrounding cells about to be sloughed. Figure 11 inset shows an area of extrusion as seen with the TEM. Accumulation of electron charges caused separation of the microvilli revealing their three-dimensional detail (Fig. 12 inset). Figure 12 shows how the microvilli appeared when examined with the TEM. Examination of the intestinal mucosa reveals the large absorptive surface area as a result of the presence of microvilli. Goblet cell orifices were not always apparent with SEM examination. TEM studies showed that there are microvilli present on the surface of goblet cells that may be capable of covering the pores except in a state of active secretion (Fig. 13A and B). There are similarities and also variations in villus shape as seen in other species compared with the chicken. In mice the villi were found to be plate-like, similar to the ones found in chicken samples (Rao and

168 R. C. BAYER, C. B. CHAWAN, F. H. BIRD AND S. D. MUSGRAVE 2 u H; >, \ s *v X V. ^N. S * ** v^*s * % X. ^Wi! tfw FIG. 13. Transmission electron micrograph of goblet cells (GC). Note the presence of microvilli on the goblet cell. Fig. 13B shows active secretion of a goblet cell.

SURFACE OF INTESTINE 169 Williams, 1972). In the terminal ileum rounded finger-shaped villi have been observed. There were some leaf-like villi found near mounds associated with Peyer's patches (Owen and Jones, 1974). These mounds were not observed in the chicken as there are no Peyer's patches. The shape of villi seen in the pig jejunum were primarily finger-like rather than plate-like (Mouwen, 1971; Waxier, 1972). Microvilli from the chicken mucosa appeared more dense than those observed in the pig (Mouwen, 1971; Waxier, 1972). This should yield a greater potential for absorptive efficiency. REFERENCES Bayer, R. C, F. H. Bird, S. D. Musgrave and C. B. Chawan, 1974. A simple method of preparation of gastrointestinal tract tissue for scanning electron microscopy. J. Animal Sci. 38: 354-356. Calhoun, M. L., 1954. Microscopic Anatomy of the Digestive System of the Chicken. The Iowa State College Press. Ames, Iowa. Grey, R. D., 1972. Morphogenesis of intestinal villi. I. Scanning electron Microscopy of the duodenal epithelium of the developing chick embryo. J. Morphol. 137: 193-214. Humphrey, C. D., and D. E. Turk, 1970. An electron central area project in poultry breeding at Purdue University. He also was entrusted with setting up the other regional programs with the title of National Coordinator. During this period he also served as consultant in poultry breeding in Puerto Rico, Egypt, Turkey, and India. In 1956 he joined the genetics staff of Kimber Farms, Fremont, California, retiring from that post in 1968. "He is a fellow of both the Poultry Science Association and the American Association for the Advancement of Science and the author of over 90 scientific papers and the book "Practical Poultry Breeding." " The Award for Distinguished Service in the Field of Agriculture was presented at a special recognition luncheon. The citation read, in part: "Dr. Don C. Warren devoted nearly a half century of work with NEWS AND NOTES (Continued from page 154) microscopic investigation of chick intestinal epithelial cells. Poultry Sci. 49: 1399. Imondi, A. R., and F. H. Bird, 1966. The turnover of intestinal epithelium in the chick. Poultry Sci. 45: 142-147. Mouwen, J. M. V. M., 1971. White scours in piglets. Vet. Path. 8: 401-413. Musgrave, S. D., R. C. Bayer, T. A. Bryan, F. H. Bird and C. B. Chawan, 1973. Bovine G. I. tract mucosa as studied by the SEM. J. Dairy Sci. 56: 1381. Owens, R. L., and A. L. Jones, 1974. Epithelial cell specialization within human Peyer's patches: an ultrastructural study of intestinal lymphoid follicles. Gastroenterology, 66: 189-203. Rao, N. S., and A. W. Williams, 1972. Normal and ischemic jejunal mucosa of mice scanning electron microscope study. J. de Microscopie, 15: 219-224. Sjostrand, F. S., 1967. Electron Microscopy of Cells and Tissues. Academic Press, New York. Spurr, A. R., 1969. A low viscosity embedding medium for electron microscopy. J. Ultrastructure Res. 26: 231-243. Takeuchi, A., and J. Zeller, 1972. Scanning electron microscopic observations on the surface of the normal and spirochette-infested colonic mucosa of the Rhesus monkey. J. Ultrastructure Res. 40: 313-324. Waxier, G. L., 1972. Lesions of transmissible gastroenteritis in the pig as determined by scanning electron microscopy. Am. J. Vet. Res. 33: 1323-1328. the domestic fowl. His 45 years of active research included eight years with the U.S. Department of Agriculture, 25 years as Poultry Geneticist at Kansas State University, and 12 years as Geneticist with Kimber Farms, Inc. of Fremont, California. "During his 25 years of research and teaching at Kansas State University, he made many contributions to the field of poultry breeding and genetics. His exhaustive research culminated in over 90 scientific papers, a comprehensive book entitled "Practical Poultry Breeding," a chapter on "Formation of the Hen's Egg" in another book, and a color film made in the 1930's on the same subject. Dr. Warren has stated that it was at Kansas State University where he and his associates became the first men to observe the fascinating process of egg formation in a live hen. (Continued on page 199)