| The ostrich is the biggest, unflyable bird existing in the world, whose significant feeding value has assumed. Because of their incomplete developmental organ and system, low resistance, the disease inducing by the change of internal and external environment and the invasion of pathogen easily occur in ostrich chicks, thus, their growth were influenced. Nutrient absorption is important at all stages of life.The digestive tract, play significant roles in the final stages of nutrient digestion and assimilation. African ostrich has longer brood time, during the brood time, the growth and development is slow, the gastrointestinal disease is serious. So, in order to improve the brooding rate and decrease gastrointestinal disease rate for African ostrich chicks, some studies have been investigated about the African ostrich, but none have investigated the morphological development of the digestive tract of African ostrich chicks. Ghrelin is a brain-gut peptide that has been isolated as an endogenous ligand for the growth hormone secretagogue receptor (GHS-R) from the rat stomach. Ghrelin is predominantly produced by the X/A-like endocrine cells in the oxyntic mucosa of the stomach. Ghrelin regulates a myriad of physiological functions including appetite regulatory effects, regulation of water intake, gastrointestinal motility and regulation of hormone release and synthesis, thus, it can influence the growth and development in vertebrates. It was proved that ghrelin consists of 28 amino acids in mammals, ghrelin consists of 26 amino acids in the chicken. Ghrelin are found in the stomach, ileum, adrenal gland, heart atrium, breast, mucosa of oral cavity, esophagus, oviduct, fat, gallbladder, lymphocyte, kidney, spleen, lymphatic, heart, lung, muscle, cardiac muscle, ovary, testis, placenta, prostate, pancreas, thyroid, skin, pituitary gland, blood vessel in animals, and the distribution are changed gradually with age. However, there have been no studies on the ghrelin in African ostrich chicks.As mentioned above, the objective of this study was to investigate the morphological structure of the digestive tracts, developmental characteristics, distribution and developmental in ghrelin in the gastrointestinal tract of African ostrich chicks by the methods of paraffin section, HE staining, histochemical, immunohistochemistry(SABC), transmission electron microscopy (TEM) and reverse transcription polymerase chain reaction (RT-PCR) methods. The main contents and the research results are as follows:1. Morphological structure of the digestive tracts of African Ostrich Chicks90 d healthy African ostrich chicks were used in the present study to investigate the morphological structure of the digestive tracts of ostrich chicks by the methods of the gross anatomy, paraffin section, HE staining, PAS staining, AgNOR staining and TEM. The results indicated that the digestive tract consists of pharyngo-oral cavity, esophagus, proventriculus, gizzard, duodenum, jejunum, ileum, cecum, colon, and rectum. African ostrich has not crop. The oral-pharyngo cavity, proventriculus, gizzard, jejunum and colon are more developed than other birds, especially in the colon, the location accounted for most of the latter part of the entire abdominal cavity. The four layers, named in order from the lumen outward, are the mucosa, the submucosa, the muscularis, and the adventitia or serosa. The esophagus has grossus plica, the developed muscularis, developed esophageal glands; there are pars glandularis and pars non-glandularis in the proventriculus and in the pars glandularis, the glandular organ of glandular stomach is made up of simple tubular gland in the lamina propria and compound tubular glands in the submucosa; complex tubular glands composed of a large number of glandular cells, minor endocrine cells and mucous cells; the muscularis mucosa of the gizzard is very obviously, it is composed of internal-longitudinal and external-ring smooth muscles. The villi of small intestine is long and has branches, but it has not central chyle vessel. There are developed intestinal gland and many aggregated lymphoid nodule in the lamina proper of the duodenum. There are not duodenal gland in the submucosa. From duodenum to ileum, it is very obvious that intestinal villi have confluens and branches, the intestinal villi of jejunum is crook and showed a "S" shape, the number of aggregated lymphoid nodule is gradually decreasing; African ostrich has a pair developed cecum and very developed colon, the epithelium mucosae is stratified columnar epithelium, and there are many goblet cells between them, there are mucosa plica, the villi are short and developed. These features of the digestive ducts are possible concerned with food habit, and decide that African ostrich has stronger ability to digest and absorb food.2. Developmental morphology of the digestive tract of African Ostrich Chicks1,45 and 90d healthy African ostrich chicks were selected from the normal group. The development of the digestive tract of ostrich chicks was detailly observed by the gross anatomy and performing histochemistry and morphometry. The results of the present study showed the body weight, length and weight of digestive tract increased with age; the weights of the esophagus, proventriculus and duodenum (relative to the body weight) peaked on day 90, the weights of the gizzard, jejunum, ileum and large intestine (relative to the body weight) peaked on day 45; the thickness of simple tubular gland and compound tubular gland of proventriculus, the gizzard glandular height, the thickness of muscularis mucosa of the gizzard, the villus height and width, and muscle thickness were positively correlated with the age of the birds. The ratio of the villus height to the crypt depth differed among the segments of the small intestine and at the different time points: the ratio of the villus height to the crypt depth in the jejunum decreased from day 1 to day 90, while that in the duodenum and ileum decreased from day 1 to day 45, and increased from day 45 to day 90. The number of goblet cells in the intestinal villi and crypts increased rapidly up to postnatal day 45 and then decreased rapidly between day 45 and day 90. The number of goblet cells in the villi was highest in the jejunum on day 1, and in the ileum on day 45; while that in the crypt was highest in the ileum on days 1 and 90, in the duodenum on day 45. These results suggest that the digestive tract develops gradually from postnatal day 1 to day 90 and that the period up to postnatal day 45 is marked by significant developmental changes in the parameters reflective of the digestive capacity, such as the weight, length, and surface area of the intestine and the number of goblet cells. Therefore, in reared African ostrich chicks, feed management should be enhanced between postnatal day 1 and day 45.3. Distribution and developmental changes in ghrelin-immunopositive cells in the gastrointestinal tract of African ostrich chicks1,45 and 90d healthy African ostrich chicks were selected from the normal group. The distribution, morphological characteristics, and developmental changes of ghrelin-producing cells in the gastrointestinal tract of African ostrich chicks were investigated using immunohistochemistry. The results of the present study showed that Ghrelin-immunopositive (ghrelin-ic) cells existed in all regions of the gastrointestinal tract. In the proventriculus, most of the ghrelin-ic cells were observed at the base of glandular lobuli, and a few ghrelin-ic cells were observed in the basal zone of proventriculus plicae. In the gizzard, ghrelin-ic cells were scattered in the glandular epithelia of crypts. In the duodenum, jejunum, ileum, cecum, colon, and rectum, ghrelin-ic cells were scattered in the epithelia of crypts and villi. Two types of ghrelin cells were found; i.e., closed-type cells and cells with triangular or elongated shapes and with their apical cytoplasmic process in contact with the lumen (open-type cells), indicating that ghrelin cells can be classified into two cell types in the entire gastrointestinal tract. The more numerous ghrelin-ic cells in the proventriculus and gizzard were small and round-shaped, so-called closed-type cells. Few ghrelin-ic cells were elongated-shaped with apical cytoplasmic processes in contact with the lumen, so-called open-type cells. On the other hand, in the duodenum, jejunum, ileum, cecum, colon, and rectum, the more numerous ghrelin-ic cells had triangular or elongated shapes with their apical cytoplasmic processes in contact with the lumen (open-type cells). Few ghrelin-ic cells were round-shaped, so-called closed-type cells. Morphometric analysis revealed that ghrelin-ip cells were localized preferentially in the proventriculus; in the duodenum, jejunum, ileum, gizzard, cecum, colon, and rectum, the cell density gradually decreased. Interestingly, in the proventriculus, the number of ghrelin-ic cells increased gradually after birth until postnatal day 45 and peaked on day 45; in the gizzard and small intestine, the number of ghrelin-ic cells increased gradually after birth until postnatal day 90 and peaked on day 90. There were virtually no differences in the number of ghrelin-ic cells from postnatal day 90 to postnatal day 334 in the stomach and small intestine. By day 45 in the proventriculus and day 90 in the gizzard and small intestine, the number of cells reached a plateau and remained steady. The number of ghrelin-ic cells in the stomach and small intestine was highest in the jejunum and proventriculus on postnatal day 1, but highest in the proventriculus on days 45,90, and 334. These results of the present study clearly demonstrate that ghrelin-ic cells exist and the number of ghrelin-ip cells increases with age in the African ostrich gastrointestinal tract from postnatal day 1 to day 90; ghrelin may be involved in gastrointestinal tract development.4. Cloning and sequence analysis of Ghrelin gene in African ostrich chicksA pair of primers was designed and synthesized according to the gene sequence of chicken Ghrelin(ID:AB075215). RT-PCR method was used to amplify Ghrelin gene from the whole RNA of proventriculus tissue in the 45 d African ostrich chicks. After being cloned in pMD-18T, the PCR segment was determined as Ghrelin cDNA. The results of the present study showed the gene was 191 bp in length and exhibited a high homology with those from chicken (95%), duck(94%), goose (84%), turkey (83%), emu(84%)and Japanese Quail(82%). Mature ghrelins consists of 28 amino acid peptide in African ostrich chicks and exhibited the homology with those from chicken (73%), duck(69.8%), goose (74.6%), turkey (73%), emu(88.9%)and Japanese Quail(68.3%). The result of the present study indicated that Ghrelin gene is highly conservative in evolution according to the homology of the gene and amino acid sequences of bird ghrelin. This study provides a foundation for further analysis of structure, expression and regulation of Ghrelin gene in African ostrich.5. The Distribution and Developmental Changes of ghrelin mRNA Expression in the gastrointestinal tract of African ostrich chicks1,45 and 90d healthy African ostrich chicks were selected from normal group. The distribution, and developmental changes of ghrelin mRNA expressio in the gastrointestinal tract of African ostrich chicks were investigated using RT-PCR. A pair of primers was designed and synthesized according to the gene sequence of chicken Ghrelin(ID:AB075215). And the total RNA of different tissues was extracted to determine the abundance of ghrelin mRNA by RT-PCR. Ninety-day-old female African ostriches were used to investigate the distribution of ghrelin mRNA expressio in various tissues, the results showed ghrelin mRNA existed in the proventriculus, gizzard, duodenum, ileum, cecum, and rectum, there were no expression in the jejunum and colon. Developmental changes in the ghrelin mRNA in the African ostrich stomach and small intestine, the results of the present study showed that ghrelin mRNA existed in the proventriculus, there were no expression in other tissue on day 1; ghrelin mRNA existed in the proventriculus, gizzard, and ileum, there were no expression in the duodenum and jejunum on day 45; ghrelin mRNA existed in the proventriculus, gizzard, duodenum and ileum, there were no expression in the jejunum on day 90 and 334.These results of the present study clearly demonstrate that ghrelin mRNA exist and the distribution of ghrelin mRNA changes with the age in African ostrich gastrointestinal tract from postnatal day 1 to day 90; ghrelin may be involved in gastrointestinal tract development.
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