Research On Early Development Of The Pigeon Small Intestine And Its Regulation By Carbohydrates

This study was conducted to evaluate the digestive capacity in domestic pigeons (Columba livia) by determining the intestinal morphology, mucosal and pancreatic enzyme activities, and mRNA expression of the digestion/absorption related genes during pre-and posthatch development. Furthermore, the nutritional regulation on intestinal development prior to hatching in pigeons by in ovo feeding, a method of supplementing exogenous nutrients into the amnion of the late-term avian embryo, was also evaluated. The main results are listed as follows:1. Early development of intestinal morphology and digestive enzyme activities in pigeonsVilli were rudimentary at12d of incubation (E12) and rapid increase of villus height and area was observed from E16. In the duodenum and jejunum, villus size continued to increase through14d posthatch (D14). Villus width increased dramatically from E14to24h after hatch (P<0.05). At day of hatch (DOH), crypts invagination was not complete throughout the small intestine, and increased intensively during the first3d posthatch (P<0.05). Villus area and crypt depth increased in parallel in the duodenum and jejunum and more slowly in the ileum (P<0.05). Enterocyte density peaked at3d after hatching in all segments. The mucosa DNA content increased linearly from E12to DOH, whereas the protein/DNA increased from D3(P<0.05). Mucosa enzyme activities were detectable for maltase, aminopeptidase-N (APN), Na+K+ATPase, and alkaline phosphatase (ALP) at E12, and for sucrase at E14. The disaccharidase mass specific activity peaked at D8and increased most dramatically in the jejunum as compared with the duodenum and ileum (P<0.05). Na+K+ATPase activity increased linearly from E12to D14, while APN changed slightly after D3(P<0.05). The ALP activity increased most dramatically before hatching (P<0.05). Intestinal total enzyme activities exhibited a steady increase after3-5d posthach, which was highly correlated with BW (P<0.05). Trypsin and chymotrypsin were detectable at E14, while amylase activity was detectable at E16. Pancreatic enzymes indicated patterns somewhat similar to those for intestinal enzymes.2. Cloning and analyzing of pigeon brush border enzymes and nutrient transporter genesThe cDNA fragments for sodium glucose transporter SGLT1, glucose transporter GLUT2, oligopeptide transporter PepTl, aminopeptidase-N APN and sucrase-isomaltase SI were isolated and cloned using reverse transcription PCR, containing946bp,762bp,946bp, and985bp and700bp nucleotides, respectively. These cDNA fragments were submitted to Genbank with accession number JN887478, JN887480, JN887476, JN887477, and JN887479, respectively. The predicted amino acid sequence for pigeon SGLT1was more than88%identical to Zebra Finch, turkey and chicken, and showed highest identical to Zebra Finch. The pigeon SGLT1amino acid sequence also showed high identical to mammalias, such as human, sheep and mice, and they were located in the same branch in phylogenetic trees. The predicted amino acid sequences for pigeon GLUT2, PepTl, APN and SI were highly identical to those of chicken, and were located in different branches in phylogenetic trees as compared to mammalias.3. The ontogeny of nutrient transporter and brush border enzymes gene expressionTo better understand the digestive capacity in pigeons, this part was conducted to evaluate nutrient transporters and digestive enzymes gene expression in small intestine and yolk sac membrane (YSM) during pre-and posthatch development. Intestine was collected at embryo d12,14and16, day of hatch, and d1,3,5,8and14posthatch. YSM was collected at embryo d12,14,16and day of hatch. The mRNA expression of each gene was assayed using real-time PCR. The examined genes were all detectable in pigeon small intestine at E12. Expression of intestinal nutrient transporters increased linearly (P<0.05) with age, whereas that of APN increased quadratically (P<0.05) and SI increased cubically (P<0.05). The APN mRNA expression showed a decline from E14to E16, and subsequent increase to D14 (P<0.05), while the greatest mRNA quantities for SI were observed at day of hatch and D8after hatch (P<0.05). Levels of PepTl mRNA were greatest in the duodenum, GLUT2and SGLT1mRNA were greatest in the jejunum, and APN were greatest in the ileum (P<0.05). The YSM expressed all the examined genes. The YSM-expressed genes decreased between embryo d16and day of hatch, whereas intestine-expressed genes increased (P<0.05).4. Effects of in ovo feeding of carbohydrates on growth and energy status in pigeonsThe optimum time for in ovo injection was identified as14.5d of incubation by determining changes of pigeon amniotic fluid volume.200fertile eggs with viable embryos were identified by number and weighed. The eggs were then randomly distributed into5groups of4replications. The injection solutions were:(Ⅰ)15g/L maltose (M)+15g/L sucrose (S),(Ⅱ)25g/L M+25g/L S,(Ⅲ)35g/L M+35g/L%S,(Ⅳ)45g/L M+45g/L S, all dissolved in7.5g/L saline. Preliminary experiments conducted in our laboratory demonstrated that sham injection (shell perforated but without solution injection) or injection of200μL0.75%saline did not affect embryo growth and development. Thus, the other group was not injected and served as the control group in this study. Results showed that, G6pase activity and serum glucose level increased from E6to DOH in all groups, whereas those of pectoral muscle and energy status decreased (P<0.05). Treatment Ⅱ increased (P<0.05) the hatchability as compared with the control. However, the hatchability decreased linearly as the carbohydrate level continued to increase (P<0.05).48h after in ovo injection, treatment Ⅰ and Ⅱ showed highest BW, while treatment Ⅱ and Ⅲ showed highest intestinal weight (P<0.05). Pectoral muscle weight in treatment Ⅱ increased significantly as compared with the control group (P<0.05) at E16. At day of hatch, treatment Ⅱ increased pigeon hatching weight and the yolk sac free BW, while the yolk sac weight decreased in treatment Ⅱ and Ⅲ (P<0.05). The weight of liver, pectoral muscle, stomach and small intestine, as well as the relative intestinal weight, increased significantly as compared with that of the control (P<0.05).48h after in ovo injection, G6pase activity decreased while serum glucose increased compared to the control (P<0.05). The liver glycogen concentration increased in treatment Ⅰ and Ⅱ at E16, and the muscle glycogen increased in treatment Ⅱ at day of hatch (P<0.05).5. Effects of in ovo injection of carbohydrates on small intestine development in pigeonsAt day14.5of incubation, fertile eggs were injected with200μL of carbohydrate solution containing25g/L maltose+25g/L sucrose, all dissolved in7.5g/L saline. Jejunal villus surface area, activity of the brush border enzymes sucrase, maltase, aminopeptidase-N and alkaline phosphatase, and mRNA expression of the digestion/absorption related genes were examined at day16of incubation and day of hatch. Results showed that in ovo injection of carbohydrates caused a villus surface area increase of38%on day16of incubation and23%on day of hatch relative to controls (P<0.05). The in ovo injected pigeons exhibited higher (P<0.05) activities of jejunal sucrase, maltase and alkaline phosphatase per gram of tissue from day16of incubation to day of hatch, compared with the controls. At16d of incubation, the mucosa DNA content increased by20%by in ovo injection as compared to the control group (P<0.05). At day of hatch, aminopeptidase-N activity per gram of tissue in embryos injected in ovo was approximately27%greater (P<0.05) than control embryos. Enhanced expressions of the jejunal SGLT1, GLUT2and APN mRNA were found at day16of incubation in embryos that received carbohydrate solution into the amniotic fluid in comparison with the control group (P<0.05).In conclusion, changes of pigeon intestinal morphology, enzyme activities, and mRNA expression of the digestion/absorption related genes were rapidly during pre-and posthatch development, although rates of development were different in the three intestinal segments. The results indicated that intestinal hydrolysis may be a determining step in digestion and young pigeons may hatch with a less mature digestive tract as compared to precocial chicks. Changes in carbohydrate-digesting enzymes activity in both small intestine and pancreas are more pronounced than for protein-digesting enzymes activity, suggesting that carbohydrate-digesting enzymes may be a determining step in pigeon intestinal development. In ovo injection of carbohydrate (25g/L maltose+25g/L sucros dissolved in7.5g/L saline) on14.5d of incubation can enhance the hatchability, hatching BW and glycogen reserves. Besides, the in ovo injection of carbohydrate improved villi size, brush border enzymes activity, and digestion/absorption-related intestinal genes mRNA expression and hence hatching intestinal maturity in pigeons. Therefore, the in ovo feeding may serve as a tool to regulate the pigeon intestinal development prior to hatching.