大口黑鲈开口摄食与转食人工配合饲料期消化系统发育特征

Early developmental characteristics of digestive system of Micropterus salmoides larvae during the first feeding and artificial formula feed adaptation

关键词:  大口黑鲈  消化系统  组织学  扫描电镜

Key words:  Micropterus salmoides  Digestive system  Histological section  Scanning electron microscopy

摘要:

本研究利用组织切片和扫描电镜技术，观察和研究了2~30日龄大口黑鲈(Micropterus salmoides)仔鱼消化系统的发育过程及组织学变化。结果显示，在水温为(23±1)℃条件下，0~4日龄仔鱼消化道初步分化，为内源性营养期；4~6日龄仔鱼消化道逐渐分化形成食道、胃和肠，胃和肠黏膜褶形成，肝胰脏细胞团出现，仔鱼具备基本摄食能力，进入混合性营养期；10~16日仔鱼消化道和消化腺结构分明，胃、幽门盲囊、肠道紧密排列，肝脏和胰脏独立，进入外源性营养期，此阶段后可逐步转食人工配合饲料。20~30日仔鱼胃腺发达，胃和肠道出现次级黏膜褶，幽门盲囊黏膜褶显著增多、增长，肝脏逐渐出现脂肪积累区，胰脏可见酶原分泌颗粒，肝胰脏组织结构近似成鱼。扫描电镜显示，30日仔鱼胃部内表皮具有丰富的网状黏膜褶，胃小凹间分布着密集的分泌孔；幽门盲囊和肠道内表面结构相似，无固定形态的黏膜褶上布满黏液细胞和分泌孔。20日龄后仔鱼具备转食人工配合饲料的能力。此外，在仔鱼开口和转食人工配合饲料过程中，部分死亡个体的胃肠组织表现出腔体扩大或皱缩，内表皮无成型的黏膜褶或黏膜层脱落，胃和肠道组织损伤。本研究可为大口黑鲈仔鱼开口和转食人工配合饲料条件的优化提供组织学基础资料。

Abstract:

Micropterus salmoides is an economically important cultured carnivorous fish in China. In recent years, owing to the development and wide application of artificial formula feed, M. salmoides production has rapidly increased and reached 470 000 tons in 2019. However, a low survival rate of M. salmoides larvae is observed during the first feeding and artificial formula feed adaptation. In this study, to better understand the artificial formula feed adaptation of M. salmoides larvae, the developmental characteristics of the digestive tract and digestive gland of the fish larvae from 2~30 dph were observed and described using histological sections and scanning electron microscopy (SEM). Moreover, the digestive tract (stomach and intestine) characteristics of certain dead larval fish during the first feeding and the transformation of artificial formula feed were investigated. For histological analysis, the larval fish (including stomach, intestine, pyloric cecum, liver, and pancreas tissues) were dehydrated with an alcohol gradient (70%, 80%, 90%, and 100%), embedded in paraffin, cut into 5 μm sections, and stained with standard hematoxylin and eosin. For SEM analysis, the stomach, pyloric cecum, and intestine of 30 dph larval fish were fixed in 2.5% glutaraldehyde solution for 12 h. Then, the tissues were fixed in 1% osmium solution for 2 h, dehydrated with gradient alcohol (70%~100%), soaked in tert-butyl alcohol for 2 h, dried with a lyophilizer, and plated with gold by ion sputtering. Finally, the images were captured using a HITACHIX-650 scanning electron microscope. At a water temperature of (23±1)°C, the larval yolk sac and oil drop gradually decreased at 0~4 dph, the digestive tract was initially differentiated, and heartbeat, blood circulation, mouth crack, esophagus, and anus were observed at 4 dph. Larval fish were in the endogenous nutrition period at this stage. In 4~ 6 dph larval fish, the esophagus, stomach, intestine, liver, and pancreas regions gradually formed, the digestive tube opened to the outside initially, and yolk sac and oil drop significantly decreased and then completely disappeared, indicating that the larval fish entered the endo-exotrophic period. At this stage, sufficient Artemia salina should be provided to induce the larval fish to open their mouth and perform first feeding. In 10~16 dph larval fish, the stomach, pylorus caecum, and intestine were closely arranged. From the surface of the intestinal cavity, the mucosa, submucosa, muscular, and serosa layers were successively presented. The size of the mucosa columnar epithelial cells and the number of goblet cells clearly increased. The masses of hepatocytes and pancreatic cells significantly increased, and a vacuole structure appeared in the hepatocytes. The pancreatic cells were arranged closely, and the blood cells and pancreatic ducts were distributed. At this stage, larval fish entered the exogenous nutrition period. From this stage onward, larval fish are better able to transform feeding artificial formula feed step by step. In 20~24 dph larval fish, the stomach gland was well developed, and the stomach wall thickness and number of stomach glands further increased. Furthermore, the mucosal folds and connective tissue of the submucosa were further differentiated, and the longitudinal mucosal folds increased and curled. In addition, secondary mucosal folds in the intestine gradually appeared and the number of mucosal folds in the pyloric cecum increased. Fatty accumulation and secretory granules were observed in the liver and pancreas, respectively, indicating that the larval hepatopancreas was similar to that of adult fish. The digestive system of larval fish was completely developed at this stage, and the larval fish had the ability to transform and adapt to artificial formula feed. No significant differences were observed in body length and body weight of M. salmoides larvae (P>0.05) during the adaptation to artificial formula feed at 12, 16, and 20 dph. However, the survival rate of 12 dph larval fish (45.59%) was lower than that of 16 (60.60%) and 20 dph larval fish (69.83%). In addition, the body length, body weight, and survival rate of M. salmoides larvae were positively correlated with the time point of feeding artificial formula feed. SEM results showed abundant polygonal reticular mucosal folds in the gastric epidermis of 30-day-old larval fish, and there were dense secretion pores between the gastric pits. Mucosa folds with fixed shapes were observed on the inner surface of the intestine, and mucous cells, secretory pores, and secretory granules were clearly observed between the mucosal folds. Interestingly, the inner surface structure of the pyloric cecum was similar to that of the intestine. A difference was the mucosa folds without a fixed shape on the inner surface of the pyloric caecum. These mucosal folds and secretory pores are important for food digestion, absorption, and excretion, indicating that the larval fish are old enough to adapt to the artificial formula feed at this point. During the first feeding and artificial formula feed adaptation, the stomach and intestine of the larval fish were incompletely developed and accompanied by tissue damage in the dead individuals. For example, the stomach and intestinal cavity were significantly shriveled, the inner epidermis did not have the molding mucous membrane fold and goblet cells, and the mucous membrane layer was cracked or absent. These results indicate that larval fish overfeed and fail to effectively digest, absorb, and expel nutrients, which ultimately results in death associated with nutritional deficiencies, intestinal blockages, and inflammation. During the process of adaptation to artificial formula feed for M. salmoides, the development of larval digestive system was investigated. The digestive system of larval fish at 4~6 dph gradually differentiated and was still in the mixed nutrition period, and sufficient A. salina should be provided to induce the larval fish to open their mouths and perform the first feeding. At 6~16 dph, the digestive system gradually developed and larval fish entered the exogenous nutrition period, and sufficient food could be provided. At 16~20 dph, the digestive system of the larval fish completely developed, and this stage is the optimal time to switch to artificial formula feed. Our study provides basic data for feeding condition optimization of M. salmoides larvae.