Effects of Exogenous Ghrelin on Duodenal Growth and Development of African Ostrich Chicks

Ghrelin is a novel endogenous natural ligand for the growth hormone secretagogue receptor, which is mainly produced by X/A-like cells in the mucosal layer of the gastrointestinal tract (Kojima et al., 1999). A small amount of ghrelin is also produced by other organs (Okuhara et al., 2018). Ghrelin has been found to be widely expressed in other tissues and organs of animals (Chen et al., 2017). A large number of studies have confirmed the inference that the wide distribution of ghrelin immunopositive cells in animals may have many biological roles (Cardona et al., 2012). Ghrelin immunopositive cells are widely distributed in the African ostrich (Wang et al., 2017a; Wang et al., 2017b; Ye et al., 2018; Zhang et al., 2018), Wang et al., (2009) showed that ghrelin immunopositive cells are mainly distributed in the mucosal layer of the gastrointestinal tract of the African ostrich chicks and the number of immunopositive cells gradually decreases from the stomach to the small intestine and then to the large intestine. The number of ghrelin immunopositive cells in the small intestine also showed significant age-related changes, peaked at 90 days and then remained stable. This suggests that ghrelin plays an important physiological role in the digestive system of African ostriches. Previous studies have shown that ghrelin had stimulatory effects on the motility of the antrum and duodenum in both fed and fasted animals (Fujimiya et al., 2011). Moreover, Ghrelin can enhance the autophagy of intestinal epithelial cells in rats with sepsis and protect the small intestinal epithelium against sepsis-induced injury (Wan et al., 2016). To our knowledge, the effects of exogenous ghrelin on the duodenal growth and development of African ostrich chicks have not yet been reported. Therefore, in this study, we injected different doses of ghrelin into African ostrich chicks via the metatarsal vein to investigate the effects of the different doses on the length, tissue structure and relevant hormones and enzymes of the duodenum of the ostrich chicks and provide a morphological basis for elucidating the effects of ghrelin on the self-renewal of small intestinal epithelial cells of the chicks. Furthermore, we wanted to provide new ideas for further research on the growth and development of the small intestine of ostrich chicks.

Animals
 
The present study was conducted at the Ostrich Institute of Yangtze University in Jingzhou, Hubei in the year of 2018-19. Seven-day-old African ostrich chicks were obtained from the Ostrich Research Institute of Yangtze University, Hubei, China. The birds had free access to water and the feed prepared according to the specifications of the Elsenburg Ostrich feed database (Brand, 2010). After 33 days, the birds were weighed and 16 African ostriches chicks (male or female) with similar weight (average weight: 3.65±0.53 kg) and growth status were selected for the experiment. The experimental procedures and ostrich treatment procedures have been approved by the Animal Protection and Welfare Committee of our institute.
 
Tissue preparation
 
The 16 ostriches chicks were divided into four groups containing four chicks each-one control and three experimental groups. The ostriches chicks were injected intravenously (metatarsal vein) with saline (control) or ghrelin (experimental) at 8 am every day for 6 days; the total injection dose was 500 µl per bird. The three experimental groups were treated with 10 µg/kg, 50 µg/kg and 100 µg/kg, respectively, of ostrich ghrelin (synthesized by Shanghai Qiangyao Biological Technology Co., Ltd.) diluted with normal saline. After 6 days of treatment, the birds were weighed and anesthetized with 20% uratan (1 g/kg; China Caoyang No. 2 Middle School Chemical Factory). The abdominal cavity of each bird was opened, The duodenum and its contents were quickly removed, the weight and duodenal length were measured and the length index was calculated. Next, each duodenum is divided into two parts: one part was washed with physiological saline, made into a homogenate and centrifuged (12000 rpm for 10 min) at 4°C and the supernatant was collected. Then, the contents of pepsin and gastrin were measured by radioimmunoassay. The other part was fixed in 4% paraformaldehyde solution and then embedded in paraffin; the paraffin blocks were sectioned (4 µm) using a Leica microtome (Nussloch Gmbh, Wetzlar, Germany) and the sections were used for hematoxylin and eosin (HE) staining.
 
Morphometric analysis
 
For each intestinal tissue sample, 3 cross-sections were prepared after the samples had been stained with hematoxylin. Further, for each intestinal cross-section, 10 intact, well-oriented crypt-villus units were selected for experiments conducted in triplicate (30 measurements for each sample). Villus height was measured from the tip to the crypt junction and the crypt depth was calculated as the depth of the invagination between and beside villi using a calibrated ocular micrometer. The ratio of villus/crypt length was also calculated. All the measurements were performed under an Olympus light microscope, using the HMIAS-2000 high-definition chromatic color medical science figure analysis program (Qianping, Wuhan, China).
 
Statistical analysis
 
Results are expressed as means ± standard errors on the mean (means ± S.E.). Statistical analysis was done using analysis of variance statistics software with Duncan’s multiple range test where appropriate. Differences of P<0.05 were considered significant.

Effects of exogenous ghrelin on the duodenum morphology of African ostrich chicks
 
The proximal part of the small intestine, i.e., the duodenum, receives pancreatic juice and bile, which is mixed with partially digested food from the stomach; it plays a vital role in food processing, both physiologically and chemically (Zhang et al., 2020). In the present study, we found that an increase in the body weight of the African ostrich chicks after the injection of  50 µg/kg and 100 µg/kg concentrations of ghrelin (P>0.05; Table 1) and the length and length index of the duodenum in the 10 µg/kg, 50 µg/kg and 100 µg/kg groups were significantly lower than those of the control group (P<0.05; Table 1) and the 100 µg/kg group yielded the lowest result. Slupecka et al., 2012 also found that different doses of ghrelin significantly decreased the length of the duodenum and middle part of the small jejunum. We studied the villus length and crypt depth of the duodenum. The data obtained in this study show that the villus length in the 10 µg/kg group was shorter than that in the control group (P>0.05). The villus length increased significantly in both 50 µg/kg and 100 µg/kg groups (P<0.05; Table 2, Fig 1) and the higher the villi and their densities, the larger the surface area for digestion and absorption. (Ohanaka et al., 2018). In addition, we observed that thecrypt depth was slightly reduced in the 50 µg/kg and 100 µg/kg groups when compared with the control group (P>0.05); the crypt depth in the 10 µg/kg group was significantly reduced (P<0.05; Table 2, Fig 1). A large number of stem cells in the crypt can differentiate into intestinal villi cells (Xia et al., 2004) and the rate of crypt cell division is revealed by changes in crypt depth that ultimately can influence the process of digestion in the small intestine (Yang et al., 2009). The villus length to crypt depth ratio represents the absorption capacity of the small intestine (Liu et al., 2020); higher the ratio, stronger the absorption and transport capacity of intestinal epithelial cells (Xie et al., 2020). In the current study, we found that, after injection of different concentrations of ghrelin, the ratio of villus length/crypt depth between each dose group increased when compared with the control group; however, it was not significant in the 10 µg/kg group (P>0.05) and the 50 µg/kg and 100 µg/kg groups showed significant differences (P<0.05; Table 2, Fig 1). These findings indicate that exogenous ghrelin can promote crypt differentiation in the duodenum and growth of intestinal villi, thereby improving the absorption of nutrients in the duodenum.
 

 

 

 
Effects of exogenous ghrelin on digestive enzymes in the duodenum of African ostrich chicks
 
The chyme of the glandular stomach and muscular stomach of poultry was mainly affected by pepsin and gastric lipase and pepsin was the main enzyme (Zhang et al., 2005). Due to the faster passage of foods through the proventriculus and muscular stomach, the digestive effects of pepsin are not strong and protein is mainly digested in the small intestine (Fan, 2003). In this study, pepsin content was higher in the duodenum of the 10 µg/kg group than in that of the control group, but the difference was not significant (P>0.05; Table 3); pepsin content was significantly lower in the 50 µg/kg and 100 µg/kg groups (P<0.05; Table 3). This showed that a high dose of ghrelin inhibited pepsin secretion in the duodenum, whereas a low dose of ghrelin might promoted pepsin secretion. Gastrin is the main hormone responsible for the stimulation of gastric acid secretion. The main biological activity of gastrin is to promote the secretion of gastric acid and pepsin, promote the growth ofgastrointestinal mucosa, contract the gastric antrum and pyloric sphincter and delay gastric emptying (Liu et al., 2012). In this study, gastrin content was significantly higher in the 10 µg/kg group than in the control group (P<0.05; Table 4). Gastrin content was higher in the 50 µg/kg group than in the control group, but the difference was not significant (P>0.05; Table 4). Gastrin content was significantly lower in the 100 µg/kg group than in the control group (P<0.05; Table 4). This showed that a low dose of ghrelin promoted gastrin secretion and a high dose of ghrelin inhibited gastrin secretion. The above results show that a low dose of ghrelin may promote the growth of duodenum mucosa in African ostrich chicks and a high dose of ghrelin may inhibit the growth of duodenum mucosa in African ostrich chicks; however, the specific effects and mechanism need to be studied further.
 

 

The results of the present study indicated that the development of the duodenum of African ostriches chicks was closely associated with the concentration of ghrelin. The high doses of ghrelin promote duodenal villus growth in African ostrich chicks, but inhibit duodenal length and length index and pepsin and gastrin levels. The low doses of ghrelin inhibit duodenal length and length index, but promote the villus length to crypt depth ratio and gastrin and pepsin levels increased in varying degrees. These data suggest that high doses of ghrelin may inhibit the growth and development of the duodenum of African ostriches chicks, while low doses of ghrelin may promote the growth of the duodenum of ostriches chicks.

We would like to thank the National Natural Science Foundation Project of China. This study was supported by the National Natural Science Foundation Project of China [grant numbers 31402152, 31672504].