MATERIALS AND METHODS

Animals and experimental design: Twenty-four, 9 month aged local goats were used in this study. This research project was conducted from March 2011 to August 2011 and approved by the local ethics committee. Goats were equally divided into control group, first treated group and second treated group. Goats in the control group (n = 8) were fed a diet consisting of 60% guinea grass and 40% concentrates which included corn, soya bean meal, calcium, minerals, vitamins and 12.8% crude protein. Goats in the first treated group were fed with 10% guinea grass and 90% palm kernel cake which include calcium, minerals, vitamins and 15.2% crude protein. Finally goats in the second treated group were fed with a diet similar to those of the first treated group but with addition of molybdenum 40 ppm and sulphur 100 ppm to the concentrates. Feeding of goats with the prepared diets was continuous for 120 days. On the 121th day, animals were slaughtered and their abdomens were dissected and then stomachs were removed from the carcasses of the goats. The abomasum of each animal was opened along its greater curvature and samples from cardiac, fundic, corpus and pyloric regions were collected and labelled. For the microscopic study, tissue samples were immediately immersed in 4% paraformaldehyde in Phosphate Buffer Saline (PBS) for 16 h and cryoprotected overnight in 30% sucrose before being sectioned at 10 μm on a cryostat (Ericson et al., 2009). For general morphology some samples were processed and stained with H and E stain, whereas, the most of the other were processed for immunohistochemistry study in which Fluorescein Isothiocyanate (FITC) technique was used.

Fluorescein isothiocyanate technique: Cryosections were hydrated in distilled water for 15 min and then immersed in 0.5% hydrogen peroxide for 30 min to inhibit endogenous peroxidase activity and to reduce lipid content to allow subsequently better penetration of the antisera (Masot et al., 2007). Sections were washed again in the distilled water and rinsed in 0.1 PBS (pH 7.4) for 3 changes each of 10 min. Sections were then incubated in the normal goat serum (diluted 1 to 20 with PBS) for 20 min at room temperature to block non-specific binding of antibodies (Ericson et al., 2009). Then sections were rinsed in 0.1 PBS for 3 changes each of 10 min. After that sections incubated for 24 h in the primary antisera at 4°C. The current primary antisera used were antisera raised in rabbits against gastrin cells (G17) which were diluted (1:1000) in antisera diluents (100 mL of PBS, 2.5 g of bovine serum albumin and 0.25 g sodium azide). Once more, sections were rinsed in 0.1 PBS for 3 changes each of 10 min then incubated in the secondary antisera (Goat anti-rabbit IgG Cy3 conjugate) diluted in antisera diluents (1:400) for 1 h at room temperature (Al-Saffar et al., 2011). Finally, sections were rinsed in 0.1 PBS for 3 changes each of 10 min and then mounted with Dako fluorescence mounting medium and visualized with fluorescence microscopy (Fang et al., 2012) using image analyzer microscope (Olympus, BX 51, Japan).

Statistical analysis: Statistical calculations were carried out with the SPSS 15.0 for windows software package. Data was expressed as Mean±SEM and analyzed with Analysis of Variance (ANOVA) for all groups and subsequently confirmed by using student t-test between each two groups. The numbers of immunoreactive gastrin cells were counted in 5 randomly selected fields in each slide of the 3 selected slides belong to each animal sample. Gastrin cells were counted in three different locations that are superficial (surface), middle and the basal (deep) parts of the gastric glands. Means of the counted G cells were compared between these three locations and in the other hand the average means of the G cells of each group was compared with those of the other groups (Table 1). The value of p<0.05 considered significant difference.

RESULTS

General morphology: The microscopic examination revealed that the wall of the abomasum consists of four layers that are inner mucosa, submucosa, tunica muscularis and the outer most serosa. In the pyloric part, the epithelium of the mucosa invaginated deeply into the lamina propria, forming the gastric pit. Pyloric glands are exclusively mucous type and empty into the bottom of gastric pit. The surface epithelial cells and gastric pits were also found to be mucous cells. The gastric pits were lined with a high columnar epithelium (Fig. 1a-1, 1b-1). The surface epithelium of cardiac and pylorus was made up of surface mucous cells and branched tubular mucous glands (Fig. 1d-2), whereas fundic and corpus were formed of branched tubular glands that contain several cell types. The epithelium rest over muscularis mucosa consists of few layers of smooth muscle fibers (Fig. 1c-4). The lamina properia and submucosa contains loose connective tissue, lymphoid cells and blood vessels (Fig. 1c-3). The outer wall of the abomasum contains layers of smooth muscles bundles (Fig. 1c-5). The outermost layer of the abomasum is the tunica serosa representing the visceral peritoneum which contains loose connective tissue, blood vessels and a covering of mesothelial cells.

Fig. 1(a-d):	Pyloric mucosa of the goat’s abomasum showed “gastric pits” (arrow), mucus-secretory simple columnar lining epithelium (1), simple tubular mucus-secretory glands (2), loose connective tissue with blood vessels in the underlining propria-submucosa (3), smooth muscle layers of muscularis mucosa (4) tunica muscularis (5), H and E, (a) X40, (b) X100, (c) X100 and (d) X200

Immunohistochemistry: Microscopic examination with the fluorescence microscope showed numerous number of positive G cells distributed throughout the length of the pyloric gastric glands in the abomasums of the control and both treated groups. Whereas, the mucosa of cardiac, fundic and the corpus were characteristically lack this type of cells. The immunoreactive G cells appeared red-golden fluorescent-stained cells which were distributed in a single pattern all over the gastric glands of the pyloric mucosa and they were concentrated significantly (p<0.05) in the basal (deeper) part compared to those counted at both middle and surface (superficial part) of these glands.

Table 1:	Number of gastrin cells in the pyloric mucosa of the control and the two treated groups of goats
*Significantly (p<0.05) lower number of gastrin cell present in the pyloric mucosa compared to those of the first and second treated groups, **Non significant (p<0.05) difference in the number of gastrin cells present in the pyloric mucosa of the first and second treated groups, #Significantly (p<0.05) higher number of gastrin cell present in the basal (deeper) part of the pyloric mucosa compared to those of the middle and surface (superficial) epithelium, -Non significant (p<0.05) difference in the number of gastrin cells present in the middle and surface parts of the pyloric mucosa in the control, first and second treated groups

The lining epithelium of the gastric glands rests on an underlining propria-submucosa layer of loose connective tissue which is interrupted by smooth muscle layers of muscularis mucosa (Fig. 2-4).

The mean number of G cells observed in the control and treated groups is well publicized in the Table 1. In each group, the average number of G cells which was counted at the basal (deeper) part of the gastric glands of the pyloric mucosa was significantly (p<0.05) higher than those observed at the middle and surface (superficial) parts of the gastric glands. The number of G cells at the basal, middle and surface part of the gastric glands of the control group was 49.63±0.64, 8.5±0.2 and 9.63±0.3, respectively. Whereas, the G cells number observed in the basal, middle and surface part of the gastric glands of the first treated group was 77.38±0.77, 11.63±0.37 and 14.25±0.4, respectively. Second treated group showed no significant changes in the number of G cell compared to those of the first group and their G cells number was 80.88±0.56, 10.88±0.34 and 13.75±0.24, respectively.

Fig. 2(a-c):

Pyloric mucosa of goat’s abomasum in the control group showed gastrin cells distributed in the surface part (thin arrow), middle part (thick arrow) and deeper part of the gastric glands (star), Loose connective tissue located in the underlining propria-submucosa which is interrupted by smooth muscle layers of muscularis mucosa (triangle), fluorescein isothiocyanate (FITC), (a) X100, (b) X200, (c) X200

Fig. 3(a-c):

Pyloric mucosa of goat’s abomasum in the first treated group showed characteristically higher number of gastrin cells compared to the control group; distributed in the surface part (thin arrow), middle part (thick arrow) and deeper part of the gastric glands (star). Loose connective tissue located in the underlining propria-submucosa which is interrupted by smooth muscle layers of muscularis mucosa (triangle), FITC, (a) X100, (b) X200, (c) X400

Statistical analysis revealed that goats of both treated groups were showed significantly (p<0.05) higher number of G cells in their pyloric mucosa compared to those observed in the control animals. The average number of G cells counted in the pyloric mucosa of the control group was 67.75±0.6, whereas, the average number was significantly higher up to 103.25±0.77 and 105.5±0.83 in the first and second treated groups, respectively.

Fig. 4(a-c):

Pyloric mucosa of goat’s abomasum in the second treated group showed obviously higher number of gastrin cells compared to the control group; distributed in the surface part (thin arrow), middle part (thick arrow) and deeper part of the gastric glands (star), Loose connective tissue located in the underlining propria-submucosa which is interrupted by smooth muscle layers of muscularis mucosa (triangle), FITC, (a) X100, (b) X200, (c) X200

Analysis with Student t-test revealed absence of mean difference (p<0.05) between both treated groups. Finally, all groups showed no significant (p<0.05) differences in their average number of G cells distributed at both middle and surface parts of the gastric glands.

DISCUSSION

The immunoreactive G and somatostatin (D) cells were considered the major endocrine cells in the stomach that are play an important role in acid secretion (Liu et al., 2005). Previous thinking indicated that the gastrin hormone and gastrin cells play a role in gastric normal diet digestion. The present study was performed to assess the effect of different diets on the numbers and distribution of gastrin cells in the abomasum of goats. The crude protein which was 12.8% in the control groups as standards feed for goat production, raised up to 15.2% in the treated groups fed with combination of Palm Kernel Cake (PKC) and grass. The selection of palm kernel cake (PKC) based on its high nutritive value and its extensive use as feed ingredients in ruminants (Chanjula et al., 2011). Current findings revealed significant effect of the diet on the mean numbers of gastrin cells in the treated goats compared to those of the control group. Results indicated that different diets particularly the protein level does affect the numbers of gastrin cells abomasum of goats. The findings were consistence with previous observations that are considered proteins and amino acids as the most powerful gastrin secretagogues (Calbet and Holst, 2004) and are the most potent stimulator of gastrin hormone (Feng et al., 2010).

The microscopic observations of the immunofluorescence technique showed that in the goat’s abomasum, immunoreactive G cells were located and distributed in the pyloric region only and they were totally absent in the cardiac, fundic and corpus regions of the abomasum. These findings were similar to those previously observed in the sheep’s abomasum in which gastrin cells were very numerous in the abomasum and duodenum but are deficient in the cardiac or fundic regions of the abomasum (Franco et al., 1993). Similarly, previous findings confirmed that the immunoreactive gastrin cells located mainly in the middle third of the pyloric mucosa in the stomach of dogs, cats and humans (Takahashi et al., 1981; Marino et al., 1994; Galan et al., 1996).

Gastrin cells are considered essential regulators of gastric acid secretion and the alterations in its relative numbers may play a key role in gastroduodenal disease (Liu et al., 2005). The importance of G cells arises from the fact that the dietary protein intake triggers the production of gastrin hormone from these cells, which subsequently stimulates the secretion of hydrochloric acid that aids the process of digestion (Mullin et al., 2009).

Current findings showed higher number of G cells located at the basal part of abomasal pyloric glands of treated goats. These findings appeared similar to those previously observed in the rat antral mucosa, which revealed the existences of numerous G cells in the basal part of the glandular area of the stomach (Varagic et al., 1996). The present data revealed significantly lesser density of the G cells at the surface and middle parts compared to those of the basal deeper part of the pyloric glands. The difference of G cell density reflects the function of the surface mucosal protection against gastric acid secretion produced during the process of digestion. Morphological examination showed a picture which was quite clear to explain this consideration as it showed highly mucous cell secretory surface epithelium in the pyloric mucosa of all groups which were in consistency with previous findings (Suprasert et al., 1999).

CONCLUSION

The current data concluded that the protein content of the diet have certain effect on the G cells number which certainly affect the gastric acid secretion and subsequently gastric function of the goat’s abomasum. The results of fluorescein technique showed prominent effect of higher protein diet indicated by the numerous G cells appearance in the pyloric mucosa.

ACKNOWLEDGMENTS

Great respect and thanks to the staff of Anatomy, Histology and Embryology Department for their great support to achieve this research.