Administration of Ethanolic Extract of Spinacia oleracea Rich in Omega-3 Improves Oxidative Stress and Goblet Cells in Broiler Chickens Infected with Eimeria tenella

Abstract

This study investigated the anticoccidial activity of spinach (Spinacia oleracea) whole-plant extract against Eimeria tenella, both in vitro and in vivo. For this purpose, one hundred 8-day-old broiler chicks of both sexes were divided into four groups (n = 25 in each group). Chicks in the first group served as the negative control (non-treated-non-infected). Chicks in the second group were challenged at 18 days old with 5 × 10⁴E. tenella sporulated oocysts. The third group was challenged with 5 × 10⁴ sporulated E. tenella oocysts at 18 days old after receiving spinach extract at a dose of 50 mg/kg at 8 days old. The fourth group received 0.2 mg/kg diclazuril (Coxiril^® 0.2%) in their diet two days before being orally infected with 5 × 10⁴ sporulated E. tenella oocysts and this continued till day 10 post-infection (PI). The growth performance, clinical symptoms, oocyst shedding, histological findings, and biochemical parameters were used to evaluate the efficacy on day 8 PI when the infection was at its peak. A gas chromatography examination revealed that omega-3 fatty acids were the main constituents of the spinach extract, followed by oleic acid, palmitic acid, and phytol, with amounts of 23.37%, 17.53%, 11.26%, and 7.97%, respectively. The in vitro investigation revealed that the spinach extract at concentrations of 10% and 5% inhibited the oocyst sporulation by 52.1% and 45.1%, respectively. The 5% concentration was selected for the in vivo trial based on the results of the in vitro study. The infected-untreated group showed high levels of OPG; lower body weight; a greater number of parasite stages; few goblet cells; decreased SOD, CAT, and GPX levels; and increased MDA and NO levels. The spinach-treated group, on the other hand, showed a significant decrease in oocyst output per gram of feces (OPG), increased body weight, decreased parasitic stages, and a nearly normal number of goblet cells. Additionally, it reduced malondialdehyde (MDA) and nitric oxide (NO), while increasing superoxide dismutase (SOD), catalase (CAT), and glutathione peroxidase (GPX). In conclusion, spinach produced significant antioxidant effects, increased body weight, reduced the number of oocysts and parasite stages in the caecum, and restored the number of goblet cells relative to those of an uninfected control. Furthermore, spinach extract inhibits the sporulation percentage of E. tenella oocysts. The ethanolic extract of S. oleracea (whole plant) contained high concentrations of fatty acids, palmitic acid, Phytol, betulin, and ursolic aldehyde, all of which are known to regulate the antioxidant pathway and modulate inflammatory processes and may be the main reason for its anticoccidial activity.

Keywords: emeriosis; histological; omega-3; oxidative stress; spinach.

Figure 1

GC-MS chromatogram of spinach extract.

Figure 2

Arrows refer to different oocyst shapes: (A) unsporulated oocysts, (B) sporulated oocysts, and (C) damaged oocysts (scale bar—20 μm, seen with a 40× lens).

Figure 3

Oocyst index of caecal content (oocyst/g). (***) The mean difference was significant at p < 0.001.

Figure 4

Cecal sections of chickens in different groups showing the number of parasitic (H&E); (A) negative control group (noninfected–untreated), (B) positive control group (infected–untreated), (C) Diclazuril group (infected–treated with diclazuril 0.2 g/kg of food), and (D) spinach group (infected–treated with spinach extract 50 mg/kg of body). Black arrows refer to parasitic stage while red arrows refer to leukocytic infiltration.

Figure 5

Cecal sections of chickens in different groups stained with alcian blue stain to determine the number of goblet cells. (A) Negative control group (noninfected–untreated), (B) positive control group (infected–untreated), (C) Diclazuril group (infected–treated with diclazuril 0.2 g/kg of food), and (D) spinach group (infected–treated with spinach extract 50 mg/kg of body weight). Black arrows refer to goblet cells.

Figure 6

Effect of diclazuril and spinach extract on levels of some oxidative stress markers in ceca of chickens in different groups. (*) the mean difference is significant, (**) Low significance, (***) moderate significance, and (****) very high significance at p-value < 0.05.

Figure 7

The experimental design depicts the entire course of the experiment, from the first day to the end.