ارزیابی تأثیر غنی‌سازی جیره با پری‌بیوتیک بر ویژگی‌های گوشت و خصوصیات لاشه جوجه‌های گوشتی پس از عفونت تجربی با گونه های آیمریا

Introduction

Due to the drastic increase of the world population, the poultry industry is considered as one of the most important yet inexpensive sources of animal protein with low cholesterol value in the human diet (Teng & Kim, 2018). On the other hand, because of the increasing trend in cardiovascular diseases, lots of researchers have paid attention to test the effectiveness of growth promoters on altering lipid metabolism (Teng & Kim, 2018). Fat metabolism in an organism under the influence of animal nutrition strategies can affect the lipid profile of muscles in monogastric animals (Grela et al., 2014). Therefore, improvement in poultry meat production quantitatively and qualitatively is crucial.

Coccidiosis is a parasitic disease caused by host-specific Eimeria species (Wondimu et al., 2019). Seven species of Eimeria (E. tenella, E. acervulina, E. maxima, E. necatrix, E. brunetti, E. mitis, and E. praecox) are found in domestic chickens. Coccidiosis brings about mortality, morbidity, diarrhea, hematochezia, poor weight gain, and dwindled feed conversion rate leading to economic losses (Elmush-araf et al., 2007; Pop et al., 2019; Sultan et al., 2019). Feed supplementation with anticoccidial drugs has been used to control the disease; however, increasing drug resistance and consumers’ tendency not to use products with drug residue and their concern about environmental contamination persuade researchers to find new and natural anticoccidial agents (Elmu-sharaf et al., 2007; Kadykalo et al., 2017; Noack et al., 2019). Accordingly, the use of medicinal plants, probiotics, and prebiotics are of the best considered ways to prevent, control, or cure coccidiosis (Wond-imu et al., 2019).

Prebiotics are non-digestible feed ingredients that convert to short-chain fatty acids (propionic, lactic, etc.) in the large intestine, invigorate the growth and/or the activity of some particular intestinal bacteria (bifidobacteria and lactobacilli), and modulate the immune system in the intestinal lumen (Nazhand et al., 2020; Sánchez-Hernández et al., 2019; Silva et al., 2020). Mannan-oligosaccharide (MOS), a compound extracted from the cell wall of the yeast Saccharomyces cerevisiae, is of the most well-known prebiotics in the food industry (Carlson et al., 2018; Elmusharaf et al., 2007; Froebel et al., 2019). MOS may prevent the adhesion of pathogenic bacteria to the intestinal mucosa and induce better utilization of diet ingredients, leading to better performance (Adhikari & Kim, 2017; Ricke et al., 2020). Prebiotics are known to extend the length of the intestinal mucosa villi, hence increasing absorption surface areas (Teng & Kim, 2018). Dietary supplementation with MOS induced a decline in oocyst production; thus, the severity of coccidiosis (which is probably due to the increase in the length of the intestinal villi) enhanced the integrity of the intestines. (Elmusharaf et al., 2007).

Regarding the global spread and high economic losses caused by coccidiosis and resistance to anticoccidial drugs, there is a crucial need to find new approaches to make up for the losses of this disease. The present study was designed to assess the changes caused by the addition of prebiotics to the feed on carcass characteristics and also chemical composition, physical characteristics, color, texture, and fatty acid profile of chicken pectoral muscles containing Eimeria species.

Materials and Methods

Birds

A total of 40 male Ross 308 broiler chicks aged one day (Amol Joojeh, Iran) were acquired from a local hatchery. Wire-floored cages were used to rear the birds from day one to six weeks. The temperature of pens was adjusted according to Partovi et al. (2019). The groups had access to water and fed ad libitum. The rations were designed according to Ross 308 nutrient recommendations provided in Table 1.

Experimental Design

The broiler chicks were randomly assigned to four groups as follows: (1) negative control (NC) fed with basal diet without coccidia challenge, (2) positive control (PC) received basal diet with coccidia challenge, (3) antibiotic group (COX) challenged and fed with coxidine, (4) prebiotic group (PRE) challenged and fed with supplemented diet by prebiotics. Infection with Eimeria species was caused with E. acervulina (2×10⁵), E. maxima (1×10⁵), and E. tenella (2×10⁵) oocysts on the 18th day (Conway & McKenzie, 2007). The oocysts were obtained from the Department of Veterinary Parasitology at the University of Tehran and confirmed according to morphological and morphometric properties in a parasitology lab at Amol University of Special Modern Technologies. On day 18, chickens were challen-ged with 1 mL of the Eimeria species mixture by oral gavage using a 24-gauge stainless steel animal feeding tube attached to a 3-mL syringe. Coxidine (Rooyan Darou, Iran) was used in this study as an antibiotic. Coxidine (sulfaquinoxaline + diaveridine) was administered in COX at 18 days of age. The procedure of adding the drug to drinking water was carried out according to Partovi et al. (2019). Chickens from PRE received basal diet supplemented with 0.05% Celmanax prebiotic (mannan-oligosaccharide and beta-glucan) at one day of age (Arm & Hammer Animal Nutrition, USA).

Table 1. The composition of basal diet

Vitamin and mineral premix supplied per kilogram of diet: vitamin A, 10000 IU; vitamin D3, 9800 IU; vitamin E, 121 IU; B12, 20 μg; riboflavin, 4.4 mg; calcium pantothenate, 40 mg; niacin, 22 mg; choline, 840 mg; biotin, 30 μg; thiamin, 4 mg; zinc sulfate, 60 mg; manganese oxide, 60 mg

Ethics

The experimental protocol was in accordance with the Animal Care Committee of Amol University of Special Modern Technologies, Iran. The ethi-cal committee number is ir.ausmt.rec.1397.09.28.

Slaughtering Procedures

Three birds from each group were killed at slaughtering age (42 days). Final body weight, weight of fresh carcass, and pectoral and thigh meat were measured. Carcasses were refrigerated at 0 to 4°C, for a day before the breast muscles were filleted. Polyethylene bags were used to pack pectoral muscles. Fecal samples were collected from the 6th to the 10th day after infection. The samples were analyzed for the presence of coccidial oocysts using a standard fecal flotation technique (Lee et al., 2011).

Physical Characteristics of Breast Meat

A pH meter (Jenway 3505, Staffordshire, UK) was applied to measure the pH value of breast meat. The pH probe was calibrated using pH 4.0 and 7.0, and calibration was repeated between samples. According to a method devised by Pastorelli et al. (2016), the drip and cooking losses were determined in percentage. The drip loss was measured by calculation of the difference between the weight of meat at 4°C before and after 24 h. To measure the cooking loss, meat was cooked at 75°C in a water bath for 60 min and then cooled down for 30 min, followed by drying. The difference between the weight of meat before and after this process is described as cooking loss.

Chemical Composition of the Pectoral Muscle

Dry matter, fat, protein, and ash of pectoral meat were measured based on directions provided by the Association of Official Analytical Chemists (AOAC, 2000a; AOAC, 2000b; AOAC, 2000c; AOAC, 2000d).

Color of the Pectoral Muscle

L (lightness), a (redness), and b (yellowness) of pectoral muscle samples were determined. A Konica Minolta Chroma Meter CR-400 (Minolta Camera Co., Osaka, Japan) was used to measure the L- (lightness), a- (redness), and b- (yellowness) values of the breast meat.

Texture Characteristics of the Pectoral Muscle

The texture characteristics of pectoral muscles were determined using a texture analyzer (Texture Pro CT V1.2 Build 9, Brookfield Engineering Laboratories, Inc., MA, USA) according to Partovi et al. (2019).

Analysis of the Fatty Acid Profile of Breast Meat

In order to analyze the fatty acid profile, the crude fat of broiler breast meat was extracted according to Nielsen (2017), and the extracted fat was methylated (IUPAC, 1987). Then, fatty acids were analyzed by gas chromatography according to Cifuni et al. (2004).

Statistical Analysis

The obtained data were subjected to the Shapiro–Wilk test for normality and Levene’s test for the homogeneity of variances. Data from the carcass characteristics of broiler chickens, physical characteristics and chemical composition, color, texture profile analysis, and fatty acid profile of breast meat in broiler chickens infected with Eimeria species were subjected to one-way analysis of variance (ANOVA) in the general linear model, and comparisons between experimental groups were assessed using Tukey’s post hoc tests via SPSS 22 (SPSS Inc., Chicago, Ill., USA). All the results were reported as mean ± standard deviations. The comparisons with P-value<0.05 were assumed as statistically significant.

Results

The results of coccidial oocyst shedding in different post-challenge sampling days are shown in Table 2. No oocysts were detected in NC. In PC, there was shedding of oocytes on days 6–10 after infection. By contrast, in COX, shedding was observed on days 6, 7, and 8 but not on days 9 and 10. The fecal oocyst counts in COX were lower compared to PC. Oocyte shedding had a decreasing trend in PRE. The chickens fed with prebiotics showed a significant reduction in the number of oocyst shedding compared to PC. The dietary supplementation with prebiotics affected carcass characteristics in broilers infected with Eimeria species (Table 3).

Significant differences were detected among all groups regarding carcass characteristics (P=0.001). PC had the least favorable carcass characteristics of broiler chickens compared to all other groups (P=0.001). Dietary supplementation with antibiotics in the infected group improved carcass characteristics significantly compared to PC. PRE had the best carcass characteristics compared to all other groups (P˂0.05).

The results of Feed conversion ratio (FCR) in Table 3 reveal that the FCR values of groups NC, COX, and PRE are lower compared to PC (P˂0.05). PRE had the lowest FCR, but the difference was not significant (P>0.05). Prebiotics were used from the first day of age and continued until the end of the experiment. However, the challenge was started at 18 days of age. It might be the cause of better weight gain and lower FCR in PRE.

Dietary supplementation with prebiotics changed the physical characteristics of pectoral muscles in broilers infected with Eimeria species (Table 3). Drip loss, pH, and cooking loss did not show any significant differences between experimental groups (P>0.05).

Table 3 depicts the effect of dietary supplementation with prebiotics on the chemical composition of breast meat in broilers infected with Eimeria species. No remarkable differences were detected among experimental groups regarding the fat, ash, and dry matter of breast meat in broilers (P>0.05). Dietary supplementation with prebiotics increased the crude protein of breast meat in broilers compared to PC (P=0.01).

Table 2. Number of oocysts per gram of faeces expressed as log ₁₀ (X+1) in experimental groups from the 6^th to the 10^th day post-infection

Data are reported as mean ± SD; n=3; The different superscripts a,b,c,d in the same column indicate significant differences (P<0.05). NC: negative control; PC: positive control; COX: positive and medicated with antibiotic Coxidine; PRE: positive and medicated with prebiotic.

Table 3. Effect of dietary supplementation with prebiotic on carcass characteristics, physical and chemical properties of breast meat in broilers infected with Eimeria species

Data are reported as mean ± SD; n=3; The different superscripts a,b,c,d in the same row indicate significant differences (P<0.05). NC: negative control; PC: positive control; COX: positive and medicated with antibiotic Coxidine; PRE: positive and medicated with prebiotic.

Table 4. Effect of dietary supplementation with prebiotic on color and texture profile analysis of breast meat in broilers infected with Eimeria species

Data are reported as mean ± SD; n=3; The different superscripts a,b,c,d in the same row indicate significant differences (P<0.05). NC: negative control; PC: positive control; COX: positive and medicated with antibiotic Coxidine; PRE: positive and medicated with prebiotic.

The consequences of dietary supplementation with prebiotics on the color of the pectoral muscle in broilers infected with Eimeria species are shown in Table 4. Infection with Eimeria species decreased the a-value that was not significant (P=0.76). Dietary supplementation with prebiotics reduced the b-value of breast meat compared to PC (P=0.04).

Table 4 shows the effect of dietary supplementation with prebiotics on the texture profile analysis of breast meat in broilers infected with Eimeria species. No notable differences existed among the experimental groups in the adhesiveness, hardness, springiness, and force break of breast meat (P>0.05).

The consequences of dietary supplementation with prebiotics on the fatty acid profile of breast meat in broilers infected with Eimeria species are shown in Table 5. The most abundant fatty acid was oleic acid (C18:1 ranging from 24.90% to 36.10%), followed by palmitic acid (C16:0 ranging from 23.03% to 27.73%) and linoleic acid (C18:2 ranging from 18.50% to 20.43%). Dietary supplementation with prebiotics decreased the amount of fatty acids 16:1 and 18:1 and monounsaturated fatty acids (MUFAs) compared to NC (P˂0.05).

Discussion

Infection with Eimeria species negatively affected carcass characteristics, which dietary supplementation with prebiotics could compensate for this problem to a considerable extent. PRE had the best carcass characteristics compared to all other groups (P˂0.05). Gomez-Verduzco et al. (2009) reported that dietary supplementation with 0.05% MOS enhanced local mucosal IgA secretion and cellular and humoral immune responses and decreased parasite excretion in the feces of chickens infected with Eimeria species.

Dietary supplementation with MOS improved weight gain and feed conversion of chickens infected with Eimeria species. This strategy could compensate for weight gain losses induced by coccidiosis (Gomez-Verduzco et al., 2009). The positive effect of prebiotics on the incidence of coccidiosis in pheasants was proved by Zabransky et al. (2016). Similarly, Angwech et al. (2019) reported that in ovo

delivery of prebiotics improved the body weight, carcass weight, breast weight, and leg weight of Kuroiler chickens infected with Eimeria species compared to the antibiotic-treated group and non-medicated group.

Barberis et al. (2015) proved the beneficial role of MOS in reducing the replication of Eimeria species and, therefore, the economic losses attributable to coccidiosis. MOS increases the villi length, competes with sporozoites for binding sites on intestinal epithelial cells, and so protects it from lesions caused by Eimeria species. Salmonella enteritidis colonization decreased in chickens given MOS-supplemen-ted feed (Fernandez et al., 2002). Fernandez et al. (2002) showed that MOS-supplemented diet increased the count of Bifidobacterium spp., Lactoba-cillus spp., and Eubacterium spp. and also decreased the count of Enterobacteriaceae and Bacteroides spp. in birds’ intestinal microflora.

The production of bactericidal or bacteriostatic substances by Bifidobacterium spp. is one of the most important mechanisms to justify the positive effect of MOS (Fernandez et al., 2002). Spring et al. (2000) showed that MOS-supplemented diet reduced Salmonella Dublin and Salmonella typhimu-rium colonization in chickens because it prevented bacteria from adhering to the gut mucosa. Some have suggested that the mechanism for the antiparasitic effect of MOS-supplemented diet could be the production of acetic acid, lactic acid, and propionic acid due to Bifidobacterium spp., pH decrease, MOS fermentation in colon and caecum of birds, removing oxygen and declined redox potential (Fernandez et al., 2002).

The results of the present study are contrary to the results stated by Abu-Akkada and Awad (2015), who showed that dietary supplementation with prebiotics (ImmunoLin) did not improve the performance of challenged chickens with E. tenella. This contradiction could be related to the difference between the Eimeria species used in the present study and that of the mentioned study (Lee et al., 2007).

The physical characteristics of the pectoral muscle in broilers infected with Eimeria species did not show any significant differences among the experimental groups. Similarly, the meat pH of Kuroiler chickens infected with Eimeria species was not affected by in ovo delivery of prebiotics (Angwech et al., 2019). Breast meat cooking loss was not affected by dietary supplementation with probiotics and prebiotics significantly (Takahashi et al., 2005).

The chemical composition of the pectoral muscle in broilers infected with Eimeria species did not have any significant differences among the experimental groups––except for crude protein that increased in infected chickens fed with dietary supplementation with prebiotics. Breast muscle is the greatest edible part and also the most valuable part of broilers’ carcass (Konca et al., 2009). Meat composition affects the nutritional value of meat and also the quality of meat products (Konca et al., 2009). The result of the present study was supported by Angwech et al. (2019), who showed that in ovo delivery of prebiotics could not affect the fat content of Kuroiler chickens.

Feed supplementation with inulin did not have any significant influence on the crude fat content of the muscle longissimus dorsi in pigs (Grela et al., 2014). Similarly, Konca et al. (2009) reported that dietary supplementation with MOS did not affect the dry matter, ether extract, and crude ash of breast meat of turkeys. Some researchers have proved that dietary supplementation with MOS improves the morphology and structure of the intestinal mucosa, activity of digestive enzymes, transportation of amino acids, caecal metabolism, decrease of ammonia concentration, and β-glucuronidase activity in the caeca (Juskiewicz et al., 2006).

Infection with Eimeria species decreased the a-value, while dietary supplementation with prebiotics reduced the b-value of breast meat compared to PC. Color is an important sensory property affecting consumer selection and acceptability of meat (Konca et al., 2009). The findings of the current study are in agreement with those of previous works showing that dietary supplementation with MOS had no influence on the L-value (lightness) of breast meat of broiler chickens and turkeys (Konca et al., 2009; Pelicano et al., 2005). According to Rajput et al. (2014), coccidiosis diminished the a-value of broiler chicken meat. This can be attributed to hemorrhage caused in the broiler intestine due to Eimeria species (McDougald & Fitz-Coy, 2008). One of the most important clinical signs of coccidiosis is anemia due to hemorrhages in the intestines and caeca, which causes carcass paleness (Kaboudi et al., 2016).

The texture profile analyses of breast meat in broilers infected with Eimeria species were not different among the experimental groups. Pelicano et al. (2005) claimed that adding prebiotics to feed had no effects on the texture properties of broilers’ pectoral muscle. These results were proved by Takahashi et al. (2005).

Dietary supplementation with prebiotics decreased the amount of fatty acids 16:1 and 18:1 and MUFA compared to NC. Similar to the results of the present study, MUFA of meat from chickens infected with Eimeria species was reduced following in ovo delivery of prebiotics (Angwech et al., 2019). This can be attributed to changes in gut microflora, hence resulting in metabolic changes caused by prebiotics (Angwech et al., 2019). Diet supplementation with water and water-alcohol extract of inulin and also root powder from chicory did not have any effect on the fatty acid composition of muscle in pigs (Grela et al., 2014). No scientific paper identical to our work determining the effects of dietary supplementation with prebiotics on the physical charact-eristics, chemical composition, texture, and fatty acid profile of broiler meat challenged with Eimeria spp. was found in scientific databases.

Dietary supplementation with prebiotics is a promising strategy with the potential to compensate for the negative effects of infection with Eimeria spp. on carcass characteristics, protein content, and color of breast meat of broiler chickens. This strategy is suitable for large-scale poultry production, especially in countries like Iran, in which coccidiosis is an endemic disease bringing about high economic losses. Further studies involving the use of different kinds of prebiotics at higher concentrations, along with larger infection doses, are necessary to assess their positive effects on other pathogens.

Acknowledgments

This research work was supported by a research grant from Amol University of Special Modern Technologies, Amol, Iran.

Conflict of Interest

The authors declare that they have no conflict of interest.