Effects of Supplemented Growth Promoters on Performance and Intestinal Morphology in Broilers Reared under Different Stocking Densities

The aim of the present study was to investigate the influence of different growth promoters on broiler performance and intestinal morphology reared under various stocking densities. A total of 900 day old (Ross-308) straight run broilers were randomly divided into fifteen treatment groups according to a completely randomized design. A 5 × 3 factorial arrangement of treatment was employed. Treatments were consisted of four growth promoters (antibiotics, prebiotics, probiotics, and symbiotics) and a control group with three stocking densities (0.046, 0.056, 0.065m2). Each treatment was replicated 6 times with 10 birds in each. Regarding growth performance, feed intake (p=0.0008), body weight (p=0.0085) and feed conversion ratio (p=0.0001) were improved with the supplementation of symbiotic in the diet whereas lower body weight was noted in the control group. In terms of intestinal morphology, villus height (p=0.0011) and villus to crypt ratio (p=0.0001) were significantly higher in the symbiotics treatment groupas compared to the control group. Moreover, birds reared under 0.065m2 stocking density showed improvement in body weight, feed conversion ratio and livability as compared to the other treatment groups. It can be concluded that the supplementation of symbiotics in the diet of commercial broiler reared under 0.065m2 stocking density had a positive influence on overall growth performance and intestinal morphology.


INTRODUCTION
Poultry products (meat and eggs) is one of the major food producing enterprise all over the world. The trend in poultry farming is changing from birds per unit area to meat production per unit area and farmer usually place more birds than standard to increase profit (Skrbic et al., 2008). However, this overcrowding exposes the birds to stress and microbial attack. The use of various antibiotics to combat this disease threat create resistance in humans as well as in the animals. The resistance which is created by bacterial action in the body proliferates via food chain into the intestinal micro flora of humans and also may transfer through plasmid to new generation. The main purpose in banning antibiotics usage in feed in numerous countries is to minimize the resistance of antibiotics and bacterial colonies into part of farm animals (Smith et al., 2007). However, the complete removal of antibiotics in feed results in poor growth and more disease attacks, therefore the scientists are working on suitable alternatives for antibiotics. Among different replacements, prebiotics, probiotics, and symbiotic are found to have direct effect on bird's performance and gut health under optimum environmental conditions (Muir et al., 2000).
The efficiency of probiotics may enhance the fusion of different strain of probiotics into the colonies of endogenous bacteria, which stimulates the growth and activities of both exogenous and endogenous bacteria (Suskovic et al., 2001). The mixture of prebiotics and probiotics as named symbiotics may apply to the synergistic effect on growth and colonies multiplication of beneficial microorganism which ultimately exert positive effect on the health of the intestine and absorption of the nutrient in the host (De Vrese & Schrezenmeir, 2008).There is a considerable biological advantage with respect to growth performance and intestinal morphology in poultry production (Awad et al., 2009). Numerous studies reported effect of different prebiotics, probiotics and their combinations on the performance of commercial broilers however their interaction with different stocking densities are still neglected and require further investigations. Therefore, the present study was conducted to evaluate the effect of alternative sources of antibiotic growth promoters and their effect on growth performance and intestinal morphology of commercial broilers reared under various stocking densities.

MATERIALS AND METHODS
In this study, 900 commercial straight run broiler chicks (Ross-308) were acquired from the commercial hatchery and allocated into 15 treatment groups. A 5 × 3 factorial arrangement of treatments were applied according to a completely randomized design. The treatments consisted of 4 growth promoters [antibiotics (lincomycine 0.15%), probiotics (bacillus subtilis 0.5%), prebiotics (mannan oligosaccharides 0.6%) and symbiotics (combination of prebiotic and probiotics 1 %)] and a control group and 3 stocking densities (0.046m 2 , 0.056m 2 , 0.065m 2 ). The treatments were replicated 6 times with 10 birds in each. The dimensions for each replicate were 0.46 m 2 , 0.56 m 2 , 0.65m 2 , respectively. Day old chicks were vaccinated against Newcastle Disease (ND) and Infectious Bronchitis (IB) at the hatchery. The chicks were reared on deep litter system where rice husk was used as bedding material with a thickness of 3 inches. In each replicate, one round feeder and three nipple drinkers were installed for ad libitum feeding and watering. During brooding, the temperature and humidity were optimized at 33±1.1 o C and 62±3% whereas, in rearing period 22-24ºC temperature, 65-70%humidity, were maintained. A 24 hours lighting (30 lux) schedule was followed while vaccines were administered according to the schedule (Table 2).

Growth Performance Traits
The body weight of each bird was recorded on a weekly basis and weight gain was calculated by using the following formula: Body Weight Gain (g) = Final Body Weight (g) -Initial Body Weight (g) Feed intake (g) was recorded on a daily basis by using the follow formula: Feed Intake (g) = Feed offered (g) -Feed residue (g) Weekly feed conversion ratio was calculated as follows: Feed Conversion Ratio = Feed Intake (g) Weight Gain (g) The mortality (%) was recorded on a daily basis if any and calculated as the number of birds died as relative to the total number of birds multiplied by 100.

Effects of Supplemented Growth Promoters on Performance and Intestinal Morphology in Broilers Reared under Different Stocking Densities
eRBCA-2019-1073

Intestinal Health
After slaughtering at 35 days of age, 5 ileum samples of each treatment group were collected to determine the Villus height (um), Crypt depth (um) and Villus to crypt ratio by the method adopted by Houshmand et al. (2011).

Statistical Analysis
The collected data were analyzed through factorial ANOVA technique. General Linear Model was used in SAS software (version, 9.1). Significant treatment means were separated through Duncan's Multiple Range (DMR)-test (Duncan, 1955). The following mathematical model was applied: Where, Y ijk = Observation of depended variable recorded on i th and j th treatment µ = Population mean T i = Effect of i th treatment (i = 1, 2, 3, 4, 5) S j = Effect of j th stocking density (j = 1, 2, 3) (TS) ij = Interaction effect between treatment and stocking density åe ijk = Residual effect associated with i th and j th treatment NID ~ 0, ó 2

Feed intake
All treatments separately as well as in interaction showed variations in feed intake (Table-3). The birds fed the control diet showed the highest feed intake whereas the birds fed symbiotic in the diet showed the lowest (p=0.0008) feed intake than that of other groups. The probable reason of these results might be that the supplementation of growth promoters like symbiotics in broiler diets enhance the biological functions of the beneficial microbes in the gut of the host birds and increase the nutrient absorption thereby decreasing the feed intake (Sara et al., 2016). Our findings are in agreement with those of Dozier et al. (2006), who reported a linear decrease in feed intake with increasing population density.
As far as the stocking density is concerned, nonsignificant differences were observed among all treatment groups. The interaction of growth promoter and stocking density indicated that the birds reared at 0.056m 2 stocking density in the control group showed the highest (p≤0.05) feed intake, while the birds supplemented with symbiotic as growth promoter and reared at 0.065m 2 stocking density showed the lowest (p≤0.05) feed intake compared to the other groups. Similar results were found by Tong et al., (2012) who observed significantly higher feed intake and growth of broilers reared at different stocking densities. Similarly, Mehmood et al., (2012a) found significantly higher feed intake in the birds reared at 0.065m 2 /bird than that of 0.056m 2 and 0.046 m 2 /bird stocking densities.

Body weight
Different treatments, including growth promoters and stocking density separately and in interaction, showed pronounced effect on the body weight (Table-3). Among the growth promoters, symbiotics showed the highest (p=0.0085) body weight, while the control group showed the lowest body weight compared to that of other treatment groups. Increased body weight in symbiotic group may likely be attributed to the beneficial effects of growth promoters, stimulating activity of one or a limited number of bacteria in the colon, and thus improves host health and body weight (Ashayerizadeh et al., 2009). The symbiotics supplementation also promoted favorable condition in the intestine for the colonization of beneficial microflora, facilitated better growth performance of broiler chicks. As far as the

Effects of Supplemented Growth Promoters on Performance and Intestinal Morphology in Broilers Reared under Different Stocking Densities
eRBCA-2019-1073 stocking density is concerned, birds reared at 0.065m 2 stocking density showed the highest (p≤0.05) body weight followed by those reared at 0.056m 2 and 0.046m 2 . Increased body weight on 0.065m 2 stocking density in the current study may be explained by the fact that more rearing space facilitated the birds to exhibit his maximum potential for faster growth rate (Sahota et al., 2012).

Feed Conversion Ratio
Overall means of feed conversion ratio (FCR) showed significant differences among the various treatment groups (Table-3). The symbiotic fed birds had improved (p=0.0001) FCR compared to that of other treatment groups. The improved FCR in symbiotic fed group might be attributed to the improved intestinal environment as it is reported that feeding symbiotic in the diet reduces the intestinal pH and increases digestive enzyme activity in gastrointestinal tract (Samli et al., 2007). Similar to these findings, Oliva Das et al.
(2016) indicated a significant improvement in FCR in birds fed different growth promoters such assymbiotic than those fed with the control diet.
As far as the stocking density is concerned, nonsignificant differences were observed among all groups. The interaction of growth promoters and stocking densities exhibited non-significant difference in feed conversion ratio. Similarly, Thitaram (2005) investigated the effect of isomalto-oligosaccharide (IMO), as a growth promoters, in a concentration of 1%, 2% and 4%, but found no differences in feed conversion ratio compared to the control group.

Mortality
Mean mortality percentage were significant among stocking density and their interaction with growth promoters (Table-3). The lowest mortality was recorded in birds reared under 0.065m 2 stocking density whereas maximum percentage was observed in 0.046m 2 . Most likely the explanation of this trend is that the mortality rate is inversely proportional to the stocking density, as the stocking density increases the mortality rate also increases (Dozier et al., 2006). In addition, birds remain under permanent stress in smaller spaces which deteriorate the immune status. However, some studies indicated that different stocking densities did not lead to the apparent differences in the incidence of mortality (Tong et al., 2012).

Villus height
Means of different growth promoters, symbiotic found significantly higher (p=0.0011) values of villus height as compared to other treatment group, but a non-significant difference was observed in stocking densities (Table-5). The interaction among different treatment groups exhibited higher villus height in symbiotics supplemented groups reared at 0.065m 2 stocking densities, while the lowest was found in antibiotics supplemented group reared at 0.065m 2 stocking density. The longer ileal villi observed in broiler chickens that were fed on diets supplemented with probiotic and symbiotic may be due to the

Effects of Supplemented Growth Promoters on Performance and Intestinal Morphology in Broilers Reared under Different Stocking Densities
eRBCA-2019-1073 enhanced short chain organic acids formation induced by probiotics. Thus the increase in the acidity in the gut, which reduces the growth of many pathogenic or non-pathogenic intestinal bacteria, reduce intestinal colonization and reduce infectious processes, ultimately decrease inflammatory processes at the intestinal mucosa, which increase villus height (Loddi et al., 2004). The results were in line with the findings of Mirza (2009) who found a significant increase in ileum villus height at 42 days as a result of symbiotic supplementation to the broiler diet.

Crypt depth
The overall means of growth promoters and stocking densities showed non-significant differences ( Table-5). However; the interaction between growth promoters and stocking density revealed significant differences which might be due to intestinal epithelial cells originated in the crypt which migrate along the surface of the villus to the villus tip and extrude the intestine within 48 to 96 hours (Potten, 1998). The shortening of the villus height and thickness of the crypt may lead to the poor absorption of nutrients and enhance the gastrointestinal secretion which ultimately found poor growth performance (Xu et al., 2003)

Villus to crypt ratio
Comparing means among growth promoters and stocking densities showed significant results in villus to crypt ratio (Table-5). Symbiotics supplemented group showed significantly higher (p=0.0001) villus to crypt ratio compared to the other groups while, stocking density remained non-significant. In interaction the probiotics supplemented group had the higher values of villus to crypt ratio reared under 0.056m 2 stocking density, while the antibiotics supplemented group showed the lowest values reared at 0.065m 2 floor space. The supplementation of symbiotics in the diet enhance the villus to crypt ratio which ultimately had the positive effect on the absorption of nutrients and increase the body weight of the birds (Catalá-Gregori et al., 2008). Similar findings are seen in a study by