The livestock sector in Asia is under pressure to adapt and expand¹. These adaptations involve mainly a shift in livestock species and functions, with the greatest change being an increase in the number of monogastric animals (pig and poultry). The poultry industry has never been under a greater pressure than at the present time². The production of poultry in Iran has been increasing annually at a rate of 8 % for the last decade reaching 1.589 million heads and 1.9 t of output³ in 2011, which is the largest in the Middle East.

The white button mushroom (Agaricus bisporus) with high content of polyphenols, ergothioneine, vitamins, minerals and polysaccharides⁴ has been demonstrated to possess valuable biological properties including antitumor, antiaromatase, antimicrobial, immunomodulatory, anti-inflammatory, and antioxidant activities^5-11. Despite the largely known positive effects on human health^12,13, there is little information about the effect of mushroom consumption in poults. However, on turkeys, it has been reported that A. bisporus, due to its polysaccharides content, has a prebiotic-like effect and improves the efficiency in digestibility and utilization of feed^14,15.

The Iranian production of mushroom and truffles has increased from 10,000 t in 1997 to 82,000 in 2011 (FAO estimate), holding in this year the 8th place in the world mushroom production³. The mushroom industry produces significant quantities of mushroom wastage that are discarded during the mushroom production process. The recycling of this by-product as feed additive for broiler production should increase the efficiency of the Iranian food industry by reducing costs.

The positive impact of antibiotic as growth promoter (AGP) is well known in the poultry industry due to the suppression of harmful microorganisms¹⁶ and to inhibition of inflammation effects¹⁷. Association of bacterial resistance with the addition of AGP in the chicken feed may be questionable^18,19 and it seems that is the precautionary principle the main reason for the banning of AGP in several countries^20,21. In this sense, the use of antibiotics not used in humans therapy and that do not cause resistance could still be an alternative. This is the case of flavophospholipol, a phosphoglycolipid antimicrobial produced by several strains of Streptomyces that is not used for treatment of human infections, which is why it can still be legally added in poultry diets in many countries¹⁶.

Previous results from our group have shown that the addition of dietary mushroom powder and flavophospholipol has a beneficial effect on growth performance as it showed positive on weight gain, feed conversion ratio and body weight at 42-d of growth (Shamsi et al. under review). The aim of this current study is to demonstrate that these previous positive effects on chicken performance are directly linked to the carcass yield. In addition, the effect on viscera weight and digestive tract measurement have also been evaluated.

This study was performed in the Poultry Farm Facilities in Ramsar city (50°40' N and 36°54' E) and the Agriculture Faculty, Islamic Azad University, Rasht Branch, Iran, during the summer (warm) season, between July-September 2013, for a total of 42 d. The experiments were approved by the Scientific Board of the Islamic Azad University, and were conducted as per International Guidelines for research involving animals²².

Three hundred (300) one-day-old male Ross 308 broilers²³ were randomly assigned to 10 treatments with three replicates per treatment. Each replicate consisted of 10 chicks housed in pens of 1.2 m2. Before the animals were placed in their pens, all the facilities were thoroughly cleaned and disinfected with 1:125 and 1:250 solution of Multifenelic. Other equipment including drinkers and feeders was disinfected with 20% Benzalkonium chloride - Germo Killer. All the windows and the ventilators were gasified with Formalex® solution.

The room temperature was adjusted to approximately 33 ºC on d 1, and then was gradually decreased to 24 ºC. The pens were fitted with electrical heaters. Lighting was provided with a program of 23L:1N (from 1900 to 2000 h). Humidity was maintained between 55 and 65 % in the early growing period, by spraying water on the floor. The chicks were vaccinated against Newcastle and Gumboro diseases. Within 24 h after vaccination, a multiple-vitamin and electrolytic solution (1:1000) was offered via drinking water to reduce stress.

Water and feed were supplied ad libitum during the entire experimental period. The nutritional requirements were based on Ross strain rearing catalogue²³. The feed was formulated to be iso-proteic and iso-energetic for all treatments. Composition of the diets and their nutrient contents are presented in Table 1. Three types of feed formulations were used during the rearing period: the starter period (1-14 d of age (DOA), grower period (15-28 DOA) and the finisher period (29-42 DOA). Feed remaining in feeders was weighed and recorded weekly.

Table 1 Feed ingredients and nutrient analysis of diets used during the starter (1-14 d of age), grower (15-28 d of age), and finisher periods (29-42 d of age). 

1 Calcium Pantothenate: 4 mg/g; Niacin: 15 mg/g; Vitamin B6: 13 mg/g; Cu: 3 mg/g; Zn: 15 mg/g; Mn: 20 mg/g; Fe: 10 mg/g; K: 0.3 mg/g.

2 Vitamin A: 5000 IU/g; Vitamin D3: 500 IU/g; Vitamin E: 3 mg/g; Vitamin K3: 1.5 mg/g; Vitamin B2: 1 mg/g.

3 SID (Standardized Ileal Digestible).

Mushrooms (A. bisporus) were obtained from a local mushroom producer (Pars Company, Ramsar, Iran). The whole mushrooms were dried at 60 ºC followed by grinding, and added to the experimental diets. Treatments were selected to determine five levels of mushroom powder (MP; i.e., 0, 0.5, 1.0, 1.5, and 2.0 g/kg), and two levels of antimicrobial flavophospholipol (Teif Azmoon Pars, Co., Tehran, Iran), 0 or 5.0 mg/kg.

At the end of the study, one bird per pen (totalling three birds per treatment) was selected based on the average weight of the group, weighed to obtain live body weight followed by killing and complete bleeding. Head, viscera and shanks were removed. The birds were dissected to remove and determine the yield of different viscera, digestive tract and carcass parts. Empty crop, empty proventriculus, empty gizzard, abdominal fat pad, thymus, spleen, bursa of Fabricius, liver, kidneys, pancreas, lungs, hearth and brain were weighed. Empty small intestine was divided into duodenum (from the gizzard outlet to the end of the pancreatic loop), jejunum (from the pancreatic loop to Meckel’s diverticulum) and ileum (from Meckel’s diverticulum to the ileo-caeco-colic junction) and these three different parts were weighted.

Length, width and diameter of different parts were measured. Empty large intestine was divided into right and left cecum, colon and rectum. These parts were weighted and measured. Relative weights were calculated as a percentage of live weight at slaughter. Carcass was left for one hour to remove excess water and allowed to sit overnight in a refrigerator at 4 ± 1 ºC using a ratio between the eviscerated weight and live weight x 100. Carcass was then portioned; breast, drumsticks, wings, neck and notarium were obtained and weighed. Data from these measurements were used to calculate the percentage of part to the carcass weight.

General Lineal Models (GLM, SPSS 15.0, Chicago, IL, USA) examined the effects of MP and flavophospholipol on carcass and viscera weights and on the measurements of the digestive tract in a 2×5 factorial arrangement using Yij_k=ì+A_j+B_k+A_jB_k+ e_ijk statistical formula. Variables found significant with GLM were tested with one-way ANOVA to find differences between treatments, (different levels of MP addition or antibiotic vs no antibiotic addition groups). Tukey’s test was used to determine disparities among the groups.

Cold carcass weight was affected by both the inclusion of the antibiotic and MP (Table 2). The same variables affected positively too the dressed carcass weight (Model F_3,26= 10.70, P<0.001, R²= 56.8 %; coefficients: MP= 7.4 ± 36.2, P<0.001, antibiotic= 212.4 ± 62.6, P<0.01, interaction between both factors= -196.7 ± 51.1, P<0.01). Inclusion of flavophospholipol in diet improved some of the measured carcass traits. In this sense, the addition of the antibiotic resulted in birds with greater breast weight as well as greater relative breast weight (Model F_3,26= 6.23, P<0.05, R²= 18.2 %; coefficient: antibiotic= 2.0 ± 0.8, P<0.05). In contrast, dietary flavophospholipol showed a negative effect relative weight of wings (Model F_3,26= 3.79, P<0.05, R²= 31.3 %; coefficients: antibiotic= -0.7 ± 0.2, P<0.01, interaction between MP and antibiotic= 0.03 ± 0.1, P<0.05) and neck (Model F_3,26= 4.98, P<0.05, R²= 15.1 %; coefficient: antibiotic= -0.2 ± 0.1, P<0.05).

Table 2 Effect of flavophospholipol (Ab, 0.0 or 5.0 mg/kg) added to diets containing mushroom powder (MP) at 0.0, 0.5, 1.0., 1.5 or 2.0 g/kg on different carcass weights (g) of male broiler chicken at 42 d of age. 

^ab Means with the same letter are not significant different (P<0.05).

Feeding rations containing MP yielded the highest breast, drumstick and neck weights (Table 2). The effect of the two variables studied on carcass viscera weights are shown in Table 3. Inclusion of flavophospholipol in diet at 5.0% failed to improve the weight of any organ weighed. On the other hand, mushroom powder showed a positive effect on the weight of thymus, spleen, bursa of Fabricius, liver and brain. Finally, the effects of the antibiotic and MP diet addition on the gastrointestinal traits are showed in Tables 4.1, 4.2 and 4.3. There was no effect of the antibiotic on any of the gastrointestinal measurements taken and MP only showed statistically significance differences in 6 out of the 27 traits measured.

Table 3 Effect of flavophospholipol (Ab, 0.0 or 5.0 mg/kg) added to diets containing mushroom powder (MP) at 0.0, 0.5, 1.0., 1.5 or 2.0 g/kg on viscera weights (g) of male broiler chicken at 42 d of age. 

^ab Means with the same letter are not significant different (P<0.05).

Table 4.1 Effect of flavophospholipol (Ab, 0.0 or 5.0 mg/kg) added to diets containing mushroom powder (MP) at 0.0, 0.5, 1.0, 1.5 or 2.0 g/kg on digestive system (weights, g and measurements, mm) of male broiler chicken at 42 d of age. 

Means with the same letter are not significant different (P < 0.05).

Table 4.2 Effect of flavophospholipol (Ab, 0.0 or 5.0 mg/kg) added to diets containing mushroom powder (MP) at 0.0, 0.5, 1.0, 1.5 or 2.0 g/kg on digestive system (weights, g and measurements, mm) of male broiler chicken at 42 d of age. 

Means with the same letter are not significant different (P < 0.05).

Table 4.3 Effect of flavophospholipol (Ab, 0.0 or 5.0 mg/kg) added to diets containing mushroom powder (MP) at 0.0, 0.5, 1.0, 1.5 or 2.0 g/kg on digestive system (weights, g and measurements, mm) of male broiler chicken at 42 d of age. 

^ab Means with the same letter are not significant different (P<0.05).

The results of the present study show that the effect of the antibiotic and MP are positive in the cold carcass weight. Although flavophopholipol had a negative effect on relative weights of neck and wings, however it had a positive effect on the eviscerated carcass weight. On the other hand, MP addition has a positive effect on carcass weight components that are economically important, e.g., breast and drumsticks. The combination of antibiotic and MP did not exert a cumulative effect on these carcass traits despite the effects shown as individual factors.

Greater weight of the immune organs (thymus, spleen and bursa) with MP suggests a better immune status of the MP treatment chicks since lymphoid organ weights reflect the body’s ability to provide lymphoid cells during an immune response. Thus, immunosuppressed or stressed birds usually have smaller lymphoid organs while higher bursa weight reflects better health status. The bursa of Fabricius has an extremely vital role in morphologic and functional integrity of the immune system. The weight of the bursa in broilers also reflects the anatomical response to immune system alteration due to stress²⁴. In accordance with our results, other authors have also reported immune-enhancing benefits in broiler chickens fed different mushroom supplements²⁵. Since the composition of mushrooms is complex and includes different proteins, such as fungal immunomodulating proteins (FIPs)²⁶, edible mushrooms should not be considered as only a simple food.

The immunostimulant activity of A bisporus has been largely demonstrated; their polysaccharide extracts express an immunostimulating effect and induce synthesis of IFN-𝛿, which alters transcription of up to 30 genes producing a variety of physiological and cellular responses²⁷. Furthermore, A. bisporus is also a good source of selenium²⁸, which has been shown to improve humoral immune activity²⁹.

The greater liver weight of chicks treated with mushroom extract could reflect the antioxidant effect of white button mushrooms^7,8,9. It has been observed that the administration of ethanoic extract of A. bisporus significantly enhanced the activities of antioxidant enzymes in liver of mice²⁵.

Broadly speaking, supplementation of MP showed a very weak effect on the different measurements of the gastrointestinal tract. It was expected greater differences since it has been showed that the mushrooms affect intestinal morphology. Thus, researchers have found that mushroom intake influences mucosal architecture resulting in greater duodenal, jejunal and ileal villus height, changes that affect the capacity of the birds to absorb nutrients from the feed¹⁵. More surprising is the fact that it was not find any positive effect of including flavophospholipol, as AGP cause changes in the gross structure of the gastrointestinal system. Most of these changes are the result of reduction in overall weight of the small intestine due more to changes in the thickness of the intestinal wall rather than changes in the intestinal length^30,31,32. Some authors have indicated that dietary inclusion of antibiotics, given as growth promoters, reduces intestine weight by thinning the intestinal wall³³ and some have found lower small intestine weight and shortening of the gut in poults treated with virginiamycin³⁴. A plausible explanation is that the negative effects of an AGP and the positive effects of the components of the A bisporus could be overlapping. Further research is needed to arrive at a definitive conclusion.

In conclusion, the use of flavophospholipol and MP had a positive effect on the growth performance of chicks. These effects were translated into greater carcass components with economic relevance such as the breast and drumsticks. Results also show an overall increment in the immune related organs which reflects a better immune status of the broilers fed MP. An increment in the liver weight was also associated with antioxidative properties of the white mushroom button. Finally, regarding the effect on gastrointestinal parameters, despite the weak influence observed, further research is needed for a definitive conclusion.