Abstract

Objective: This experimental study was conducted to investigate the effects of diets and enzyme supplementation on the live weight (LW), feed intake (FI), feed conversion ratio (FCR), viability, leg bone quality and meat yield traits of broilers. Materials and Methods: In a 2×2 factorial experiment, having two diet types [corn-soya meal (CSM) based and corn-wheat-soya (CWS) based] and two enzymes levels (with or without), Broiler chicks (n = 112; Ross308) were assigned to four dietary treatments (CSM-, CSM+, CWS- and CWS+) with four replicates, seven chicks per replicate in a CRD. The chicks were fed on the ready-made broiler diet up to 25 days, after that formulated diets were supplied the birds ad libitum up to 45 days. All the diets were iso-caloric and iso-nitrogenous in nature and supplemented with or without enzymes in mash form. Results: Data revealed that diets (CSM- and CWS-) had no significant effect (p>0.05) on the LW, FI and FCR of broilers but enzymes (CSM+ and CWS+) increased (p<0.5, p<0.01) the LW and FI on day 45. Enzyme, diet and their interaction had no influence (p>0.05) on the viability, latency-to-sit (LTS), gait-scoring (GS) and bone traits of broilers. Diet and their interaction had no influence (p>0.05) on the bone quality traits of broilers except for enzyme. The mineral concentration (Ca%) and other bone traits were increased (p<0.05) by enzymatic diets. The results of dressing percentage, drumstick weight, thigh weight, breast weight, wing weight, back weight, shank weight and neck weight percentage of broilers were unaffected (p>0.05) by enzyme, diet and their interaction. Enzyme and diet had no influence (p>0.05) over the breast weight but its interaction influenced (p<0.05) the breast weight (%) of birds. Giblet and back weights (%) were influenced (p<0.05) by the diet only. Conclusion: It could be concluded that broilers might respond positively to enzymatic diets at the later stage of production.

INTRODUCTION

Poultry is now emerging industry across the globe. The main goal of poultry integrators is to achieve optimum production with low investment. This trend is intensifying and putting force over the poultry geneticists and nutritionists to explore alternative policies for profitable poultry production. Different feeding strategies such as reduction of specific nutrient content with addition of various supplements in poultry diets have been potential to increase the productivity of broiler and reduction of feed cost. For the ban of animal by-products and indiscriminate uses of antibiotics in animal nutrition by European Union^1,2, poultry nutritionists and researchers are looking for alternative feed supplements to enhance poultry productivity. One such strategy of reducing feed cost, could be the uses of exogenous multi-enzymes in vegetable based diet for boosting poultry production.

Research focusing on the bird's endogenous enzymes³ suggests that the young birds might be limited in the types and amounts of enzymes necessary to utilize a high carbohydrate and vegetable protein diet at an early stage, thus affecting nutrient digestibility. For this reason, there is a potentiality of using exogenous enzymes in the diets that might improve digestibility of the feeds as well as performance of the birds. Inclusion of enzymes to diets enables the birds to degrade anti-nutrient feed components, in particular, non-starch polysaccharides and phytate. Enzymes are found to promote the breakdown of starch, cell walls, storage proteins and anti-nutritional factors⁴. The purpose of adding exogenous enzymes to poultry feeds is to reduce the effects of the anti-nutritional factors of ingredients that are present in greater or lesser amounts in the diet. Non-nutritive feed additives such as exogenous enzymes and direct-fed microbial are a means to overcome the antinutritional components of soy products by improving bird performance^5,6,7, overall nutrient availability and potentially immune and intestinal health⁸.

The use of commercial enzymes in poultry feeds has evolved greatly in the last few years, based on the efficacy of the new products and a better understanding of the relationship between enzyme activity and substrates available⁹. Recent studies have also supported that the addition of multiple enzyme preparation (Natuzyme) which has carbohydrase, protease and phytase activities, might be helpful in terms of growth performance and intestinal activity in diets based on all-vegetable feed ingredients^6,10,11.

Enzymes have been approved for use in poultry feed because they are natural products of fermentation and therefore, pose no threat to the animal or the consumer.

Enzymes not only enable livestock and poultry producers to economically use new feedstuffs but will also prove to be environmentally friendly, as they reduce the pollution associated with animal production. Since the use of antibiotics in feed will be restricted in the future, there will be growing interest in using enzyme as a bioactive compound for improving gut health. However, very limited research works have been done regarding enzyme supplement in allvegetable diet and other nutrients. Enzyme supplement in vegetable diet might be economical or could have potential to improve the productivity and decrease production cost. Moreover, consumers might be benefitted to find organic, healthy and nutritious or quality poultry products, as the birds would be reared on vegetable sourced diets with enzyme only without using any antibiotics as growth promoters. Considering the above, the present study was undertaken to investigate the efficacy of enzymes on the gross responses, bone quality and meat yield traits of broilers fed enzymesourced diets.

MATERIALS AND METHODS

Enzyme preparation: A multiple enzyme preparation, Natuzyme (Bioproton Pty Ltd., Sunnybank, Australia) is a powder form composed of xylanase (10,000,000 unit kg^-1), cellulase (5,000,000 unit kg^-1), β-glucanase (1,000,000 unit kg^-1), pectinase (140,000 unit kg^-1) from Trichoderma reesei and Trichoderma longibrachiatum. It also contains protease (6,000,000 unit kg^-1) and phytase (500,000 unit kg^-1) from Aspergillus niger and α-amylase (1,800,000 unit kg^-1) from bacillus subtilis.

Animal husbandry: Day-old Ross male broiler chicks (n = 112; 46.34±0.27 g) were procured from a local commercial hatchery and used for conducting this trial from hatch to 45 days. The chicks were weighed initially and were immediately distributed randomly into four dietary treatments (details are shown below) in a 2×2 factorial arrangement, each treatment replicated four times with 7 birds per replicate. The birds were reared in brooder cages, in an open-sided house up to 45 days weeks. For the first two days the birds were provided with a temperature of 33°C. The temperature was then gradually reduced by 1 or 2°C every 1 or 2 days until the chicks were 19 days old at which point the temperature was maintained at 24°C for the rest of the trial. Eighteen hours of lighting and 6 h darkness per day were provided throughout the trial period except for first week only and at this period continuous lighting (23 h light : 1 h darkness) program was maintained for the chicks.

Diet formulation: Four experimental diets (CSM-, CSM+, CWS- and CWS+) were formulated with maize, wheat, rich polish and vegetable oil as the main energy sources, along with soybean meal as the main protein sources (Table 1). The diets were formulated exclusively with the ingredients of plant origin with or without addition of multi-enzymes (Natuzyme). Two basal diets (CSM- and CWS-) were formulated with soybean meal along with basal grains including other stuffs as shown in Table 3. These were fed as such (CSM- and CWS-) or supplemented with microbial enzymes (Natuzyme, Bioproton Pty Ltd., Sunnybank, Australia) to create diets CSM+ and CWS+. Natuzyme is a multi-enzyme (containing amylase, xylanase, β-glucanase, cellulase, pectinase, protease and phytase activities) supplemented to the basal diet at the rate of 0.4 g kg^-1. All the test diets were iso-caloric and iso-nitrogenous in nature and supplemented with or without exogenous cocktail enzymes. Bird was fed ready-made starter broiler diet for the first 25 days and then finisher or formulated diet in mash form was used to feed the broilers entire trial period from the rest of the trial period (26-45 days).

Data and sample collection: Mortality of birds were recorded as it occurred, while body weight and feed intake were recorded weekly for the calculation of body weight gain and feed conversion ratio (FCR) was corrected for mortality. Viability was calculated from mortality of birds per replicate cage. On day 45, two birds were selected randomly per treatment and killed humanely to obtain the right tibia bone samples for measurements of leg bone characteristics (bone length, bone weight, width of bone) and mineral concentration (Ca and P). Meat yield (dressed yield, breast weight, thigh weight, drumstick weight, shank weight, giblet weight, back weight) characteristics were also recorded on the last day of experiment. Feed samples were also collected prior supplying the birds to assess the nutritive value of the feeds. Cost benefit was calculated at the last of the trial period.

Latency-to-sit (LTS) and gait-scoring tests: On day 35, leg bone development was assessed through LTS and gait-scoring tests. The LTS tests were performed according to the method of Berg and Sanotra¹², with some modifications. Birds were tested in pairs in a luke-warm water. The test is based on the fact that bodily contact with water is an aversive experience for broiler chickens. Standing time is therefore positively correlated to the strength of leg bone. Two birds per replicate were used to carry out this test. Plastic tub containing warm water (32°C) at a depth of 3 cm was used to carry on the test (Fig. 1). The water temperature was checked every 10 min with a thermometer to ensure the correct temperature was maintained and it was checked again before another batch of birds was tested. Hot or cold water was added to raise or lower the water temperature if needed. Two birds from each replicate were selected randomly from the cage and put into the water tubs. The tubs were covered with mesh lids to prevent birds from flying out. The birds were observed and left to stand in the tubs of water for up to 10 min. The time elapsed before the birds first attempted to sit down in the water was recorded in seconds with the aid of a stopwatch. If any bird was still standing after 10 min, the test was terminated.

The gait-scoring test conducted followed the 3-point scoring system developed by Kestin et al.¹³ and Webster et al.¹⁴. To conduct the test, two birds were randomly selected from each replicate group and allowed to walk freely on the floor. The birds were then scored by visual observation against a number of criteria as described in Table 3.

Samples processing and analyses: Feed: Four feed samples from finisher diets were collected from the formulated diets prior feeding the birds. The samples were processed by grinding with the help of mortar and pestle and then mixed thoroughly for lab analyses. About 500 g of each diet of starter and finisher were taken and sent to the lab for further analysis. Each analysis was done three times for each sample to minimize technical errors. The samples were tested for dry matter (DM %), moisture (%), crude Protein (CP%), crude fiber (CF%), ether extract (EE%) and ash using standard laboratory procedure¹⁵. Dry matter was determined by oven dry method. Crude protein was recovered by Kjeldahl process. Ether Extract was quantified by Soxhlet apparatus. Ash was measured by igniting the pre-ashing sample on a muffle furnace at a temperature of 600°C for four to 6 h. The feed analytical report is shown in Table 2.

Bone processing and characteristics : The collected tibia bones were boiled for 10 min in deionized water, to remove all the soft tissue and defatted. After that, bone characteristics such as bone length and head width were measured using digital callipers (Mitutoyo, Japan) and the weight was recorded. After taking the measurements of the bone traits, the samples were ground and put on a muffle furnace at a temperature of 600°C for 4 h to make ash. The bone ash weight of samples was recorded. After that, the ash was analyzed for bone mineral concentration (Ca and P only), by atomic absorption and spectrophotometry, respectively.

Statistical analyses: All data were subjected to analyze by the GLM procedure using Minitab statistical software¹⁶. The significance of differences between means was tested using the Duncan multiple-range test. Statistical significance was considered at p≥0.05.

RESULTS

The gross responses and livability of broiler chickens: The results of body weight (BW), feed intake (FI) and feed conversion ratio (FCR) of broiler chickens fed vegetable based diet supplemented with exogenous multi-enzyme were shown below in Table 4. The data showed that diet and its interaction (Diet×Enzyme) had no significant (p>0.05) effect on the BW, FI and FCR of broiler chickens except for enzyme. Increased (p<0.05) BW and FI were found in the broilers fed diet supplemented with enzyme (CSM+, CWS+) compared to others diets (CSM-, CWS-) without affecting (p>0.05) FCR on day 45, respectively. Highest live weight and FI were recorded with the enzyme supplemented diets and the lowest live weight and FI were recorded at CWS diet without enzyme supplementation from 25-45 days. The diet, enzyme and their interaction had no influence (p>0.05) on FCR of broiler in this study.

Livability (%) of broiler chicken fed test diet: The livability (%) of broilers was not influenced (p>0.05) by diet, enzyme and its interaction, as shown in Fig. 2. It implies that the diet, supplemented diet and their interaction had a similar effect on the growth, development and mortality of broilers. The livability of broiler chickens was not affected by the supplementation of enzyme in the diet in the current study. Latency-to-sit (LTS) and gait-scoring (GS) test: The results of LTS and GS tests denote that diet, enzymes and their interaction had no (p>0.05) effect on the LTS and GS of broiler chickens on day 35, as shown in Table 5.

Meat yield traits of broiler chickens: The results of meat yield characters i.e dressing yield, drumstick weight, thigh weight, breast weight, shank weight, giblet weight and back weights (%) of broilers chickens fed vegetable diets are shown in Table 6. The data show that diet, enzyme and its interaction (Diet×enzyme) had no significant effect (p>0.05) on these parameters such as dressing (%), drumstick weight (%), back weight (%), shank weight (%) and thigh weight (%)of broilers chickens. Diet has influenced (p<0.05) the giblet and back weight only. Interaction (Diet×enzyme) significantly (p<0.05) increased the breast weight of broiler chickens. Breast weight was poorly increased (p<0.097) by enzyme supplemented diets.

Bone (right tibia) characteristics and mineral concentration (Ca and P) of broiler chickens: The results of leg bone characteristics (e.g. bone length, bone weight, bone width and bone ash) and bone minerals (Ca, P) were shown in Table 7. Data shows that bone length (BL), bone weight (BW), bone width and bone ash (BA) were influenced by enzyme significantly (p<0.05). Diet and the interaction of diet×enzyme had no effect (p>0.05) on these bone traits such as bone length (BL), bone weight (BW), bone width and bone ash (BA) of broiler chickens. Diet and the interaction of diet×enzyme had no effect (p>0.05) on the Ca and P contents of tibia bone. Enzyme had (p<0.05) impact on the Ca contents of tibia bone and no impact (p>0.05) on P content of tibia bone.

DISCUSSION

The gross responses of broiler chickens fed vegetable based diets supplemented with exogenous enzyme: The gross responses of broiler in terms of feed intake (FI), body weight (BW) and feed conversion ratio (FCR) are the most important criteria for evaluating the growth performances of broilers. These criteria i.e., FI, BW and FCR etc., are considered as the key performance indicators (KPI) for assessing the broiler's potentiality in regard to growth responses. Results of the current study showed that, body weight of broiler was increased by the supplemental exogenous enzyme offered to the birds. The improved body weight of broilers fed on enzyme supplemented diet might be as a result of increased feed consumption. Further, the increased feed consumption of broilers fed on supplemented diets could be probably due to rapid growth rate of broiler chickens and therefore, more nutrients are required to ensure their faster growth. In addition, the increased feed intake of broilers might be due to higher fibre digestion as the vegetable feeds are rich sources of fibre. Initially, birds have filling effect in gut by fibre intake. After fibre digestion, chicks are able to increase feed intake to meet their nutrient requirements¹⁷. Moreover, the faster growth rate of broilers, might be due to greater feed intake.

Moreover, enzyme supplementation of diets might enhance the availability of certain nutrients, including trace minerals (e.g. Mn, Cu, Zn), which are known to promote greater feed intake of broiler chickens¹⁸, enabling them to reach their full growth potential. It is reported that addition of microbial enzymes in diets might facilitate the broiler chicks to degrade the anti-nutritive factors such as NSP, in addition with promoting the breakdown of starch, cell walls and storage proteins4. Moreover, Lazaro et al.¹⁹ reported that digest a viscosity might be reduced by exogenous enzymes, which could facilitate the improved contact between endogenous enzymes and nutrients, thereby improve the digestibility of the nutrients (amino acids).

Numerically lower FCR value was observed in the broilers fed diet supplemented with enzyme compared to diet without enzyme. It implies that birds fed on supplemental diets appears to be a bit more efficient in converting feed to meat. The improved feed consumption and body weight might be a result of better feed efficiency of broilers fed on supplemented diet, as is evinced in current study. Results of the current study are agreed with the findings of previous researchers^20,21,22. Cowieson²³ and Zanella et al.⁶ reported that exogenous enzymes can increase the feeding value of soy and other grain products, when fed to broilers, leading to increased growth performance and efficiency.

Livability of broiler chickens: The livability (%) of broilers was not influenced by diet, enzyme and its interaction, as observed form the current study. It can be assumed from the findings that for the growth and development of broiler chickens, basal diet, supplemented diet and their interaction had no effect on the livability of broiler chickens. Further, it implies that supplementation of exogenous enzyme in the broiler diets has no detrimental impact on the growth and survivability of the broiler chickens. Several studies demonstrated that enzyme could stimulate the natural immune system of poultry^24,25,26, which in turn could stimulate the similar livability of broiler chickens regardless of using enzymes in the diet. Hashemi and Davoodi²⁷ reported that enzymes improve the weight of immune organelles such as burse and spleen which can promote the immune situation and efficacy of health and livability. Freitas et al.²⁸ reported that using protease enzyme in the diet had no effect on the mortality of birds.

Meat yield traits of broiler chickens: The meat yield traits of broiler chickens were unaffected by enzyme supplemented diets in this study. This might be due to similar growth responses of broilers fed on enzyme supplemented diet. The breast weight was found to be a bit improved by enzyme supplemented diets. The results obtained contradict with the findings of previous researchers^11,29.

Bone (right tibia) characteristics and mineral concentration (Ca and P) of broiler chickens: Leg problems or disorders in broilers are an important welfare and economic issues for the poultry industry. Leg deformities in broilers due to genetic, nutritional and growth factors could result in decreased feed intake and body weight. Thus, feed optimization might be a strategy to decrease the severity of leg lesions in broilers³⁰.

In this study, all bone traits were influenced by dietary enzyme supplementation. The calcium percentage in bones was increased by supplementing enzymes, although LTS and GS tests depicted negligible influence. Dietary enzymes could help to release more nutrients from the feed for better growth and development of bones in broilers. Our results agree with the Sohail and Roland³¹, who stated that enzyme (Phytase) supplementation significantly increases body weight, bone mineral content, bone density and livability of broilers.

According to Ravindran et al.³², dietary phytase has been reported to increase mineral (Ca, P) retention and its concentration in serum and tibia of broilers. Phytate, being a strong acid, can form various salts with essential minerals, thus, reducing their solubilities and absorption³³. When phytate is hydrolyzed by microbial phytase, it could release all mineral constituents, myo-inositol and inorganic phosphate³⁴.

CONCLUSION

It is concluded that broilers respond positively to the diets supplemented with exogenous enzymes in terms of increased feed intake, body weight and bone quality. Thus, enzyme supplementation might enhance nutrient availability and production potential of broilers affecting cost significantly.

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Article made possible through the contribution of M. A. Hossain et al.

The responses of exogenous enzymes (multi-enzymes) on the productivity and stage of production of broilers fed vegetable-sourced diets