Crude enzymes supplementation in fibrous diet improves performance of commercial broilers

Abstract

A total of 280, 1-day-old mixed sex broiler chicks were randomly allotted to seven dietary treatment groups. Basal diet was formulated with high fibrous contents (6.72%). Increasing levels of cell-free fermentation broth containing cellulases were mixed in basal diet of groups B, C, D and E (equivalent to 1.2×10⁴ IU, 2.4×10⁴ IU, 3.6×10⁴ IU and 4.8×10⁴ IU cellulases/kg, respectively). Where as in diet of group F, fermentation broth containing 3×10⁴ IU protease and 1.2×10⁴ IU cellulases were added per kilogram. In diet of group G fermentation broth containing 3×10⁴ IU protease and 2.4×10⁴ IU cellulases were added per kilogram. Significant improvement (P<0.05) in weight gain and feed conversion ratio (FCR) was observed in all treatment groups. Combination of protease and cellulases in diet was proved better in first 4 weeks. However, effects of cellulases were equally good (but at higher dose) in last 2 weeks compared to the groups supplemented with mixture of cellulases and protease. Increasing doses of enzymes reduced the viscosity of ingesta and also in vitro-simulated digested feed. Vent pasting seen at its maximum in control group was also reduced to zero with decreasing severity under the effect of increasing enzyme levels. Combined use of indigenously produced cellulases and protease was successful to tackle problems of high fibrous broiler diet.

Keywords:

• protease
• cellulases
• broiler diet
• enzyme supplementation
• weight gain

1. Introduction

Over the last two decades, beneficial use of exogenous enzymes in poultry diets has been proven. New dimensions of enzyme utilisation are being explored by poultry nutritionists to address feed-related problems. Digestion and removal of toxic effects of non-starch polysaccharides (NSPs) present in both viscous and non-viscous grains has been shown by Meng and Slominski (2005) and Rafuse et al. (2005). Similarly, use of phytase (Cowieson and Bedford 2009) for better utilisation of phytate and reduced phosphorus excretion are success story in poultry nutrition. Effective utilisation of vegetable protein sources for release of digestive amino acids and energy from fibre therein, use of unconventional feed materials, improvement in gut microflora, meat quality, immunity and slaughter characteristics after enzyme supplementation, symbiotic use of enzymes and substitution of antibiotics with enzymes are the study topics of today (Choct 2006; Cowieson 2010).

The main objective of this study was to test efficacy of indigenously produced protease and cellulases in broiler diet. Coon (1989) reviewed different factors affecting amino acid availability in poultry feed stuff, which provided sufficient justification for the use of exogenous protease. Chicks in early stage of development may not digest many nutrients due to lack of certain digestive enzymes (Iji et al. 2001). The use of protease was also investigated in broiler diets by Angel et al. (2011), Freitas et al. (2011) and Olukosi et al. (2007).

Poultry feed ingredients of interest for enzymatic treatments are cereal grains (maize, wheat, sorghum, rye, oat and barley) and vegetable proteins such as sunflower, guar, rape, canola meals and maize gluten. Least cost formulation for broiler diets compels to increase the rate of inclusion of cheaper or abundantly available low-quality ingredients, and thus nutritionist would have to rely on exogenous enzymes in future.

2. Materials and methods

2.1. Enzymes

Cellulases and protease were produced in shake flask broth cultures from indigenous soil actinomycetes (Larik 2010). Cellulases in supernatant were analysed by the method of Bailey and Nevalainen (1981). Protease was analysed by the method of Long et al. (1981). Supernatant fluid from various batches thus produced was pooled and stored at 4°C as separate solutions of protease and cellulases. Final quantification of enzyme levels was done in pooled samples before use in broiler diet.

2.2. Experimental chicks, housing and diets

A total of 280, 1-day-old mixed sex broiler chicks were obtained from a commercial hatchery and randomly allotted to seven dietary treatment groups (40 chicks per treatment). In each treatment group, chicks were color marked to get four replicates (10 chicks/replicate). For first 3 days, chicks were fed on diet without enzyme supplementation. From 4th day onwards, chicks were weighed and enzyme supplementation was started. Feed and water were supplied ad libitum. Diet was formulated with high fibrous contents (6.72%, Table 1). Fermentation supernatant containing 1.5×10⁴ IU of protease/ml and 0.3×10⁴ IU of cellulases/ml in separate solutions was added to feed of different treatment groups. No enzyme addition was done in feed of group A (control). Feed of groups B, C, D and E was supplemented with fermentation supernatant at the rate of 4, 8, 12 and 16 ml/kg to provide 1.2×10⁴ IU, 2.4×10⁴ IU, 3.6×10⁴ IU and 4.8×10⁴ IU of cellulases, respectively. Where as in feed of group F, mixture of protease and cellulases (2 ml+4 ml equivalent to 3×10⁴ IU protease and1.2×10⁴ IU cellulases, respectively) and in feed of group G, mixture of protease and cellulases (2 ml+8 ml equivalent to 3×10⁴ IU protease and 2.4×10⁴ IU cellulases, respectively) were added per kg. All mixings were done on a daily basis (morning and evening).

Table 1. Composition of basal broiler diet.

Ingredients	Quantity (%)
Corn	50.0
Fish meal	2.5
Rice polish	8.64
Sunflower meal	15
Guar meal	3.97
Soybean meal	4.4
Corn Gluten 60	2.18
Canola meal	7.0
Limestone	0.9
Dicalcium phosphate	1.43
Lysine	0.4
Premix	0.7
Methionine	0.08
Salt	0.28
Coccidiostat	0.02
Molasses	2.50
Calculated analysis
Metabolisable energy	2750 kcal/kg
Crude protein	19.03
Sodium	0.18
Fibre	6.72
Total ash	7.34
Methionine	0.45
Methionine+cystine	0.791
Linoleic acid	0.18
Calcium	0.9
Available phosphorus	0.47
Lysine	1.15
Tryptophan	0.2

2.3. Viscosity measurements

Effect of crude enzyme supplementation on viscosity of ingesta of birds of control and all treatment groups were measured by the method described by Bedford and Classen (1992) using Brookfield Digital Viscometer (Model: LVDV-E, P40 adaptor). On day 43, three chicks from each group were slaughtered, and ingesta samples were squeezed from intestinal portion between Meckel's diverticulum and 4 cm above the ileocecal junction. The pooled samples were immediately frozen at −20°C. Feed was also simulated to in vitro digestibility conditions (Bedford and Classen 1992), and effect of enzyme addition was noted on viscosity.

2.4. Statistical analysis

The data on weight gain and feed conversion ratio (FCR) were subjected to ANOVA and Duncan's test. Regression analysis (quadratic model) on weight gain of groups A–E was also done using statistical software SPSS 15.0 version (2006). The data on viscosity parameter are of mean of triplicate samples.

3. Results

3.1. Enzyme activity

For 1 ml of fermentation supernatant, 0.3×10⁴ IU cellulases and 1.5×10⁴ IU of protease were quantified.

3.2. Growth performance of chicks

Significantly high values of weight gain and significantly low values of FCR were observed in all treatment groups as compared to control at days 9, 16, 23 and 30. There was no significant difference in average weight gain in control as compared with groups B, C and D at day 37. Average weight gain of group E was significantly higher than that of groups A and B, and lower than that of group G at day 37. Average weight gain of group G was the highest among all other treatment groups at day 37. The FCR of group G was significantly less than that of groups D, F, B, C and A at day 37. Average weight gain of control group was significantly less, and FCR was significantly higher than all other treatment groups at day 43 (Table 2). Regression (quadratic fit) analysis also revealed significant effect of cellulases on weight gain (groups A–E, Table 3, Figure 1).

Figure 1.  Comparison of observed and predicted response of effect of cellulases addition in feed on body weight gain of broiler chicks through regression equation (quadratic model).

Note: Wt=weight in grams. Group 1=group A: control (no enzyme added); Group 2=group B: 4 ml (1.2×10⁴ IU cellulases)/kg feed; Group 3=group C: 8 ml (2.4×10⁴ IU cellulases)/kg feed; Group 4=group D: 12 ml (3.6×10⁴ IU cellulases)/kg feed; Group 5=group E: 16 ml (4.8×10⁴ cellulases)/kg feed; Group 6=hypothetical group 20 ml (6.0×10⁴ cellulases)/kg feed; Group 7=hypothetical group 24 ml (7.2×10⁴ cellulases)/kg feed; Group 8=hypothetical group 28 ml (8.4×10⁴ cellulases)/kg feed; Group 9=hypothetical group 32 ml (9.6×10⁴ cellulases)/kg feed; Group 10=hypothetical group 36 ml (10.8×10⁴ cellulases)/kg feed.

Table 2. Comparison of weight gain and FCR of broiler chicks fed at different levels of cellulase and combination of cellulase and protease.

		Treatment groups
Parameter	Age (days)	A	B	C	D	E	F	G	SDM	SEM
W (g)	9	140.9^f	163.8^e	176.6^c	179.6^c	183.2^b	172.7^d	194.1^a	0.01	0.002
FCR	9	1.00^a	0.84^b	0.76^d	0.76^d	0.75^d	0.79^c	0.71^f	16.05	3.03
W (g)	16	360.5^f	366.7^d	378.8^c	376.6^c	388.4^b	367.1^d	397.0^a	0.01	0.001
FCR	16	1.44^a	1.38^b	1.34^c	1.35^c	1.31^d	1.40^b	1.30^e	12.6	2.4
W (g)	23	633.0^f	665.2^e	680.9^d	728.8^b	730.2^b	696.2^c	751.1^a	0.01	0.001
FCR	23	1.71^a	1.40^b	1.40^b	1.50^d	1.45^d	1.52^c	1.41^e	69.8	40.1
W (g)	30	1026.0^f	1065.1^e	1080.5^d	1115.2^b	1140.2^a	1095.1^c	1128.1^b	0.01	0.001
FCR	30	1.80^a	1.70^b	1.70^b	1.62^d	1.60^e	1.61^c	1.60^e	37.9	7.2
W (g)	37	1484.2^c	1500.3^c	1517.0^bc	1540.9^b,c	1565.8^b	1526.1^b,c	1626.1^a	0.01	0.001
FCR	37	1.91^a	1.90^a,b	1.90^a,b	1.90^b,c	1.80^d,c	1.83^a,b,c	1.72^d	55.8	10.6
W (g)	43	1914.0^e	1968.9^d	1984.1^c,d	2021.8^b,c	2070.2^a	2003.1^c,d	2044.1^a,b	0.01	0.001
FCR	43	2.12^a	2.05^b	2.04^b	1.99^c,d	1.95^e	2.02^b,c	1.97^d,e	53.6	10.1
^a–gMeans in a row lacking common superscript letters differ significantly (P≤0.05), Duncan's test.
Note: Treatment group A: control (no enzyme added);
treatment group B: 4 ml (1.2×10^4 IU cellulases)/kg feed;
treatment group C: 8 ml (2.4×10^4 IU cellulases)/kg feed;.
treatment group D: 12 ml (3.6×10^4 IU cellulases)/kg feed;
treatment group E: 16 ml (4.8×10^4 cellulases)/kg feed;
treatment group F: 2-ml protease solution+4-ml cellulase solution/kg feed equivalent to 3×10^4 IU protease and1.2×10^4 IU cellulase, respectively;
treatment group G: 2-ml protease solution+8-ml cellulase solution/kg feed equivalent to 3×10^4 IU protease and 2.4×10^4 IU cellulase, respectively.

Table 3. Regression analysis (quadratic model) of the effect of cellulase addition in feed on body weight gain of broiler chicks.

	BW at day 9	BW at day 16	BW at day 23	BW at day 30	BW at day 37	BW at day 43
P	0.01	0.085	0.046	0.011	0.000	0.024
R ^2	0.99	0.92	0.96	0.99	1.00	0.98
Regression equation	y=114+31.4 group −3.6 group^2	y=353.8+7.5 group −0.14 group^2	y=595.2+38.7 group −2.2 group^2	y=997+32.01 group −0.7 group^2	y=1472.6+9.9 group+1.7 group^2	y=1886.8+32.7 group+0.7 group^2
Group=Treatment group and allotted numbers.
1=group A: control (no enzyme added); 2=group B: 4 ml (1.2×10^4 IU cellulases)/kg feed; 3=group C: 8 ml (2.4×10^4 IU cellulases)/kg feed; 4=group D: 12 ml (3.6×10^4 IU cellulases)/kg feed; 5=group E: 16 ml (4.8×10^4 cellulases)/kg feed.

3.3. Viscosity reduction and vent pasting

With increasing doses of enzymes, decreases in viscosity values were observed in both in vitro and in vivo experiments (Table 4). Marked vent pasting was observed in control group, whereas there was no vent pasting in groups E and G. Maximum to mild vent pasting was observed in decreasing severity in groups A, B, C, D and F.

Table 4. Effect of enzyme addition on viscosity of feed after in vivo and in vitro digestion.

Treatment group	In vivo (viscosity of ingesta (CP^a)	In vitro (viscosity of feed (CP^a)
A	1.089	1.297
B	0.691	0.891
C	0.673	0.841
D	0.613	0.801
E	0.521	0.712
F	0.641	0.823
G	0.559	0.781
^aCentipoises.
Note: Treatment group A: control (no enzyme added); treatment group B: 4 ml (1.2×10^4 IU cellulases)/kg feed; treatment group C: 8 ml (2.4×10^4 IU cellulases)/kg feed; treatment group D: 12 ml (3.6×10^4 IU cellulases)/kg feed; treatment group E: 16 ml (4.8×10^4 cellulases)/kg feed; treatment group F: 2-ml protease solution+4-ml cellulase solution/kg feed equivalent to 3×10^4 IU protease and1.2×10^4 IU cellulases, respectively; treatment group G: 2-ml protease solution+8-ml cellulase solution/kg feed equivalent to 3×10^4 IU protease and 2.4×10^4 IU cellulases, respectively.

4. Discussion

Improvement in FCR and weight gain in all treatment groups at all age intervals due to the enzyme supplementation is reflective of effective degradation of cellulosic substrates in feed by the exogenous enzymes. Despite non-significant difference in weight gain and FCR values among some treatment groups of similar age, improvement in weight gain and FCR was linear with increasing doses of enzymes. The results are in agreement with the findings of Kaczmarek et al. (2009), Hua and Kang-Ning (2008), Olukosi et al. (2007) and Cowieson and Adeola (2005).

The lines generated from observed weight and predicted weight (through regression equation) were overlapping (Figure 1) till 43 days of age. Regression (quadratic fit) also reveals that dose of cellulases used in group E (4.8×10⁴ IU/kg feed) was the best for optimum results (weight gain) up to first 3 weeks, and hypothetical curves (Figure 1) with higher cellulases doses were either static or declining during this period. However, curves with hypothetical increasing doses of cellulases showed linear increase in weight gain from 4th to 6th week, which is logical and achievable to certain extent during this period (Figure 1). This analysis paves way to further the farm trials with high enzyme doses (identical to hypothetical doses used for this analysis) during finishing stage. From the data, it can also be inferred that the effect of protease addition along with cellulases was markedly useful up to the age of 23 days. The dose of cellulases can be reduced to half to obtain same results if a small amount of protease is added (Table 2). The cause of synergistic effect of protease and cellulases can be hypothesised that cellulases first acted on outer fibrous material thus exposing protein molecules. Chicks require more protein at starter age which was better fulfilled in groups F and G than in groups of cellulases addition alone (B, C, D and E) leading to superior performance of groups F and G. However, the effect of protease addition started to decline after 23 days, and addition of cellulases alone worked equally good but at higher doses in last 2 weeks. Since equal number of treatment groups (cellulases and combination of cellulases and protease) were not available in this experiment for analysis, further experiments are needed to optimise the dose combination of protease and cellulases (for a variety of substrates).

NSPs are degraded under the action of cellulases. A gradual decrease in viscosity of feed (both in vitro and in vivo) under the action of enzymes is reflective through decrease in vent pasting in treatment groups with increasing levels of enzymes (Slominski et al. 2006; Costa et al. 2008; Glamocic et al. 2011).

It is concluded that the indigenously produced enzymes were highly effective in crude form for effective utilisation of plant cellulose by broiler chicks. The enzymes are capable to perform commercially after partial or complete purification.

Acknowledgements

The authors appreciate financial support of Higher Education Commission of Pakistan.

Additional information

Notes on contributors

Habib-ur-Rehman

Present address: Habib-ur-Rehman, Poultry Research Institute, Shamsabad, Rawalpindi, Pakistan; Pir Bux, Department of Microbiology, Shah Abdul Latif University, Khairpur, Sindh, Pakistan