Crude Protein Equivalence Value of a Multi-Enzyme Product for 28-and 42-Day-Old Broilers

In order to estimate the crude protein (CP) equivalence value of Natuzyme-p (NP) enzyme by using regression response equations, two experiments were carried out using Ross (308) broiler chicks. Graded levels of dietary CP (while amino acids levels were kept constant) and NP enzyme were used to derive the regression equation in the first experiment. Four levels of dietary CP and NP enzyme were fed to 160 feather-sexed male broiler chicks during the starter (0-28 d of age) and grower (28-42 d of age) period. Each diet was offered to four replicates of five chicks in a completely randomized design. Results obtained in experiment one failed to fit a regression equation between BW, dietary CP levels and NP enzyme. In experiment two, graded levels of CP changed along with the levels of lysine (Lys), Met+Cys and threonine (Thr). Regression equations between BW and dietary CP and NP enzyme were derived. Nonlinear and linear equations were generated for enzyme and CP. Based on an assessment of r2 and P value, nonlinear equations were used to determine enzyme equivalence. The derived regression equations of body weight for CP were set to be equal with those obtained for NP and were solved; enzyme equivalence value for CP was calculated by subtracting the obtained value from CP content of basal diet. Crude protein equivalence value of NP at 28 and 42 d of age was estimated to be 0.96 and 0.38 %, respectively.


INTRODUCTION
Several benefits of the supplementation of broiler diets with mixtures of exogenous enzymes have been reported.They may improve nutrient digestibility, reduce nitrogen and phosphorus excretion, allow the use of alternative feed ingredients, reduce variations in the nutritional quality of feed ingredients, and reduce the incidence of wet litter that commonly happens when diets rich in viscous grains are fed (Yu & Chung 2007).However, determining nutrient equivalence values of enzymes is a requisite for precise feed formulation.Although P equivalence values of microbial phytase are well established (Cohelho & Kornegay 1996), corresponding data on amino acid and CP release with added phytase and multi-enzyme products are limited.The lysine equivalence of phytase was determined (Ravindran et al., 2001), but the effect of phytase alone or together with other exogenous enzymes on the other amino acids is of considerable practical significance and needs to be quantified to enable its inclusion in least-cost diet formulation.
Estimates of nutrient equivalence value of exogenous phytase using regression equations were reported in pigs (Jendza, 2006;Radcliff, 2006) and broilers (Jendza, 2006;Adedokun, 2004).Zaghari et al. (2008) determined the crude protein equivalence of a multi-enzyme product (containing phytase: 1500 U/g, beta-glucanase: 700 U/g, alpha-amylase: 700 U/g, cellulose: 6,000 U/g, pectinase: 700 U/g, xylanase: 10,000 U/g, lipase: 30 U/g and protease: 3,000 U/g) and showed that it improved protein utilization.Khalaji et al. (2011) determined the standardized ileal threonine equivalence values of the same enzyme mentioned above.The results of their experiment showed that addition of 0.5 g of this enzyme blend released, in average, 0.1% threonine from anti-nutrient protein complexes.

General procedure
Two experiments were carried out to estimate crude protein equivalence of the enzyme blend Natuzyme-p for broilers.Equivalence CP values of the product were estimated in two experiments.In each experiment, 160 feather-sexed male Ross 308 broilers were submitted to the dietary treatments between 0-28 (starter) and 28-42 (grower) days of age.
Broilers with similar initial body weight were allotted to battery cages (55×85 cm, 0.94 m2 /bird).Each cage was equipped with a feeder and drinker.A 24 h lighting program was employed, and feed and water were provided ad libitum.Soft plastic wires were placed on the cage floor to minimize foot lesions.

Crude Protein Equivalence Value of a Multi-Enzyme Product for 28-and 42-Day-Old Broilers
The experiments were carried out according to a completely randomized design.Four replicates of five birds each were fed graded CP levels (four treatments) or product levels (four treatments) in each experiment.Feed intake and body weight were recorded at 28 and 42 d of age, and then feed conversion ratio (FCR) was calculated.Relative carcass, breast meat, gizzard, and abdominal fat pad weights were determined at slaughter.

Experiment 1
The objective of experiment 1 was to estimate CP equivalence values of the enzyme product when broilers were 28 and 42 days old.Graded CP levels were supplied in the starter (16, 18, 20 and 22 %) and grower (14, 16, 18 and 20%) diets.Amino acid levels were kept similar between diets with different CP levels by adding synthetic amino acids, so that the only source of variation in this experiment was dietary CP level.Graded levels of the enzyme product (0.15, 0.25, 0.35 and 0.45 g/kg) were added to the basal diets (14 and 16 % CP in starter and grower diets, respectively).

Experiment 2
Because in experiment 1, the regression equations between BW (or FCR) and CP levels were not fit to estimate CP equivalence value due to a possible deficiency of essential amino acids, another experiment was conducted to reevaluate CP equivalence of the  2).However, the ratios between CP and amino acids were kept constant.Graded levels of the enzyme product were added to the basal diets, which contained 16% CP, 0.95% Lys, 0.65 % Met +Cys and 0.59 % Thr in starter period and 14% CP, 0.76% Lys,0.56 % Met +Cys and 0.49 % Thr in grower period.

Statistical Analysis
Data were analyzed by the general linear models procedure of the SAS software (SAS Institute, 2003) with cage as experimental unit.Linear and quadratic effects of CP and enzyme product on body weight gain, feed conversion ratio and breast, thighs and abdominal fat weight percentage were tested using orthogonal polynomials.Linear and nonlinear response functions of body weight, feed conversion ratio and immune response that best fit the data were derived for CP and product levels.The models used were: Linear function: Where Y=the responsedmeasurement and X=CP levels (percentage of diet) or multi-enzyme product (g per kg of diet).Nonlinear or linear response equations with the highest r 2 value for added threonine and the equations for added multi-enzyme product were set to be equal and were solved using the procedure described by Yi et al. (1996).

Experiment 1
Performance and carcass data of broiler chicks fed different levels of CP and NP are presented in Table 3. Different dietary CP levels significantly influenced BW and FCR at 28 and 42 d of age.Even with similar levels of amino acids, diets with higher levels of CP resulted in higher BW at 28 and 42 d of age.Furthermore, as dietary levels of CP increased, broilers utilized their feed more efficiently compared to their counterparts received low CP diets, which deposited significantly more abdominal fat and also presented significantly lower breast meat yield compared with those fed higher dietary CP levels.The addition of different levels of the enzyme product did not significantly affect performance or carcass parameters.No linear and nonlinear equations between NP enzyme levels and BW fit, and therefore the results obtained in this experiment were not used to estimate CP equivalence values of the enzyme product.

Experiment 2
The influence of different levels of dietary CP and amino acids on broiler performance (BW and FCR) and carcass parameters (carcass, abdominal fat, gizzard and breast meat relative weights) are presented in Table 4.At 28 and 42 d of age, broilers fed higher protein and amino acid levels were significantly heavier than their counterparts fed low-density diets.The higher BW of these birds was associated with a more efficient utilization of the feed consumed.Although different levels of the tested enzyme product had no significant effect on broiler BW or FCR at 28 and 42 d of age, the equations fit these data and were used to estimate equivalence.In experiment two, enzyme supplementation resulted a numerical improvement in BW and FCR.Nonlinear response equations between graded levels of CP and the enzyme product with BW at 28 and 42 d of age are presented in Table 5.Crude protein equivalence values of Natuzyme-p enzyme during starter and grower period were estimated as 0.96 and 0.38 % respectively.

DISCUSSION
It has been shown that enzyme complexes with multiple enzyme activities are able to improve broiler performance by increasing nutrient digestibility by interactions that occur among several enzymes.Zallena et al. (1999) and Meng & Slominski (2005) reported higher protein and amino acid availability in corn-soybean meal diets supplemented with enzyme blends.The addition of different levels of the tested enzyme product to a low CP diet failed to influence either BW or FCR at any stage of broiler lifespan.In the first experiment, it seems that there was no influence of the tested product on broiler performance because the basal diet (which contained NP) was only deficient in CP when amino acids levels were kept at the recommended levels.Numerous studies have shown similar performance between broilers fed low CP diets supplemented with synthetic amino acids with those fed on diets with sufficient levels of CP (Par et al., 1991;Han et al., 1992;Namrud et al., 2008).Denbow et al. (1995), Harper et al. (1997) and Ravindran et al. (2001) used R 2 as the criterion for selecting either linear or non-linear regression models  to describe the changes in certain response criteria to graded levels of phytase and supplemental inorganic phosphorous.The R 2 values of linear and nonlinear models generated from the growth performance data in this experiment were lower than those observed in literature (Adedokun et al., 2004;Denbow et al., 1995;Ravindran et al., 2001;Yi et al., 1996).The differences may be attributed to the duration of the study and bird age.Denbow et al. (1995), Yi et al. (1996) and Ravindran et al. (2001) conducted 21 d trials, whereas the duration of our trial was 42 d.Adedokun et al. (2004) applied only linear regression equation and obtained higher R 2 values using phytase.However, the duration of their study was 14 d after allowing ad libitum access to a standard P-adequate starter diet for 7 days.However, in the present study, there were four weeks more, which allowed for higher variance as birds grew.Zaghari et al. (2008) showed the positive effect of a multi-enzyme product (containing phytase: 1500 U/g, beta-glucanase: 700 U/g, alpha-amylase: 700 U/g, cellulose: 6,000 U/g, pectinase: 700 U/g, xylanase: 10,000 U/g, lipase: 30 U/g and protease: 3,000 U/g)on CP, which only difference to the blend used in the present experiment was phytase activity.
In the current experiment the only source of variation between treatments was dietary CP level, while in previous experiment CP levels varied along with other nutrients, including AME, non-phytate P, and amino acids.Different responses may be therefore due both to different methodology and enzymes employed in the current experiment.
Based on the results obtained in the first experiment, the second experiment was designed so that CP levels varied together with amino acid levels.As expected, different dietary CP and amino acid levels resulted in significantly different performance responses at 28 and 42 d of age.These observations are consistent with those previously reported (Cheng, 1997;Kidd et al., 2004).The addition of different NP enzyme levels to the diet deficient both in amino acids and CP failed to influence BW and FCR at 28 and 42 d of age.As birds age, their amino acid requirements begin to decrease (Gao, 2007).It seems that the reduced amino acid requirements in later stages of broilers' lifespan are the primary reason for lack of response to NP enzyme addition.The lack of influence of exogenous enzymes on broiler performance during later growth stages (Zaghari et al., 2008) or in old birds (Denbow, 1995) is well documented.

Crude Protein Equivalence Value of a Multi-Enzyme Product for 28-and 42-Day-Old Broilers enzyme
product, changing essential and non-essential amino acids (including Lys, Met+Cys and Thr; Table

Table 3 -
Effects of dietary treatments on performance and carcass parameters (Exp. 1)

Table 4 -
Effects of dietary treatments on performance and carcass parameters (Exp.2)Means in the same column with no common superscript differ significantly (p<0.05).