EVALUATION OF THE " ENZYMMIX F " PRODUCT AS AN ADDITIVE FOR BROILERS REARED IN AN ANTIBIOTIC-FREE SYSTEM

This experiment was undertaken to compare the effects of using the Enzymmix F product with a Brazilian enzyme blend in diets with different nutritional levels. A total of 1,080 male broiler chicks were distributed into a randomized-block design with a 3 × 2 factorial arrangement consisting of three enzyme complexes (Enzyme Blend, Enzymmix F, and the combination of both) and two nutritional levels (regular and reduced). We evaluated the birds’ performance in the periods of 1 to 21 and 1 to 42 days of age. The yields of carcass and cuts and intestinal integrity were evaluated at 45 days of age. During the analyzed periods, no significant difference was observed for the performance parameters considering the “enzyme” factor. However, the reduction in the nutritional level of the diet resulted in inferior performance in both periods. There was no statistical difference for the yields (%) of carcass and cuts. Enzymmix F provided the greatest villus height in the jejunum segment. The use of the Enzyme Blend in the nutritionally reduced diet led to a lower crypt depth. In conclusion, Enzymmix F increased the weight of wings (g) and the villus height in the jejunum.


INTRODUCTION
The expansion of poultry farming in the last decades has driven the search for excellence in this sector.According to Vargas et al. (2017), the nutrition area has stood out for discovering numerous alternatives to increase production based on the optimization of the use of dietary nutrients and reduction of feed costs.These functions are commonly attributed to exogenous enzymes, which play an even more important role in alternative broiler production systems such as the antibiotic-free system.As stated by Thorpe and Beal (2001), the use of exogenous enzymes offsets the reduction in animal performance caused by the restricted use of antibiotics in the diet.Poultry are the species that most benefit from the use of exogenous enzymes, which is mainly because of their very short digestive system that does not allow for enough digestion time (Mavromichalis, 2012).
According to Barbosa et al. (2014), enzymes act as biological catalysts, and their adoption enables a greater utilization of diet nutrients such as phosphorus, calcium, amino acids, and energy, resulting in better production efficiency, greater economy in feeding, and benefits to the environment.These last two benefits partly stem from the possibility of working with less dense diets, considering that enzyme supplementation elevates the nutritional value of ingredients and improves the degree of precision in their formulation (Ravindran, 2013).
Within this perspective and considering the nutritional relevance of including exogenous enzymes in poultry diets, the present study was conducted to compare the effect of the Enzymmix F product with that of a Brazilian enzyme complex, in diets with different nutritional levels, on the performance parameters, yields of carcass and cuts, and intestinal integrity of broilers.

MATERIAL AND METHODS
This study was carried out according to the ethical principles in animal experimentation and was approved by the local Research Ethics Committee.
The experiment started in July 2017 and took place on the Serra Dourada farm, located in the municipality of Ipeúna -SP, Brazil.The study involved 1,080 oneday-old Cobb MX male chicks with a live weight of 47.83 ± 3.07 g vaccinated against Marek's disease, fowlpox, infectious bursal disease, and coccidiosis.Birds were housed in thirty-six 1.0 × 2.5 m (2.5 m²) cages at the rate of 30 birds/cage, corresponding to a density of 12 birds/m².Each cage had an individual water-and feed-supply system; these were available to the animals ad libitum.Temperature (ºC) and ventilation were controlled by curtain management and activation of fans and a misting system whenever necessary.Two data loggers (Hobo® H08-00X-02) were installed in the shed to measure the temperature and humidity in the facility.The chicken litter (6-mm thickness) used in the experiment was made of powdered pine bark.
The lighting program adopted was that recommended by Humane Farm Animal Care (2009), considering that the birds were kept in a certified production unit regarding the animal welfare norms.This unit was also certified for not using antibiotics, chemotherapeutics, or animal ingredients in the diets.Following these guidelines, the diets were formulated based on corn and soybean, according to the composition of feedstuffs presented by Rostagno et al. (2011).The feeding program was divided into five phases: starter (1-7 days), developer (8-21 days), grower 1 (22-28 days), grower 2 (29-35 days), and finisher (36-45 days).
Three enzyme complexes (Enzyme Blend, Enzymmix F, and the combination of both) and two dietary nutritional levels (regular and reduced) were evaluated.The analyzed treatments are described in Table 1.The experiment was analyzed as a 3 × 2 factorial arrangement consisting of three enzyme complexes and two dietary nutritional levels (Tables 2 and 3), constituting six treatments (Table 1) that were distributed into randomized blocks with six replicates of 30 birds each.
It should be noted that a control treatment (without enzyme) was not established due to previous knowledge of the positive effect of the Brazilian enzyme complex on broiler performance.Thus, this product was used as the control in this research.
The Enzyme Blend is a Brazilian commercial product that contains two active enzymes [glucanase (860 UV/ kg) and xylanase (1,250 UV/kg)] produced by a single non-genetically modified fungus (Talaromyces versatilis).
Enzymmix F is a European enzyme complex registered for use in animal production that contains four mediumsized enzymes: pepsin A (65-90 U/mL), trypsin (80-100 U/mL), amylase (60-90 U/mL), and triacylglycerol lipase Evaluation of the "Enzymmix F" product as an additive for broilers reared in an antibiotic-free... (60-90 U/mL).This product was obtained from an invertebrate (Eisenia foetida) culture, from which the enzymes were extracted.
The inclusion levels and forms of administration of the products complied with the recommendations of the manufacturers.Enzymmix F was provided in the water available in the drinkers at the rate of 1 mL of product for every 1,000 mL of water.The Enzyme Blend was provided as an additional supplement in the diet, at the rate of 0.050 kg/t.Production-performance parameters were evaluated in the periods of 1 to 21 and 1 to 42 days of age.Body weight (BW) gain was calculated as the difference between the final and initial weights for each investigated period.Birds were weighed per pen (experimental unit).Feed intake was calculated as the difference between the total feed supplied and orts remaining at the end of each feeding phase.Feed conversion ratio (FCR) was calculated as total feed intake divided by weight gain, corrected for mortality (MO), as proposed by Sakomura and Rostagno (2007).Mortality was recorded daily.The production efficiency index was calculated using the following equation: PEI = ((BW*(100 MO))/ (Age*FCR))*100.
At 45 days, when the slaughter of free-antibiotic chickens on the farm under study occurs, six birds 10% heavier or lighter than the average weight of the unit were slaughtered per treatment for an assessment of intestinal integrity, whole-carcass yield (%) and absolute weight (g) and yield (%) of cuts.The carcass yield was determined as the ratio between the weight of the eviscerated carcass and the live-bird weight, after an 8-h feed-deprivation period.The yields of cuts (breast, drumstick + thigh, and wing) were also determined; these were weighed and their yields were calculated relative to the eviscerated-carcass weight.
The studied variables' data were subjected to a Shapiro Wilk normality test and analysis of variance.Means were compared by Tukey's test at the 5% significance level.

RESULTS AND DISCUSSION
Mean values for maximum, average, and minimum temperature and humidity recorded during the experimental period are exhibited in Figure 1.
No significant difference was observed for the performance parameters considering the "enzyme" factor (Tables 4 and 5) in the periods of 1 to 21 and 1 to 42 days of age of the birds.However, the reduction of  Evaluation of the "Enzymmix F" product as an additive for broilers reared in an antibiotic-free...     the dietary nutritional levels led to lower performance (p<0.05)compared with the use of regular levels.This lower performance is characterized by the higher feed intake and worse feed conversion observed in both periods (Tables 4 and 5).Moreover, at the end of the experimental period, a lower (p<0.05)production efficiency index was recorded for this treatment (Table 5).There was no interaction effect between enzyme and nutritional level of the diet on the evaluated performance parameters.
The lack of differences in the periods of 1 to 21 and 1 to 42 days of age of the birds indicates that the product tested here (Enzymmix F) has similar efficiency to that of the Brazilian product.At the same time, it also reveals a lack of synergism between the studied enzyme complexes, which, as previously mentioned, have distinct origins and enzymes.Dalólio et al. (2016) also did not find significant differences in bird performance using an enzyme complex at different concentrations.Those authors suggest that by adopting additional supply, an inclusion method used for Enzyme Blend, differences can be reduced, consequently reducing the likelihood of significant differences.Other authors, however, observed an improvement (p<0.05) in the feed conversion of broilers fed corn and soybean mealbased diets supplemented with an enzyme complex under the same inclusion method (Pucci et al., 2010).
The nutritional reduction of the diets was not offset by the effect of the enzyme complexes under study.This result corroborates other authors who reported effects of diet density on broiler performance despite enzyme supplementation (Bonato et al. 2004).In practical terms, this result demonstrates that for the products tested here to be used without performance losses, these levels should be reduced to no more than 100 Kcal ME/kg apparent ME, 3% limiting AA, and 2 units of crude protein.
There was no statistical difference for the yields of carcass and cuts as a function of the use of enzymes.However, a more in-depth analysis of the data revealed that the wing was lighter in the treatment containing the Enzyme Blend (Table 6).It should be noted that although there is a higher demand for this cut, the processing industry tends to value mostly the "breast" cut because of its added value.This cut, in turn, did not differ statistically across the treatments, but in absolute terms the breast weight was higher in the treatment using Enzymmix F (Table 6).
For most of the quantified variables, the enzyme complexes were efficient to the point that no significant difference was observed between the nutritional levels of the diets; once again, the exception was the wing weight (Table 6).Variations in the yield of this cut are commonly attributed to sex and line (Hellmeister Filho, 2003), which are factors that did not vary across the treatments, in this study.
Our data corroborate those reported by other authors who also did not find differences in the yields of carcass and primal cuts (breast, drumstick, and thigh) as affected by the use of enzyme complexes (Torres et al., 2003;Carvalho et al., 2009).The use of the same ingredient base (corn and soybean) for the formulation of the diet as well as the same line of the birds likely contributed to this result.Differences in yields of carcass and cuts are more common when different raw materials (Geron et al., 2015;Frank et al., 2016), lines (Api et al., 2017;Silva et al., 2017) and diet physical forms (Oliveira et al., 2011) are used.
With respect to the intestinal-quality data, there was a statistical difference for villus height in the jejunum, the most important segment for nutrient absorption (Uni, 2004).In this segment, an interaction effect between the studied factors was also observed on crypt depth (Table 7).
The Enzymmix F treatment provided a greater villus height than the Enzyme Blend and Enzymmix F + Enzyme Blend treatments (Table 7), suggesting a positive effect of the use of Enzymmix F on this parameter, although such benefit did not lead to improved production performance at the end of the experimental period (Table 5).This greater villus height is believed to be associated with the use of Eisenia foetida as raw material, an organism known to produce meals with high levels of glutamic acid (Ignacio, 1993), which, as stated by Solta (2009), increases the height and weight of villi in the duodenum and jejunum of broilers.
The decomposition of the interaction (p<0.05) between the "enzyme" and "nutritional level" factors shows that the use of the diet with nutritional levels reduced by 2 units of crude protein, 3% amino acids, and 100 kcal energy led to a lower crypt depth in the treatment including the Enzyme Blend (Table 8).The balance between cell renewal and cell loss enables the maintenance of the villus size and the crypt depth (Mariorka et al., 2002).Thus, because a greater villus ns = not significant by Tukey's test at 5%; CV = coefficient of variation; DWG = daily weight gain; FCR = feed conversion ratio, PEI = production efficiency index.

Figure 1 -
Figure 1 -Temperature and humidity during the experimental period.

Table 5 -
Effect of the "enzyme" and "nutritional level" factors on performance variables in the period from 1 to 42 days of age Different lowercase letters in the column differ in each factor by Tukey's test at 5%.

Table 4 -
Effect of the "enzyme" and "nutritional level" factors on performance variables in the period from 1 to 21 days of age