Biscuit Residue in the Nutrition of Laying Hens: Effects on Animal Health, Performance and Egg Quality

Background: Corn and soybean meal are common ingredients used in poultry feed in order to supply the demand for energy and protein, respectively. Also, these ingredients directly influence the final price of the diets, and consequently, the final cost of production. A major problem is associated to the oscillation of these grains in some months of the year. Therefore, there is a need to search for nutritionally and economically viable alternatives to mitigate this problem. The aim was to evaluate whether the addition of biscuit residue could partially replace the use of corn in the feed of laying hens in order to obtain economic viability without impairment on production, egg quality and animal health. Materials, Methods & Results: The experiment was completely randomized and each repetition was considered one experimental unity. One hundred Hy-line brown 48 week-old chickens were allocated in cages and divided into five groups with four repetitions each, allocating five animals per cage. The group T0 was composed by animals that received the basal diet, i.e. without biscuit residue. The other groups were composed by increasing levels of biscuit residue to substitute corn i.e., group T7 (7% of biscuit residue), T14 (14% of biscuit residue), T21 (21% of biscuit residue) and T28 (28% of biscuit residue). Productive performance of the animals, egg quality, as well as analysis of blood parameters related to lipid metabolism, carbohydrates and proteins of birds were measured at the beginning of the experiment (day 0) and at the end of each cycle (days 21, 42 and 63 of experiment). Among the performance variables, only feed conversion was altered by biscuit residue, i.e., the feed conversion was lower in the groups that received the residue when compared to the control. The replacement of corn by biscuit residue did not affect laying rate (P > 0.05). Regarding egg quality, a less intense coloration was observed in the eggs of the treatments that received biscuit residue compared to the control group (P < 0.05), but without difference between treatments. Seric levels of uric acid were higher in the treatments that received biscuit residue in the days 21 and 42 compared to the control group, effect not seen in the third cycle (day 63). The cost ($) of feed reduced as the levels of inclusion of the biscuit residue in the diet were increased, with the control diet costing $ 0.244/kg, while 28% of biscuit inclusion lower its cost to $ 0.189/kg (22.3% lower). The final cost to produce a dozen eggs from each cycle was on average of $ 0.524, 0.415, 0.441, 0.397 and 0.332 for 0, 7, 14, 21 and 28% inclusion, respectively. Discussion: Residues of biscuit is widely used to feed swine, with positive results as an ingredient to attend the demand for energy. Even though the Brazilian Tables for Poultry and Swine include biscuit residue as a possible ingredient, there is a lack of scientific research in laying hens. These study was verified that the inclusion of biscuit residue (18.936%) is feasible, since it favors the productive performance of the animals, without adverse effects on their health; in addition, the inclusion of biscuit residue reduces dietary costs, as the cost of egg production. Whereas animal feed accounts for a large part of the production cost, the reduction in feed costs without impairment on animal productivity, is a key factor in animal farming. At certain times of the year, food can account for 80% of the production costs and consequently it influences the final price of the product in the market. In our study, we observed a significant reduction on total costs of diets that received biscuit residue, which occurred because a part of the energetic portion of the diet was supplied by the biscuit residue, and not by corn, which influenced the final price.


INTRODUCTION
Brazilian egg and broiler meat production have representativeness importance, placing the country at the seventh position in the world. The overall Brazilian egg production is destined to the domestic market, since its consumption tends to increase over the years [9]. The egg is a good nutritionally complete with high biological value, exerting several benefits for human health when consumed at necessary quantities [5]. Despite this, it is a proteic food of lower market value, and due to this reason its production must be extremely efficient in order to maintain market competitiveness [16]. In order to achieve high productivity, it is important that have all production variables well aligned, and among them the animal nutrition is the main one, which directly influences the animal production and health.
Corn and soybean meal are common ingredients used in poultry feed in order to supply the demand for energy and protein, respectively [7]. Therefore, there is a need to search for nutritionally and economically viable alternatives to mitigate this problem [22]. According to the literature, biscuit residue provides a large portion of the energetic fraction of the diet, providing also protein and amino acids, as lysine at values very similar to that found in corn. Thus, this residue was studied to understand the mechanism of utilization and exploitation of the nutrients of this by-product [2]. In this sense, Corassa et al. [6] revealed that biscuit residue (15%) could improve the performance of piglets, enhancing body weight and feed consumption, which could be related to better palatability. Therefore, the objective of this study was to evaluate whether the inclusion of biscuit residue in the nutrition of laying hens as a replacement of corn could cause a positive effect on animal production and health, as well as economic viability.

Experimental design
The experiment was conducted in an experimental poultry farm at the Universidade do Estado de Santa Catarina (UDESC), located in Chapecó city, Southern of Brazil during three production cycles of 21 days each.

Biscuit residue
Biscuit residue was donated and increasing amounts of it were used according to its chemical composition, in addition to other ingredients, as cited in the Brazilian Table for Poultry and Swine [19].

Experimental design
The experiment was completely randomized and each repetition was considered one experimental unity. One hundred Hy-line brown 48 week-old chickens were allocated in cages and divided into five groups with four repetitions each, allocating 5 animals per cage. The group T0 was composed by animals that received the basal diet, i.e. without biscuit residue. The other groups were composed by increasing levels of biscuit residue to substitute corn i.e., group T7 (7% of biscuit residue), T14 (14% of biscuit residue), T21 (21% of biscuit residue) and T28 (28% of biscuit residue). The diets were formulated according to the specific nutritional requirements as shown in Table  1 [19].

Analysis of biscuit residue and animal diets Nutritional composition
Analyses of dry matter (DM), mineral matter (MM), ethereal extract (EE), crude protein (CP), neutral detergent fiber (NDF) and acid detergent fiber (ADF) were performed following the method described by Silva and Queiroz [21] in biscuit residue and feed samples.

Determination of total phenolic compounds (TPC) and antioxidant activity by the elimination of radicals by DPPH
For extraction, 0.5 g of each sample was dissolved in 50 mL of distilled water. The mixture was placed in an ultrasonic bath (70 W) for 3 h and remained in the dark for 3 more hours. The supernatant was filtered with quantitative filter paper ('Whatman' # 40) and stored in a 100 mL volumetric flask wrapped in aluminum foil. After extraction, extracts were stored in Eppendorf tubes and maintained at -80 ºC until analysis [1,12]. Quantification of TPC was performed by Folin-Ciocalteu colorimetric method modified by Bonoli et al. [3]. An aliquot of each diluted sample was mixed with 0.5 mL of Folin-Ciocalteu reagent and was stirred for 1 min. Next, 2 mL of sodium carbonate (20 %) was added into the mixture and stirred for 30 s. After 2 h of incubation, the absorbance was read at 750 nm in relation to the blank. The standard curve was prepared by solutions of gallic acid in methanol. The concentration of TPC was obtained using an equation derived from the standard curve of gallic acid (expressed in mg of gallic acid equivalent per g of dry sample; mg EGA/g). Data were shown as the mean ± SD of triplicates. Evaluation of free radical scavenging activity in the extracts was determined by the antioxidant reduction capacity of DPPH radical according to Brand-Williams et al. [4]. Five dilutions of each extract were prepared in test tubes and 0.3 mL of each diluted extract was added to 2.7 mL of DPPH radical (40 µg/mL). After incubation for 1 h in the dark, the absorbance was read at 515 nm Free radical scavenging activity was reported as the IC 50 (µg/ mL), which is defined as the antioxidant concentration required to eliminate 50% of the DPPH present in the test solution. All tests were performed in triplicate and IC 50 values were reported as mean ± SD of triplicates.

Body weight
Two animals per repetition were individually weighted in the beginning of the experiment and at the end of the productive cycle using a digital balance.

Egg production (%)
Daily, the eggs were collected and counted from each replicate of the treatments. Thus, at the end of each cycle, it was possible to calculate egg production (%).

Egg mass (g/bird/day)
At the end of each productive cycle, the eggs were identified and individually weighted. Based in this data, the egg mass was obtained by the formula: Egg mass = mean weight (g) x percentage of egg production (%).

Feed consumption (g/bird/day)
Experimental diets were weighted and provided to the animals using specific feeders and the amount of feed consumed in each period of 21 days was recorded. These results reflect the average of consumed feed for both cycles.
Feed conversion (kg/kg and kg/dp) The feed conversion (FC) per kg of egg was obtained by the average of feed intake in the experimental period divided by the egg mass, i.e., the amount of feed consumed to produce one kilogram of egg. To calculate feed conversion per dozen eggs produced, the quantity of food consumed in the period was divided by quantity of eggs produced multiplied by 12.

Analysis of egg quality
At the end of each cycle, fresh eggs were collected to analyze: egg weight, specific gravity [11]. Shell resistance was evaluated using the equipment TA.XT. PLUS 1 , and results were expressed in Kgf. Shell thickness was evaluated after washing and drying during 48 h using a digital pachymeter, and the results were expressed as the average of the three-point measure in the egg (low, middle and high). Also, it was calculated the Haugh unit, yolk index and quantity of albumen in relation to egg weight and bark weight, as well as percentage of albumen and yolk [10]. Brightness (L), intensity of red (a) and intensity of yellow (b) were performed using an electronic colorimeter. Yolk and albumen pH were determined by using a specific equipment (Testo 205) 2 , and the intensity of yolk color was evaluated using a 16-color fan.

Blood collection and serum biochemistry
Blood collection was performed in two animals per repetition, totalizing eight animals per treatment at four moments: the beginning of the experiment (day 0) and at the end of each cycle of 21 days each (days 21, 42 and 63 of the experiment). The animals were contained manually, and the blood was collected from ulnar vein using insulin syringe. The blood was stored into tubes without anticoagulant to obtain serum after centrifugation (850 g during 10 min) and stored at -20ºC until analysis.
Serum levels of alkaline phosphatase, triglycerides, cholesterol, uric acid, total protein, glucose and albumin were measured using colorimetric commercial kits (Analisa) 3 in the semi-automated (BioPlus 2000) 4 . Levels of globulin were obtained by the formula: total protein -albumin.

Statistical analysis
The data were submitted to descriptive analysis, followed by the evaluation of the normality of the data by the Shapiro Wilk test. Data without normal distribution (biochemical variables) were transformed to log. The data were submitted to analysis of variance (ANOVA) followed by Tukey test. A regression analysis was performed to determine the appropriate inclusion levels of biscuit residue. It was considered significant when P < 0.05.

Feed composition
Biscuit residue has high values of crude protein and ethereal extract (Table 1), which becomes adequate to compose the energetic and protein portions in the animal diet. Also, it was evident that increasing amounts of biscuit residue also increased ethereal extract levels.
Total phenolic compounds (TPC) of biscuit residue was 0.50 mg/100 g of DM (dry matter). An increase on TPC was observed in the treatments T7 (0.047), T14 (0.079), T21 (0.102) and T28 (0.20) mg of TPC/100 g of DM when compared to T0 (0.0). Antioxidant activity against the DPPH -IC 50 radical was not observed in the biscuit residue and in the diets, that is, the analyzed samples showed no IC 50 .

Performance
Average egg weight, CA/Dp, egg mass, and percentage of egg production did not significant differ between groups (P > 0.05; Table 2). Feed conversion per kg of produced egg (CA/Kg) was lower in the groups that received biscuit residue compared to the control group (P < 0.05). Considering the values of CA/Kg, the regression analyses indicated that the ideal value of biscuit residue in the diet is 18.936%. No significant difference was observed between groups regarding body weight (P > 0.05). However, the weight gain was higher in the period of 0 to 63 days in the treatment T21 and T28 compared to T0 (Table 3).

Egg quality
The results for egg quality were showed in Table 3. The values of egg color related to red intensity ("a") was lower in the groups T21 and T28 (first to third cycle) compared to T0, while the same result was found in the third cycle for the group T14. The percentage of yolk in relation to egg weight was higher in the groups that received biscuit residue compared to the control group (P < 0.05). Calculated gravity, bark resistance, bark thickness, L and b intensity, yolk and albumen pH, Haugh unit, yolk index, bark %, yolk % and albumen % did not differ between groups in three cycles (P > 0.05; Table 3).

Serum biochemistry
Results of serum biochemistry were showed in Table 4. Serum levels of uric acid was higher in the groups T21 and T28 compared to the control group in the two first cycles (P < 0.05). There was no significant difference between groups regarding serum alkaline phosphatase, glucose, triglycerides, cholesterol, total proteins, albumin and globulins (P > 0.05; Table 4).

Economic analysis
Feed cost was considerably lower when biscuit residue was added, as shown in Table 5. Also, a reduction in the cost of feed per kg of egg produced and per dozen of eggs was observed, contributing to lowering production costs, leading to a difference in the cost of feed per dozen produced of $ 0.50 (T0%) for $ 0.33 (T28%), which represents a reduction on 22.3 % of the total cost of productions.

DISCUSSION
Residues of biscuit is widely used to feed swine, with positive results as an ingredient to attend the demand for energy [13]. In our study, the productive efficiency of laying hens was positively affected when biscuit residue was used, since a significant reduction on feed conversion per kg of egg was observed. Therefore, its inclusion has applicability on poultry nutrition once it improved feed conversion and did not affect egg production.
The weight gain was higher during all experimental period in laying hens that received 21 and 28% of biscuit residue in the diet compared to the control       ... (...conclusão) group, which can be direct related to increased ethereal extract content in the diets with biscuit residue. The weight gain represents the efficiency in the use of energy from the diet by the animals, i.e., the animals deposited more energy in the corporal tissues [20]; however, it did not cause impairment on egg production. Despite that, we have to consider the possibility that, at in the long term, this effect could bring losses to animal production. Regarding egg quality, the egg yolk coloration altered with the presence of biscuit residue, i.e., the red intensity lower (lighter yolk). The yolk coloration is dependent on the carotenoid content, and corn has a significant content of carotenoids that provides good yolk coloring [17]. This fact explains the reduction on yellow coloration of the yolk of treatments with crescent levels of biscuit residue in the diet, since the amount of corn decreased. It is important to highlight that one of the ingredients of the biscuit is wheat flour, a major component, which shows a different profile of carotenoids. Thus, in many cases it is necessary the incorporation of pigments in the diet in order to suppress this coloring effect and provide better acceptance by consumers, since them mistakenly associated yolk coloration with higher nutritional egg value [5].
The reason for higher yolk production in relation to egg weight found only in the group T28 can be explained by the elevated EE content in the biscuit residue. According to Peebles et al. [14], sources and levels of fat in the diet can affect the egg fatty content. These authors revealed that increased dietary energy decrease the percentage of albumin, and the use of corn oil instead the animal fat (poultry), decreases egg shell percentage.
The serum uric acid levels increased in the groups that received 21 and 28% of biscuit residue compared to the control group, which can be related to an imbalance on levels of amino acids. According to Ross et al.
[18], the acceptable values for uric acid in brown Leghorn chickens ranged from 0.27 to 4.93, which are lower when compared to laying hens that received biscuit residue in the first two productive cycles. Higher levels of protein in the diet leads to higher amino acid imbalance, and consequently, lower use of the protein ingested, as well as higher elimination of uric acid in the environment [15]. Thus, it is necessary to maintain the nutritional balance of diets using ingredients of known bromatological composition, especially when alternative ingredients are used, as is the case of biscuit residue.
Whereas animal feed accounts for a large part of the production cost, the reduction in feed costs without impairment on animal productivity, is a key factor in animal farming. At certain times of the year, food can account for 80% of the production costs [8] and consequently it influences the final price of the product in the market. In our study, we observed a significant reduction on total costs of diets that received biscuit residue, which occurred because a part of the energetic portion of the diet was supplied by the biscuit residue, and not by corn, which influenced the final price. Thus, the presence of biscuit residue reduced feed costs without impacting productivity and animal performance. Regarding the control diet (without wholemeal biscuit) there was a reduction in the diet cost of 5.3; 10.7; 17.1; and 22.3% for the diets of the treatments T7, T14, T21 and T28, respectively. The decrease in production cost involved with feed directly reflected in feed costs to produce one kilogram of egg and one dozen of eggs, as well on feed conversion, which was lower in these groups. The potential of cost reduction from the inclusion of biscuit residue in the laying hens diet can be considered a nutritionally viable alternative, as well as economically advantageous. However, more research is needed to prove the use of biscuit residue in other ages and raising conditions, since this practice is empirically carried out in the field.