Olive pulp and exogenous enzymes feed supplementation effect on the carcass and offal in broilers: a preliminary study

Agriculture 2020, 10, 359; doi:10.3390/agriculture10080359 www.mdpi.com/journal/agriculture Article Olive Pulp and Exogenous Enzymes Feed Supplementation Effect on the Carcass and Offal in Broilers: A Preliminary Study Peyman Sayehban 1, Alireza Seidavi 1,*, Mohammad Dadashbeiki 2, Ahmad Ghorbani 3, Wagner Azis Garcia de Araújo 4, Alessandra Durazzo 5, Massimo Lucarini 5, Paolo Gabrielli5, Besma Omri 6,7, Luiz Fernando Teixeira Albino 8, Eliana B. Souto 9,10 and Antonello Santini 11,* 1 Department of Animal Science, Rasht Branch, Islamic Azad University, Rasht 41335-3516, Iran; [email protected] 2 Department of Veterinary Science, Rasht Branch, Islamic Azad University, Rasht 41335-3516, Iran; dadashb[email protected] 3 Guilan Agricultural and Natural Resources Education and Research Center, Rasht, 14445-41996, Iran; [email protected] 4 Instituto de Educação, Ciência e Tecnologia Norte de Minas Gerais; 39480-000 Januária, MG, Brazil; [email protected] 5 CREA-Research Centre for Food and Nutrition, Via Ardeatina 546, 00178 Rome, Italy; [email protected] (A.D.); [email protected] (M.L.); [email protected]ov.it (P.G.) 6 Laboratory of Improvement and Integrated Development of Animal Productivity and Food Resources, Department of Animal Production, Higher School of Agriculture of Mateur, University of Carthage, 1054 Carthage, Tunisia; [email protected] 7 National Agronomy Institute-Tunis, University of Carthage, 1054 Carthage, Tunisia; 8 Department of Animal Science, Universidade Federal de Viçosa; 36570-000 Viçosa, MG, Brazil; [email protected] 9 Department of Pharmaceutical Technology, Faculty of Pharmacy, University of Coimbra (FFUC), Pólo das Ciências da Saúde, 3000-548 Coimbra, Portugal; [email protected] 10 CEB - Centre of Biological Engineering, University of Minho, Campus de Gualtar 4710-057 Braga, Portugal 11 Department of Pharmacy, University of Napoli Federico II, Via D. Montesano 49, 80131 Napoli, Italy * Correspondence: [email protected] (A.S.); Tel.: +98-911-331-3073; [email protected] (A.S.); +39-81-253-9317 Received: 16 July 2020; Accepted: 12 August 2020; Published: 14 August 2020 Abstract: Nowadays, there is an increasing interest in the exploitation and valorization of agricultural food waste and by-products. At the same time, the growing demand by markets worldwide, especially in Africa and Southeast Asia, can justify the growing interest in the use of byproducts for the poultry industry. Olive pulp is one of the most interesting by-products of olive tree farming (typical of the Mediterranean area), being a good source of many biologically active compounds with antioxidant, antifungal, and antibacterial properties. The presence of processed olive pulp in the diet showed to be effective in increasing the weight of specific carcass and offal traits. This work aims at studying olive pulp as a feed supplement in poultry nutrition, by focusing on the effects on broiler carcass and offal. Olive pulp (OP) is one of the by-products of olive tree farming, being the residue of olive cake after it is dried. To evaluate the effects of OP in a diet supplemented with different levels of a commercial enzyme (ENZ) blend on broiler carcass and offal traits, three hundred male broiler chicks (Ross 308 lineage; one-day-old) were divided into ten treatment groups according to a completely randomized design. The treatments diets contained: unprocessed OP (50 g/kg, 100 g/kg, 50 g/kg with ENZ, 100 g/kg with ENZ), processed OP (50 g/kg, 100 g/kg, 50 g/kg with ENZ, 100 g/kg with ENZ), and control groups (without OP, and without OP Agriculture 2020, 10, 359 2 of 17 with ENZ). The OP processing increased breast percentages in broilers. Supplementation with ENZ did not change any of the studied carcass or offal trait values. The presence of OP (50 g/kg) in broiler diets increased the eviscerated carcass, leg, and neck percentage values. The presence of processed OP (50 g/kg) in the diet showed to be effective in increasing the weight of specific carcass and offal traits. Keywords: by-products; feed processing; feedstuffs; olive pulp; poultry; supplements. 1. Introduction Olive pulp (OP) is the remainder of olive cake (raw material resulting from olive oil extraction) after the cake is dried. It is also one of the most interesting by-products of olive tree farming, being a good source of several biologically active compounds with antioxidant, antifungal, and antibacterial properties [1–5] with a great nutraceutical potential [6–14]. The use of by-products from different vegetal origins to supplement feed is, on the other hand, attracting growing interest due to environmentally friendly use, efficacy, and sustainability, other than avoiding the necessity to dump potentially useful and valuable by-products of the agro food system [15,16]. Nonetheless, attention should be given to potential secondary metabolites present in the olive [17–20] adopting the necessary precaution in the field to avoid their presence [21]. The OP is considered a good source of protein, fat, calcium, copper, and cobalt, but it is low in its nutritive value (energy, digestible proteins, and minerals) and it has a high lignin content. It is also poor in some metals, e.g., phosphorus, magnesium, and sodium, but it contains reasonable levels of manganese and zinc [22–24]. The ripening stage at harvest interferes with pectic polysaccharides, which are found in the OP cell walls due to the presence of calcium chelating dimers, thereby changing the nutritional value of this by-product [25].Whereas in the past, the use of crop residues and by-products as alternatives to cereals–soybean meal-based rations for broilers diet was not successful, mainly due to the high fiber content and poor digestibility, now it is increasing in interest [26–28]. Some exogenous enzymes may be added to broiler diets containing these by-products as an aid for fiber digestion (carbohydrases) or phytic phosphorus solubilization (phytase), thereby reducing their negative effects on broiler production [29]. Lavelli and Bondesan [30] observed an increase in the total secoiridoid polyphenol (antioxidant, antimicrobial, and anti-inflammatory compounds) content and antioxidant activity in extra virgin olive oil when the fruits were predestoned. Most of these compounds are effective antioxidants; according to Kidd [31], antioxidant substances can reduce cellular free radical damage and improve the broilers’ immunology, performance, and carcass. A recent study by Debbou-Iouknane et al. [32] investigated the in vitro anticoccidial effect of olive pulp (Olea europaea L var. Chemlal) extract on the destruction of Eimeria spp. Oocysts isolated from infected chickens. The findings of this study showed that phenolic compounds of OP extract tested separately possess an anti-Eimeria spp. effect. The recent study of Papadomichelakis et al. [33] on the effects of dietary dried olive pulp inclusion on growth performance and meat quality of broiler chickens, showed that broiler chickens utilize dried olive pulp (DOP) supplemented diets more efficiently when dietary DOP inclusion is increased gradually with age, i.e. by using a combination of grower and finisher diets with a maximum of 25 and 50 g DOP/kg, respectively. The dietary addition of olive cake to broilers up to the level of 150g/kg did not affect performance parameters [34]. Zarei et al. [35] also reported that the inclusion of up to 86g/kg of olive pulp in the diet of laying hens had no negative effects on production parameters. Other researchers found positive effects of the nutritional use of olive pulp. Abo Omar [36] reported an increase in broiler feed intake (and a decrease in feed efficiency) with the inclusion of about 60g of olive pulp/kg diet. This author related this high feed intake to the fiber content of the olive pulp and the consequent increase in passage rate in the gastrointestinal tract. However, the dietary addition of 75g/kg of OP has a negative effect on weight gain, according to Rabayaaet al. [37]. On the other hand, the feasibility of including olive pulp up to the level of 160g/kg in broiler diets was reported [38]. The Agriculture 2020, 10, 359 3 of 17 use of enzymes to improve the nutritional value of olive pulp was studied in laying hens, but no positive effects were observed on production or egg quality parameters [39]. Considering this context, further studies evaluating the carcass and offal of commercial broilers fed diets with OP seems to be needed, and for this reason, the objective of this preliminary study was to assess the effect of different dietary levels of processed and unprocessed OP with enzyme supplementation on carcass and offal of broiler chickens during a 6-week trial. 2. Material and Methods Experimental protocols were approved by the Animal Care Committee of the Islamic Azad University, Iran (process #17–33–5–9013; 93–12–7). Three hundred day-old Ross 308 male broilers (Aviagen; New Bridge, Scotland, UK; 35805) were divided into 30 groups of 10 birds. Each cage (10 chickens) was assigned to a specific dietary treatment group. The experimental design had a total of 10 treatments (10 birds/treatment), and three replicates per treatment, as follows:  50 p: processed OP (50 g/kg) without enzyme blend;  50 p + ENZ: processed OP (50 g/kg) with enzyme blend (50 mg/kg);  100 p: processed OP (100 g/kg) without enzyme blend;  100 p + ENZ: processed OP (100 g/kg) with enzyme blend (50 mg/kg);  50 u: unprocessed OP (50 g/kg) without enzyme blend;  50 u + ENZ: unprocessed OP (50 g/kg) with enzyme blend (50 mg/kg);  100 u: unprocessed OP (100 g/kg) without enzyme blend;  100 u + ENZ: unprocessed OP (100 g/kg) with enzyme blend (50 mg/kg);  Ctrl: control diet without OP and without enzyme blend;  Ctrl + ENZ: control diet without OP with enzyme (50 mg/kg) blend. The birds were housed in cages (dimensions: 1.25 × 1.25 m; floor area: 0.15 m2 per bird), which were located in a thermostatically-controlled curtain side-wall poultry barn. The cage floors were covered with paper roll litter, and the birds remained in the cages for the duration of the experiment, which ended at their 42nd day of age. A two-phase feeding schedule, which consisted of starter (1– 21 days) and grower (22–42 days) feed periods, was adopted in this experimental study. Table 1 and Table 2 report the ingredients and chemical composition of diets used during the starter period and the finishing period, respectively. The diets met or exceeded the Ross 308 catalog recommendations for the starter and grower phases [40]. Agriculture 2020, 10, 359 4 of 17 Table 1. Feed ingredients and chemical composition of diets used during the starter period (1st –21st days of age, as-fed basis). Processed Olive Pulp (OPp) Unprocessed Olive Pulp (OPu) 503 p 4 50 p + ENZ 5 100 p 100 p + ENZ 50 u 6 50 u + ENZ 100 u 100 u + ENZ Ctrl 7 Ctrl +ENZ Ingredients (g/kg) Processed OP 50.00 50.00 100.00 100.00 0.00 0.00 0.00 0.00 0.00 0.00 Unprocessed OP 0.00 0.00 0.00 0.00 50.00 50.00 100.00 100.00 0.00 0.00 Enzyme 0.00 0.05 0.00 0.05 0.00 0.05 0.00 0.05 0.00 0.05 Corn 507.30 507.30 456.60 456.60 482.50 482.50 407.00 407.00 558.00 558.00 Soybean meal 370.60 370.60 370.60 370.60 377.20 377.20 383.70 383.70 370.70 370.70 Soybean oil 30.00 30.00 32.10 32.10 47.60 47.60 67.40 67.40 27.80 27.80 Wheat bran 0.10 0.05 0.10 0.05 0.10 0.05 0.10 0.05 0.10 0.05 Dicalcium phosphate 19.30 19.30 19.60 19.60 19.40 19.40 19.70 19.70 19.00 19.00 Limestone 10.90 10.90 9.10 9.10 11.50 11.50 10.30 10.30 12.70 12.70 Vitamin mixture 1 3.00 3.00 3.00 3.00 3.00 3.00 3.00 3.00 3.00 3.00 Mineral mixture 2 3.00 3.00 3.00 3.00 3.00 3.00 3.00 3.00 3.00 3.00 Salt 2.30 2.30 2.10 2.10 2.40 2.40 2.40 2.40 2.50 2.50 Sodium bicarbonate 1.50 1.50 1.50 1.50 1.50 1.50 1.50 1.50 1.50 1.50 DL-Methionine 1.40 1.40 1.50 1.50 1.40 1.40 1.50 1.50 1.30 1.30 Lysine hydrochloride 0.60 0.60 0.80 0.80 0.40 0.40 0.40 0.40 0.40 0.40 Total 1000.0 1000.0 1000.0 1000.0 1000.0 1000.0 1000.0 1000.0 1000.0 1000.0 Nutrient analysis (g/kg) Dry matter 903.20 903.20 904.90 904.90 904.80 904.80 908.20 908.20 901.50 901.50 Metabolizable energy (kcal/kg) 3.025 3.025 3.025 3.025 3.025 3.025 3.025 3.025 3.025 3.025 Crude protein 230.00 230.00 230.00 230.00 230.00 230.00 230.00 230.00 230.00 230.00 Ether extract 59.00 59.00 65.20 65.20 74.70 74.70 96.80 96.80 52.70 52.70 Linoleic acid 27.90 27.90 27.90 27.90 36.40 36.40 44.80 44.80 27.90 27.90 Crude fiber 45.80 45.80 64.90 64.90 50.70 50.70 74.70 74.70 26.70 26.70 Calcium 10.50 10.50 10.50 10.50 10.50 10.50 10.50 10.50 10.50 10.50 Phosphorus 7.40 7.40 7.30 7.30 7.30 7.30 7.30 7.30 7.40 7.40 Available phosphorus 5.00 5.00 5.00 5.00 5.00 5.00 5.00 5.00 5.00 5.00 Potassium 9.50 9.50 9.90 9.90 9.40 9.40 9.50 9.50 9.20 9.20 Chlorine 1.80 1.80 1.80 1.80 1.90 1.90 1.90 1.90 1.90 1.90 Manganese (mg/kg) 474.27 474.27 474.11 474.11 475.36 475.36 476.25 476.25 474.44 474.44 Sodium 0.16 0.16 0.16 0.16 0.16 0.16 0.16 0.16 0.16 0.16 Zinc (mg/kg) 383.69 383.69 383.21 383.21 385.60 385.60 387.01 387.01 384.17 384.17 Agriculture 2020, 10, 359 5 of 17 Choline (mg/g) 1.59 1.59 1.56 1.56 1.59 1.59 1.56 1.56 1.62 1.62 Folic acid (mg/kg) 2.19 2.19 2.18 2.18 2.21 2.21 2.20 2.20 2.21 2.21 Arginine 14.80 14.80 14.60 14.60 14.90 14.90 14.90 14.90 15.00 15.00 Glycine 9.20 9.20 9.10 9.10 9.30 9.30 9.20 9.20 9.40 9.40 Serine 11.00 11.00 10.90 10.90 11.10 11.10 11.00 11.00 11.20 11.20 Gly + Ser 20.20 20.20 20.00 20.00 20.40 20.40 20.20 20.20 20.60 20.60 Histidine 5.90 5.90 5.80 5.80 5.90 5.90 5.80 5.80 6.00 6.00 Isoleucine 9.30 9.30 9.20 9.20 9.40 9.40 9.30 9.30 9.40 9.50 Leucine 18.90 18.90 18.40 18.40 18.90 18.90 18.40 18.40 19.40 19.40 Lysine 12.70 12.70 12.70 12.70 12.70 12.70 12.70 12.70 12.70 12.70 Methionine 4.70 4.70 4.70 4.70 4.70 4.70 4.70 4.70 4.70 4.70 Cysteine 3.60 3.60 3.60 3.60 3.60 3.60 3.60 3.60 3.70 3.70 Met + Cys 8.30 8.30 8.30 8.30 8.30 8.30 8.30 8.30 8.40 8.40 Phenylalanine 10.60 10.60 10.40 10.40 10.60 10.60 10.50 10.50 10.80 10.80 Tyrosine 8.70 8.70 8.60 8.60 8.80 8.80 8.70 8.70 8.90 8.90 Phe + Tyr 19.30 19.30 19.00 19.00 19.40 19.40 19.20 19.20 19.70 19.70 Threonine 8.40 8.40 8.30 8.30 8.40 8.40 8.30 8.30 8.50 8.50 Tryptophan 3.00 3.00 3.00 3.00 3.00 3.00 3.00 3.00 3.00 3.00 Valine 10.20 10.20 10.10 10.10 10.30 10.30 10.10 10.10 10.40 10.40 1Vitamin A: 3,600,000 IU/kg; Vitamin D3: 800,000 IU/kg; Vitamin E: 7200 IU/kg; Vitamin K3: 800 mg/kg; Vitamin B1: 720 mg/kg; Vitamin B2: 2640 mg/kg; Vitamin B3 (Calcium pantothenate): 4000 mg/kg; Vitamin B5 (Niacin): 12,000 mg/kg; Vitamin B6: 1200 mg/kg; Vitamin B9 (Folic acid): 400 mg/kg; Vitamin B12: 6 mg/kg; Vitamin H2 (Biotin): 40 mg/kg; Choline: 100,000 mg/kg; Antioxidant: 40,000 mg/kg; Excipient: 1 mg/kg. 2 Mn: 39,680 mg/kg; Fe: 20,000 mg/kg; Zn: 33,880 mg/kg; Cu: 4000 mg/kg; I: 400 mg/kg; Se: 80 mg/kg; Choline: 100,000 mg/kg; Excipient: 1 mg/kg.; 3 OP=50 g/kg.; 4 Diet with OPp; 5 Diet with ENZ; 6 Diet with OPu; 7 Control diet without OP. Table 2. Feed ingredients and chemical composition of diets used during the finishing period (22nd–42nd days of age, as-fed basis). Processed Olive Pulp (OPp) Unprocessed Olive Pulp (OPu) 503 p 4 50 p + ENZ 5 100 p 100 p + ENZ 50 u 6 50 u + ENZ 100 u 100 u + ENZ Ctrl 7 Ctrl +ENZ Ingredients (g/kg) Processed OP 50.00 50.00 100.00 100.00 0.00 0.00 0.00 0.00 0.00 0.00 Unprocessed OP 0.00 0.00 0.00 0.00 50.00 50.00 100.00 100.00 0.00 0.00 Enzyme 0.00 0.05 0.00 0.05 0.00 0.05 0.00 0.05 0.00 0.05 Corn 547.60 547.60 496.80 496.80 522.60 522.60 447.10 447.10 598.20 598.20 Soybean meal 323.20 323.20 323.20 323.20 329.80 329.80 336.30 336.30 323.30 323.30 Soybean oil 42.30 42.30 44.50 44.50 60.00 60.00 79.80 79.80 40.20 40.20 Wheat bran 0.10 0.05 0.10 0.05 0.10 0.05 0.10 0.05 0.10 0.05 Dicalcium phosphate 17.00 17.00 17.30 17.30 17.10 17.10 17.40 17.40 16.70 16.70 Limestone 8.70 8.70 6.90 6.90 9.30 9.30 8.20 8.20 10.50 10.50 Vitamin mixture1 3.00 3.00 3.00 3.00 3.00 3.00 3.00 3.00 3.00 3.00 Mineral mixture2 3.00 3.00 3.00 3.00 3.00 3.00 3.00 3.00 3.00 3.00 Salt 2.30 2.30 2.10 2.10 2.50 2.50 2.40 2.40 2.50 2.50 Agriculture 2020, 10, 359 6 of 17 Sodium bicarbonate 1.50 1.50 1.50 1.50 1.50 1.50 1.50 1.50 1.50 1.50 DL-Methionine 1.10 1.10 1.20 1.20 1.10 1.10 1.20 1.20 1.00 1.00 Lysine hydrochloride 0.20 0.20 0.40 0.40 0.00 0.00 0.00 0.00 0.00 0.00 Total 1000.0 1000.0 1000.0 1000.0 1000.0 1000.0 1000.0 1000.0 1000.0 1000.0 Nutrient analysis (g/kg) Dry matter 90.36 90.36 90.53 90.53 90.52 90.52 90.85 90.85 90.19 90.19 Metabolizable energy (kcal/kg) 3150.0 3150.0 3150.0 3150.0 3150.0 3150.0 3150.0 3150.0 3150.0 3150.0 Crude protein 21.00 21.00 21.00 21.00 21.00 21.00 21.00 21.00 21.00 21.00 Ether extract 7.24 7.24 7.87 7.87 8.82 8.82 11.02 11.02 6.62 6.62 Linoleic acid 3.49 3.49 3.49 3.49 4.34 4.34 5.18 5.18 3.49 3.49 Crude fiber 4.49 4.49 6.39 6.39 4.97 4.97 7.38 7.38 2.58 2.58 Calcium 0.90 0.90 0.90 0.90 0.90 0.90 0.90 0.90 0.90 0.90 Phosphorus 0.68 0.68 0.68 0.68 0.68 0.68 0.67 0.67 0.68 0.68 Available phosphorus 0.45 0.45 0.45 0.45 0.45 0.45 0.45 0.45 0.45 0.45 Potassium 0.87 0.87 0.90 0.90 0.85 0.85 0.87 0.87 0.84 0.84 Chlorine 0.18 0.18 0.17 0.17 0.18 0.18 0.18 0.18 0.19 0.19 Manganese (mg/kg) 471.74 471.74 471.58 471.58 472.83 472.83 473.72 473.72 471.91 471.91 Sodium 0.16 0.16 0.16 0.16 0.16 0.16 0.16 0.16 0.16 0.16 Zinc (mg/kg) 381.73 381.73 381.25 381.25 383.63 383.63 385.05 385.05 382.21 382.21 Choline (mg/g) 1.48 1.48 1.45 1.45 1.48 1.48 1.46 1.46 1.51 1.51 Folic acid (mg/kg) 2.04 2.04 2.02 2.02 2.05 2.05 2.05 2.05 2.06 2.06 Arginine 1.33 1.33 1.31 1.31 1.34 1.34 1.34 1.34 1.35 1.35 Glycine 0.84 0.84 0.83 0.83 0.84 0.84 0.84 0.84 0.86 0.86 Serine 1.00 1.00 0.99 0.99 1.01 1.01 1.00 1.00 1.02 1.02 Gly + Ser 1.84 1.84 1.82 1.82 1.85 1.85 1.84 1.84 1.88 1.88 Histidine 0.54 0.54 0.53 0.53 0.54 0.54 0.53 0.53 0.55 0.55 Isoleucine 0.84 0.84 0.83 0.83 0.85 0.85 0.84 0.84 0.85 0.85 Leucine 1.75 1.75 1.71 1.71 1.75 1.75 1.70 1.70 1.80 1.80 Lysine 1.11 1.11 1.11 1.11 1.11 1.11 1.11 1.11 1.11 1.11 Methionine 0.42 0.42 0.42 0.42 0.42 0.42 0.42 0.42 0.42 0.42 Cysteine 0.33 0.33 0.32 0.32 0.33 0.33 0.32 0.32 0.34 0.34 Met + Cys 0.75 0.75 0.74 0.74 0.75 0.75 0.74 0.74 0.76 0.76 Phenylalanine 0.96 0.96 0.95 0.95 0.97 0.97 0.96 0.96 0.98 0.98 Tyrosine 0.79 0.79 0.78 0.78 0.80 0.80 0.79 0.79 0.81 0.81 Phe + Tyr 1.75 1.75 1.73 1.73 1.77 1.77 1.75 1.75 1.79 1.79 Threonine 0.76 0.76 0.75 0.75 0.76 0.76 0.76 0.76 0.77 0.77 Tryptophan 0.27 0.27 0.27 0.27 0.27 0.27 0.27 0.27 0.27 0.27 Valine 0.94 0.94 0.92 0.92 0.94 0.94 0.93 0.93 0.95 0.95 1Vitamin A: 3,600,000 IU/kg; Vitamin D3: 800,000 IU/kg; Vitamin E: 7200 IU/kg; Vitamin K3: 800 mg/kg; Vitamin B1: 720 mg/kg; Vitamin B2: 2640 mg/kg; Vitamin B3 (Calcium pantothenate): 4000 mg/kg; Vitamin B5 (Niacin):12,000 mg/kg; Vitamin B6: 1200 mg/kg; Vitamin B9 (Folic acid): 400 mg/kg; Vitamin B12: 6 mg/kg; Vitamin H2 (Biotin): 40 mg/kg; Choline: 100,000 mg/kg; Antioxidant: 40,000 mg/kg and 1 mg/kg Excipient. 2 Mn: 39,680 mg/kg; Fe: 20,000 mg/kg; Zn: 33,880 mg/kg; Cu: 4000 mg/kg; I: 400 mg/kg; Se: 80 mg/kg; Choline: 100,000 mg/kg; Excipient: 1 mg/kg.; 3 OP=50 g/kg;.4Diet with OPp; 5Diet with ENZ; 6Diet with OPu; 7 Control diet without OP. Agriculture 2020, 10, 359 7 of 17 The OP was obtained by washing fresh olive fruit with water and milling them. Table 3 reports the chemical composition of two types of olive meal used in the experimental procedures. After these steps, the olives were added to hot water (80 °C) and centrifuged. At this stage, the water–oil emulsion was removed from the milled olive, which we named “olive cake” (OC). In the next step, -tocopherol (antioxidant) and an antifungal toxin-binder (zeolite adsorbent) were added to the OC, which was then dried with hot air (70 °C), thereby resulting in the OP. Olive processing consisted of passing the milled fruits through a sieve (pore diameter: 1.5 mm). During this process, parts of the stones (seeds) were removed to produce “partly destoned” OP. In the diets with enzyme (ENZ), this product (50 mg/kg) was added to the OP (Natuzyme P50®; Sydney, Australia). The nutritional chemical analysis of processed (OPp) and unprocessed (OPu) dried OP was performed [41]. Table 3. Chemical composition of two types of olive meal used in the experiment (as-fed basis). Types of Olive Meal Dried Processed Olive Pulp (Partly Destoned)* Original Dried Unprocessed Olive Pulp Dry matter (g/kg) 934.50 935.70 Metabolizable energy (kcal/kg) 2980.00 1250.00 Crude protein (g/kg) 107.30 71.10 Crude fiber (g/kg) 256.00 350.00 Neutral detergent fiber (α-amylase) (g/kg) 716.00 744.00 Acid detergent fiber (g/kg) 550.00 584.00 Ash (g/kg) 85.00 62.00 Crude fat (g/kg) 130.00 85.00 Calcium (g/kg) 8.20 6.10 Phosphorus (g/kg) 0.70 0.60 Soluble sugars (g/kg) 1.70 1.40 Starch (g/kg) 9.70 10.50 Total polyphenols (g/kg) 3.70 1.90 Total tannins (g/kg) 22.90 17.90 * Seeds partially removed. At the age of 42 days (after 4 h-fasting for complete evacuation of the gut), one bird from each replicate were randomly selected, weighed, and euthanized (stunned and slaughtered). In the slaughter method, stunning by electronarcosis and subsequent bleeding (by the jugular court) were used. The averages of these birds were calculated and used as one experimental unit. The experimental units were used to measure carcass yield and offal characteristics. Birds were fully defeathered via the dry method. Feet were separated from the carcass at the tibiotarsal joint. Neck, wingtips, gut, and liver were removed, and the carcass was weighed (cold carcass weight, after chilling). Economically relevant parts of the carcass and offal were separated. First, breast muscle (including the skin and sternum) was dissected free from the carcass. Legs (thighs and drumsticks were dissected by disarticulation at the hip joint and dissection of tissue from the iliac bone. All abdominal fat (including that around the rectum, gizzard, and proventriculus) was collected. Various parts of the carcasses were dissected and weighed separately. All parts, including the head, breast, wings, neck, thighs and drumsticks (legs), gizzard, heart, liver, lung, and abdominal fat, were weighed and weight recorded. The total weight of all dissected parts was related to the whole eviscerated carcass. Relative percentage ratios were calculated according to the following equation: 100 x (weight of component(s)/eviscerated carcass weight). Data analysis of variance used the two-way ANOVA procedure. Data were submitted to twoway analysis of the variance [42]. The following equation was applied: Yijk= μ+Ai+Bj+Ck+ABij+ACik+BCjk+ABCijk+eijkl (1) where μ = general average, Ai = olive pulp levels, Bj = effect of processing, Ck = effect of enzyme supplementation, ABij = effect of the interaction between olive pulp levels and processing, ACik = effect of the interaction between olive pulp levels and enzyme supplementation, BCjk = effect of the interaction between olive pulp processing and enzyme complex interaction effect, ABCijk = effect of Agriculture 2020, 10, 359 8 of 17 the interaction among olive pulp levels, olive pulp processing, and enzyme supplementation, and eijkl = incidental residual effect of observation. After the statistical differences were confirmed, the general linear model (PROC GLM) was used, and the differences between means (p ≤ 0.05) were assessed via Duncan’s multiple range test. 3. Results Higher values for OP did not affect the following characteristics: live body weight (LW), defeathered body weight (DW), full abdomen carcass weight (FC), empty abdomen carcass weight (EC), eviscerated carcass weight (ECr), breast weight, thigh and drumstick weight (legs), wing weight, and relative breast and wing weights (p >0.05; Table 4). The relative breast weight was increased by OP processing (partial destoning) (p>0.05; Table 4). However, a significant difference, which was not found in the values for LW, DW, FC, EC, relative ECr, breast, wing, relative wing, thigh and drumstick, and relative thigh and drumstick weights was due to the OP processing (p >0.05) as shown in Table 4. Agriculture 2020, 10, 359 9 of 17 Table 4. Carcass traits of broilers fed diets containing different amounts of processed (OPp) and unprocessed (OPu) olive pulp, with and without enzyme (ENZ), in the period 1–6 weeks of age. LW (g) DW (g) FC (g) EC (g) ECr (%) BrW (g) BrWr (%) TDW (g) TDWr (%) WW (g) WWr (%) Diets OPp (50 g/kg) 2770 a 2464 a 2228 a 1743 a 78.3 a 842 a 34.2 a 718 a 29.2 a,b 95 a 3.87 a OPp (50 g/kg) + ENZ 2752 a 2476 a 2295 a 1752 a 76.3 a 857 a 34.5 a 680 a 27.5 b 108 a 4.35 a OPp (100 g/kg) 2807 a 2482 a 2315 a 1763 a 76.2 a 857 a 34.5 a 707 a 28.5 b 110 a 4.40 a OPp (100 g/kg) + ENZ 2782 a 2456 a 2288 a 1738 a 75.9 a 847 a 34.4 a 690 a 28.1 b 106 a 4.33 a OPu (50 g/kg) 2910 a 2564 a 2365 a 1847 a 78.0 a 862 a 33.5 a 845 a 32.8 a 112 a 4.34 a OPu (50 g/kg) + ENZ 2952 a 2572 a 2368 a 1800 a 76.1 a 849 a 33.0 a 749 a 29.2 a,b 111 a 4.32 a OPu (100 g/kg) 2827 a 2490 a 2278 a 1687 a 74.0 a 801 a 32.1 a 673 a 27.0 b 105 a 4.22 a OPu (100 g/kg) + ENZ 3033 a 2635 a 2433 a 1780 a 73.2 a 842 a 32.0 a 735 a 27.9 b 107 a 4.04 a Without OP 2797 a 2447 a 2215 a 1674 a 75.6 a 775 a 31.7 a 672 a 27.5 b 100 a 4.06 a Without OP + ENZ 2872 a 2642 a 2348 a 1820 a 77.5 a 902 a 34.2 a 681 a 25.7 b 109 a 4.16 a Standard errors ±114 ±99 ±90 ±70 ±1.4 ±39 ±0.9 ±35 ±0.9 ±6 ±0.20 LW: Live body weight; DW: Defeathered body weight; FC: Full abdomen carcass weight; EC: Empty abdomen carcass weight; ECr: Eviscerated carcass; BrW: Breast weight; BrWr: Relative breast weight; TDW: Thigh and drumstick weight; TDWr: Relative thigh and drumstick weight; WW: Wing weight; WWr: Relative wing weight. a,b Means (± standard errors) within each column of dietary treatments with no common superscript differ significantly at p ≤0.05. 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