Effect of olive meal and supplemental enzymes on performance traits, blood biochemistry, humoral immunity response and caecal microbiota of broilers

S. Sateri, A. Seidavi, M. Bouyeh, P. Neumann, M. Kutzler, V. Laudadio, F. Loperfido & V. Tufarelli 1 Department of Animal Science, Rasht Branch, Islamic Azad University, Rasht, Iran 2 Department of Animal and Rangeland Sciences, Oregon State University, Corvallis, OR, USA 3 Section of Veterinary Science and Animal Production, Department of DETO, University of Bari ‘Aldo Moro’, Valenzano, Bari, Italy


Introduction
Using food by-products in livestock feeding is common practice for producers to reduce feed costs (Laudadio & Tufarelli, 2011).The OM is a by-product of the olive oil mill extraction process.It is rich in lipids (73% oleic acid, 13% palmitic acid, and 7% linoleic acid), making it an economic ingredient for the livestock industry (Ranalli et al., 2002;Tufarelli et al., 2013).The fatty acid composition of poultry diet is highly effective not only in successful broiler chicken rearing, but also in the lipid composition of meat (Wood et al., 2008;Cherian, 2011).
Research on the effects of feeding OM in broiler diet is limited.However, studies investigating the use of OM in poultry diets may provide an opportunity to increase its popularity (Zarei et al., 2011;Zhang et al., 2013;Cayan & Erener, 2015).A preliminary study using OM as a feedstuff for broiler chickens reported that feeding diets containing up to 9% OM had no negative effect on growth performance and significantly increased the daily fibre intake in birds (Zangeneh & Torki, 2011).Because OM is the remnant of the plant after the oil has been extracted, this by-product is high in non-digestible fibre.This fibre fraction is characterized by β-1-4 bonds, which are not well digested by monogastric species (Tufarelli et al., 2007;Zarei et al., 2011).When high fibre diets are used, feed producers can add supplemental enzymes such as β-glucanase, phytase or hemicellulase to the diet to increase fibre digestibility.Adding enzymes greatly increases the amount of nutrients that avian digestive tracts can obtain from these feed by-products (Campbell & Bedford, 1992;Canoğullari et al., 1999;Adeola & Cowieson, 2011;De Vries et al., 2012;O'Neill et al., 2014).
Therefore, the objective of this study was to evaluate the effects of feeding broiler diets with various levels of OM with or without supplemental enzymes.The authors hypothesized that feeding a diet containing OM with supplemental enzymes might improve growth performance and carcass traits of broilers without affecting their blood biochemistry, humoral immunity response, and caecal microbiota.

Material and Methods
A 42-day experiment was conducted using one-day-old male Ross-308 broiler chicks according to the institutional ethical norms of the Faculty of Agriculture, Islamic Azad University (Rasht Branch, Iran).The poultry house was located in Karaj City, Iran.The mean temperature inside the experimental facility was maintained between 29 and 31 °C from 1 to 7 days, 27 and 29 °C from 8 to 14 days, and from 24 to 26 °C from 15 to 42 days of the rearing period.Relative humidity was maintained at 55-65%.Broilers were reared under 20 hours of light per day in 1.2 × 1.5 m cages.Rearing conditions were similar to all treatments.Broilers were allowed free access to fresh water and fed ad libitum.BW, BWG, and FI were determined weekly.Broilers were routinely vaccinated against endemic avian diseases in Iran.To reduce their stress in the 24-hour period after each vaccination, a multi-vitamin + electrolytes solution was added to their drinking water at a dilution of 1:1000.
Chicks (n = 300) were randomly assigned to ten dietary treatments, which varied in concentration of OM (0, 2, 4, 6, and 8%, respectively) and the addition of an enzyme supplement.The OM was purchased from a local company.Aflatoxin analysis of OM was below the normal level, revealing up to 2.86 ppb of aflatoxin B and up to 3.27 ppb of total aflatoxin.Broiler nutritional requirements were based on Ross-308 nutrition specifications (SVIAGEN, Scotland, UK).All diets under the two rearing periods were formulated to have the same energy and protein levels.The composition of diets is reported in Table 1.The enzyme supplement Natuzyme P50 ® was added to half of the tested diets.This supplement contained phytase (100,000 U/g), β glucanase (700 U/g), α-amylase (700 U/g), cellulase (6,000 U/g), pectinase (700 U/g), xylanase (10,000 U/g), lipase (30 U/g), and protease (3,000U/g), as well as trace amounts of amyloglucosidase, hemicellulase, pentosonase, acid phytase, and acid phosphatase.There were ten birds per replicate and three replicates per treatment.Each replicate was considered an experimental unit.18.70 1 Vitamin A: 5000 IU/g; vitamin D3: 500 IU/g; vitamin E: 3 mg/g; vitamin K3: 1.5 mg/g; vitamin B2: 1 mg/g; 2 Calcium pantothenate: 4 mg/g; niacin: 15 mg/g; vitamin B6: 13 mg/g; Cu: 3 mg/g; Zn: 15 mg/g; Mn: 20 mg/g; Fe: 10 mg/g; K: 0.3 mg/g Samples of blood (5 ml) were collected from broilers' brachial vein into EDTA tubes at 16,30,33,34,40, and 42 days of age.Broilers were fasted for four hours prior to collection of blood on day 42.Blood samples were centrifuged at 3000 rpm ×20 min.Plasma was collected and stored at -20 °C until analysis.Humoral immune responses were measured from samples collected on days 16 and 34 for Newcastle disease virus (NDV), on day 30 for avian influenza (AI), and on day 40 for infectious bronchitis virus (IBV) and infectious bursal disease (Gumboro) (Pourhossein et al., 2015).
Antibody titers against NDV and AI were measured in birds using the haemagglutination inhibition test as described by Cunningham (1971).Antibody titers against IBV and Gumboro were measured with commercial ELISA kits (Bio-check BV, Gouda, Holland) in accordance with the manufacturer's instructions.The absorbance of ELISA controls and samples was read at 405 nm using a plate reader (Bio-Tek Instruments Inc. ELX 800; Winooski, VT, USA), as described by Pourhossein et al. (2015).
At the end of the feeding period (42 days), 20% of the broilers were slaughtered, and the weights of full and empty carcass, and defeathered BW were recorded, as well the weight and yield of meat cuts (breast, drumsticks and wings), abdominal fat, head, gizzard, heart, and neck.
Caecal microflora samples (~1 g) were collected and transferred into sterile tubes with phosphatebuffered saline (PBS) (9 ml) and shaken for approximately 30 min.Serial dilutions (10 , respectively) were made, and agarose plates were inoculated with 100 μl of each dilution.Nutrient agar plates were incubated in aerobic conditions at 37 °C for 48 hours to determine total aerobic bacteria counts.For isolation of Lactobacilli sp., dilutions were cultured in de Man Rogosa Sharpe agar (MRS agar) under anaerobic conditions at 37 °C for 72 hours.Moreover, dilutions were cultured for coliforms in MacConkey agar in aerobic conditions for 24 hours.At the end of the incubation periods, the number of colony forming units (CFU) was counted for each dilution and media.Bacterial counts were reported as log 10 of CFU per 1 g of caecal content (Abbasi et al., 2015).
All data were analysed by ANOVA using a 2 × 5 factorial arrangement with five OM levels (0, 2, 4, 6, and 8%, respectively) and two enzyme treatments (0 and 0.005%), using the two-way ANOVA procedure (SPSS, Chicago, IL, USA).Duncan's post hoc test was used if the initial test was significant at P ≤0.05.The observations were considered statistically significant if P ≤0.05.

Results and Discussion
The results of the present study are summarized in Tables 2-8.The addition of the supplemental enzymes to broiler diet had minimal impact on the evaluated parameters.In particular, there were no significant differences among dietary groups on growth performance in terms of final BW, average daily weight gain, FI and FE (Table 2).These results agree with a previous study by Rabayaa et al. (2001), that found no negative impact of OM in diet on broilers' performance traits.
At day 42, the full and empty carcass weight, as well as the carcass yield, did not differ (P >0.05) in broilers fed diets with added OM or supplemental dietary enzymes (Table 3).These results are in agreement with those of Omar (2005), who found that adding up to 10% of olive pulp in broiler diet had no significant effect on carcass characteristics.The same author also reported that the level of olive pulp in diet had no influence on the weight of edible (i.e.liver, heart, gizzard) or inedible (i.e.oesophagus, crop, proventriculus) organs.Moreover, as reported in Table 4, the weight and yield of breast, drumsticks and wings were not different (P >0.05) when broilers were fed different levels of OM and enzyme or their combination.
Similar to the findings of Omar (2005), Sateri et al. (2014) and Al-Harthi (2017), the current study found that the yield of head, gizzard, heart, and neck, as well abdominal fat, did not differ (P >0.05) in broilers fed various levels of OM with or without the addition of the enzyme supplement (Table 5).a,b means within each column of dietary treatments with no common superscript differ significantly at P < 0.05 At day 42, circulating total cholesterol was higher (P <0.05) in broilers fed the diet containing 2% OM without the supplemental enzyme compared with those fed 4% OM and the enzyme (Table 6).
However, there were no differences (P >0.05) among treatment groups for blood LDL and HDLcholesterol, triglycerides, total protein, albumin, glucose and uric acid.These results agreed with similar studies that examined the changes in blood biochemistry parameters in laying hens fed diets with varying concentrations of olive pulp with or without -mannanase enzyme supplementation (Zangeneh & Torki, 2011;Sayehban, 2015;Sayehban, 2016).
NDV antibody titers did not differ (P >0.05) in any of the groups after either of the vaccinations (Table 7), or for AI antibody titers.However, IB antibody titer was significantly (P <0.05) higher in birds fed 2% and 4% OM in diet with supplemental enzymes, compared with that containing 6% OM with supplemental enzyme and the control group (without OM or supplemental enzymes).In addition, IBD (Gumboro) antibody titers were highest (P <0.05) in birds fed 4% OM diet including the enzyme supplement compared with broilers fed 6% and 8% OM without enzyme supplement.This is the first study to examine the effects of OM addition to broiler diet on caecal microbiota (Table 8).Extensive research is available on the effects of diet on the biodiversity of the caecal microbiota in broiler chickens.In healthy chickens, the caecal microbiota plays an important role in nutrient assimilation and in the prevention of pathogenic bacteria colonization (Apajalahti, 2005).This is important because these pathogens can spread to humans through the consumption of meat that has been contaminated during slaughter.In poultry, in addition, prophylactic antibiotics are widely used to improve growth performance (Allen & Stanton, 2014), but this practice can result in the development of a reservoir of antibiotic resistant bacteria, which can affect public health (Zhou et al., 2012).
Previous studies have focused on the addition of supplemental enzymes, yeast and other probiotics and, depending on the study, some beneficial effects were found (Dhama et al., 2015;Rezaei et al., 2015;Alefzadeh et al., 2016).In the current study, the numbers of aerobic, lactic acid-producing, and coliform bacteria did not differ (P >0.05) among experimental groups, demonstrating the safety of adding OM to both animal and human populations.

Conclusion
In conclusion, a broiler diet containing 4% OM with the addition of an enzyme supplement resulted in an appropriate combination to support broiler growth performance and carcass characteristics without negatively affecting blood biochemistry, humoral immunity response, and caecal microbiota.The findings in this study support the literature, which suggests OM as a suitable ingredient for commercial broiler diets.Moreover, adding OM by-products to poultry diet could provide an economic and environmentally friendly alternative to traditional diets.Thus, this study identified that the inclusion of dietary enzyme supplements is not necessary to achieve optimal productive performance of birds fed a diet containing 8% (or less) OM.

Table 1
Ingredients and nutrient analysis of diets fed to broilers in starter and

Table 2
Growth performance of broilers fed diets containing olive meal (OM) and enzyme or their interaction

Table 3
Carcass traits at 42nd day of age in broilers fed diets containing olive meal (OM) and enzyme or their interaction Meat cuts at 42nd days of age of broilers fed diets containing olive meal (OM) and enzyme or their interaction a,b Means within each column of dietary treatments with no common superscript differ significantly at P < 0.05Table 4

Table 5
Carcass traits at 42nd day of age of broilers fed diets containing various levels of olive meal (OM) and enzyme or their interaction

Table 6
Effect of diets containing olive meal (OM) and enzyme or their interaction on blood parameters of broiler chickens

Table 7
Immune response (log 10 ) after vaccination of broilers fed diets containing various levels of olive meal (OM) with and without enzyme or their interaction Means within each column of dietary treatments with no common superscript differ significantly at P <0.05

Table 8
Microflora of cecum (log 10 CFU) at 42nd day of age of broilers fed diets containing various levels of olive meal (OM) and enzyme or their interaction