Prebiotics offered to broiler chicken exert positive effect on meat quality traits irrespective of delivery route

Prebiotics offered to broiler chicken exert positive effect on meat quality traits irrespective... ABSTRACT Elimination of antibiotic growth promoters from poultry production has encouraged intensive search for relevant alternatives. Prebiotics are proposed as efficient replacements to stimulate colonization/expansion of beneficial microflora in chickens. The aim of this study was to deepen the knowledge on the effect of prebiotic administration on slaughter performance and meat quality traits of broiler chickens by evaluating different routes of their delivery (in ovo vs. in-water vs. in ovo + in-water). At d 12 of incubation, 1,500 eggs (Ross 308) containing viable embryos were randomly allotted into 4 groups and injected in ovo with 0.2 mL solution containing: 3.5 mg/embryo BI (Bi2tos, trans-galactooligosaccharides); 0.88 mg/embryo DN (DiNovo, extract of Laminaria spp.); 1.9 mg/embryo RFO (raffinose family oligosaccharides) and 0.2 mL physiological saline (C). All prebiotics increased final BW compared to C group (P < 0.01), irrespective of delivery route. The prebiotics injected in ovo (T1) or in ovo combined with in-water supplementation (T2) increased carcass weight as compared with in-water group (T3), while T3 had the lowest carcass yield compared to the other groups. All prebiotics increased breast muscle weight and yield (P < 0.01), as well as fiber diameter (P < 0.05). Ultimate meat pH was lower (P < 0.01) in T3 than in T2 group. Meat from chickens treated with prebiotics showed a lower redness index, while lightness and yellowness were not affected by the treatments. Saturated fatty acid (SFA), polyunsaturated fatty acid (PUFA) and n-3 fatty acids contents were higher (P < 0.01), and monounsaturated fatty acid (MUFA) level was lower (P < 0.01) in prebiotic groups compared with C group. Nutritional indexes (n-6/n-3, PUFA/SFA ratio and thrombogenic index) displayed favorable human health-promoting values in the meat of chickens which were treated with prebiotics, irrespective of delivery route. Muscle cholesterol content was not affected by prebiotics. In conclusion, this study has shown that prebiotics can exert positive effects on growth of broiler chickens, carcass and meat quality traits, irrespective of delivery route. INTRODUCTION World production of poultry meat is expected to increase by over 181 million tonnes by 2050 (Alexandratos and Bruinsma, 2012). Such a raise in poultry production will likely be powered by continued genetic progress resulting from the work of primary breeders. However, a great number of scientific evidence suggests that the intense genetic selection is linked with the elevated incidence of histological and biochemical changes to muscle tissue that lead to different types of myopathies, which adversely affect the poultry meat industry (Petracci et al., 2015; Kuttappan et al., 2016; Baldi et al., 2018). In addition, new antibiotic-resistant bacteria are emerging and spreading globally, while known poultry pathogens like Campylobacter and Salmonella are still defined as a major food borne threat for consumers. In fact, European countries have implemented bans on the use of growth-promoting antibiotics, while in the USA the Center for Veterinary Medicine of the US FDA (Food and Drug Administration) recently issued a “Guidance for Industry”, which describes requirements for label claims and recommended restrictions on usage of antibiotics in food producing animals (FDA, 2012). Pre- and pro-biotics are proposed as one of the alternatives to antibiotic growth promoters, to prevent enteric diseases and improve performance in poultry. Prebiotics are non-digestible components of feed derived from sugars, including raffinose, galactooligosaccharides and β-glucans (Sobolewska et al., 2017). Recently, the prebiotic is defined as “a non-digestible compound that, through its metabolization by microorganisms in the gut, modulates composition and/or activity of the gut microbiota, thus conferring a beneficial physiological effect on the host” (Bindels et al., 2015). Numerous probiotic and prebiotic products are commercially available and are conventionally supplemented in-feed or in-water at first hours/days post hatching. There are substantial inconsistencies regarding efficacy of bioactives, which are found in the reported broiler chicken feeding trials (Waldroup et al., 2003; Bozkurt et al., 2009; Kim et al., 2011; Nabizadeh, 2012; Fowler et al., 2015). These discrepancies could be related to a number of conditions, which namely are: the physiological state of the bird, contaminated hatchery area, microbiota balance in the animal gut, disadvantageous interaction among various dietary additives (i.e., oligosaccharides, coccidiostatics, probiotics), different chemical structure and composition of the prebiotics used, mode of administration and duration of the treatment, doses of biological active substances applied as well as the amount of feed or water intake, including the water quality (Gaggìa et al., 2010; Huyghebaert et al., 2011; Nabil Alloui et al., 2013; Ducatelle et al., 2015; Bednarczyk et al., 2016; Sugiharto, 2016). Therefore, the major challenges are: (i) to observe the evident effect of prebiotics under fully controlled rearing condition, (ii) to optimize the method of prebiotic delivery. During the last years, we have tested different prebiotics (commercial type and in-house extracted) and their synergistic combinations with probiotics (synbiotics) in field and laboratory studies (Pilarski et al., 2005; Bednarczyk et al., 2011, 2016; Maiorano et al., 2012, 2017; Dunislawska et al., 2017). To be effective, these compounds have to be administered in fully controlled conditions, as early as possible. In our earlier study (Villaluenga et al., 2004) we have determined the 12th day of incubation for prebiotic injection into the air cell of the incubated egg, as the optimal time to provide stimulation of embryonic microflora with no harm done to the incubating eggs. At this time the chorioallantoic membrane is highly vascularized and allows transferring of the prebiotic from the air cell into the circulatory system and further to the developing intestine. Alike in the current trial, also in the previous one (Bednarczyk et al., 2016; Maiorano et al., 2017) we have offered to broiler chickens a broad range of bioactive formulations, including: i) commercially available prebiotics, such as DN (DiNovo, BioAtlantis Ltd., Tralee, Co. Kerry, Ireland), extract of Laminaria spp. containing laminarin and fucoidan and BI (Bi2tos, Clasado Ltd., Sliema, Malta), a non-digestive trans-galactooligosaccharides (GOS) from milk lactose digested with Bifidobacterium bifidum NCIMB 41,171; ii) in-house extracted prebiotic (RFO—raffinose family oligosaccharides) from lupin (Lupinus luteus) seeds (Ciesiolka et al., 2005). We hypothesized that the combination of different modes of prebiotic administration: injection in ovo (into the air chamber of the egg on the 12th d of incubation), followed by additional application of prebiotic in drinking water, during the first week of chicken rearing, may exhibit a synergistic effect not only on growth performance (Bednarczyk et al., 2016) but also on meat quality traits. Only few publications which report the effect of prebiotic administration on meat quality traits of broiler chickens can be found (Maiorano et al., 2012, 2017). None of them included the analysis of fatty acid (FA) composition. Some authors proved that prebiotics can alter lipid metabolism (Letexier et al., 2003) and enhance the ratio of polyunsaturated fatty acid (PUFA) to saturated fatty acid (SFA) in chicken meat (Zhou et al., 2009; Velasco et al., 2010), which is beneficial to human health. The aim of this study was to deepen the knowledge on the effect of prebiotic administration on slaughter performance and meat quality traits of broiler chicken by evaluating different routes of their delivery (in ovo vs. in-water vs. in ovo and in-water combined). MATERIALS AND METHODS Birds and Experimental Design Eggs (60 g average weight) were obtained from single 32-wk-old breeder flock (Ross 308) and incubated in a commercial broiler hatchery (Drobex-Agro Sp. z o.o., Solec Kujawski, Poland). Prior to the injection, the eggs were candled to select only the ones containing viable embryos. At d 12 of incubation, 1500 eggs containing viable embryos were randomly allotted into 4 experimental groups (375 eggs/group). Prebiotic solutions of each experimental group contained different doses of bioactives: 3.5 mg/embryo BI (Bi2tos, Clasado Ltd., Sliema, Malta); 0.88 mg/embryo DN (DiNovo, BioAtlantis Ltd., Tralee, Co. Kerry, Ireland); 1.9 mg/embryo RFO. The above mentioned prebiotic solutions were used to compare effects of different routes of delivery: (T1) in ovo injection, (T2) in ovo injection combined with in-water delivery, and (T3) in-water delivery. Control group (C) was injected in ovo with physiological saline only and did not receive any prebiotic in-water. As for in ovo treatments, the eggs were injected in ovo with 0.2 mL of selected prebiotic solution, which was deposited in the air cell followed by immediate sealing of the eggs. After hatching, the chicks were sexed and 600 males (42.0 ± 2 g average weight) were randomly assigned to 10 experimental groups (60 males/group): T1 (DN, BI and RFO), T2 (DN, BI and RFO), T3 (DN, BI and RFO) and C. Birds were grown to 42 d of age in collective cages (n = 6 replicate cages, 10 birds in each cage). Chicks from T1 group were raised without any additional supplementation with prebiotics in water. T2 and T3 groups were supplemented in-water with respective prebiotic (DN, BI or RFO) for the first 7 d of life. Those animals received 12 mL of the prebiotic dissolved in water per pen (20 mg of prebiotic/mL). Birds were reared according to the regulations and permission of the local Ethical Commission (decision No.22/2012 21.06.2012) and in accordance with the animal welfare recommendations of European Union directive 86/609/EEC, in an experimental poultry house Drobex-Agro (Solec Kujawski, Poland) that provided good husbandry conditions (e.g., stocking density, litter, ventilation). Animals were fed ad libitum the standard commercial feed mixtures (Table 1): starter (d 1–21), grower (d 22–35), finisher (d 35–42). In the same trial, along the rearing period, in vivo performance and mortality were recorded as described by Bednarczyk et al. (2016). Table 1. Composition and nutrient content of diets.   Starter  Grower  Finisher    (1–21 d)  (22–35 d)  (36–42 d)  Ingredient, g/Kg   Wheat  26.73  29.19  30.66   Maize  30.00  30.00  30.00   Soybean meal  32.50  28.20  25.33   Canola seeds  5.00  6.00  7.00   Soybean oil  2.10  1.33  1.80   Lard  -  2.00  2.50   NaCl  0.30  0.30  0.28   Mel stern  1.09  0.95  0.85   Phosphate 1-Calcium  1.15  0.94  0.63   DL-methionine  0.25  0.18  0.13   L-lysine  0.32  0.32  0.27   L-threonine  0.06  0.09  0.05   Vitamin—mineral premix1  0.50  0.50  0.50  Calculated composition   ME, MJ/kg  12.47  12.97  13.39   CP, % DM  22.00  20.50  19.50   Lysine, % DM  1.35  1.25  1.15   Methionine, % DM  0.57  0.49  0.43   Methionine + Cystine, % DM  0.95  0.85  0.78   Ca, % DM  0.90  0.80  0.70   P, % DM  0.40  0.35  0.28    Starter  Grower  Finisher    (1–21 d)  (22–35 d)  (36–42 d)  Ingredient, g/Kg   Wheat  26.73  29.19  30.66   Maize  30.00  30.00  30.00   Soybean meal  32.50  28.20  25.33   Canola seeds  5.00  6.00  7.00   Soybean oil  2.10  1.33  1.80   Lard  -  2.00  2.50   NaCl  0.30  0.30  0.28   Mel stern  1.09  0.95  0.85   Phosphate 1-Calcium  1.15  0.94  0.63   DL-methionine  0.25  0.18  0.13   L-lysine  0.32  0.32  0.27   L-threonine  0.06  0.09  0.05   Vitamin—mineral premix1  0.50  0.50  0.50  Calculated composition   ME, MJ/kg  12.47  12.97  13.39   CP, % DM  22.00  20.50  19.50   Lysine, % DM  1.35  1.25  1.15   Methionine, % DM  0.57  0.49  0.43   Methionine + Cystine, % DM  0.95  0.85  0.78   Ca, % DM  0.90  0.80  0.70   P, % DM  0.40  0.35  0.28  1Provided per kilogram of diets: vitamin A 12,500 IU, vitamin D3 - 4500 IU, vitamin E 45 mg, vitamin K3 3 mg, vitamin B1 3 mg, vitamin B2 6 mg, vitamin B6 4 mg, pantothenic acid 14 mg, nicotinic acid 50 mg, folic acid 1.75 mg, choline 1.6 g, vitamin B12 0.02 mg, biotin 0.2 mg, Fe 50 mg, Mn 120 mg, Zn 100 mg, Cu 15 mg, I 1.2 mg, Se 0.3 mg, fitase 500 FTU. View Large Table 1. Composition and nutrient content of diets.   Starter  Grower  Finisher    (1–21 d)  (22–35 d)  (36–42 d)  Ingredient, g/Kg   Wheat  26.73  29.19  30.66   Maize  30.00  30.00  30.00   Soybean meal  32.50  28.20  25.33   Canola seeds  5.00  6.00  7.00   Soybean oil  2.10  1.33  1.80   Lard  -  2.00  2.50   NaCl  0.30  0.30  0.28   Mel stern  1.09  0.95  0.85   Phosphate 1-Calcium  1.15  0.94  0.63   DL-methionine  0.25  0.18  0.13   L-lysine  0.32  0.32  0.27   L-threonine  0.06  0.09  0.05   Vitamin—mineral premix1  0.50  0.50  0.50  Calculated composition   ME, MJ/kg  12.47  12.97  13.39   CP, % DM  22.00  20.50  19.50   Lysine, % DM  1.35  1.25  1.15   Methionine, % DM  0.57  0.49  0.43   Methionine + Cystine, % DM  0.95  0.85  0.78   Ca, % DM  0.90  0.80  0.70   P, % DM  0.40  0.35  0.28    Starter  Grower  Finisher    (1–21 d)  (22–35 d)  (36–42 d)  Ingredient, g/Kg   Wheat  26.73  29.19  30.66   Maize  30.00  30.00  30.00   Soybean meal  32.50  28.20  25.33   Canola seeds  5.00  6.00  7.00   Soybean oil  2.10  1.33  1.80   Lard  -  2.00  2.50   NaCl  0.30  0.30  0.28   Mel stern  1.09  0.95  0.85   Phosphate 1-Calcium  1.15  0.94  0.63   DL-methionine  0.25  0.18  0.13   L-lysine  0.32  0.32  0.27   L-threonine  0.06  0.09  0.05   Vitamin—mineral premix1  0.50  0.50  0.50  Calculated composition   ME, MJ/kg  12.47  12.97  13.39   CP, % DM  22.00  20.50  19.50   Lysine, % DM  1.35  1.25  1.15   Methionine, % DM  0.57  0.49  0.43   Methionine + Cystine, % DM  0.95  0.85  0.78   Ca, % DM  0.90  0.80  0.70   P, % DM  0.40  0.35  0.28  1Provided per kilogram of diets: vitamin A 12,500 IU, vitamin D3 - 4500 IU, vitamin E 45 mg, vitamin K3 3 mg, vitamin B1 3 mg, vitamin B2 6 mg, vitamin B6 4 mg, pantothenic acid 14 mg, nicotinic acid 50 mg, folic acid 1.75 mg, choline 1.6 g, vitamin B12 0.02 mg, biotin 0.2 mg, Fe 50 mg, Mn 120 mg, Zn 100 mg, Cu 15 mg, I 1.2 mg, Se 0.3 mg, fitase 500 FTU. View Large Slaughter Surveys At 42 d of age, 7 birds per cage were randomly chosen (42 birds per treatment), individually weighed (after a fasting period of 12 h), and slaughtered. After evisceration, the hot carcass weight (without head and feet) was recorded, and carcass yield was calculated. In addition, the breast muscle was removed from all carcasses and its percentage based on hot carcass weight was calculated. After 24 h of refrigeration, the Pectoral muscle (PM) pH was measured using a portable pH meter (FiveGo, Mettler-Toledo AG, Schwerzenbach, Switzerland) equipped with a penetrating glass electrode. At the same time, tri-stimulus color coordinates (lightness, L*; redness, a*; yellowness, b*) were detected on the PM using a Chroma Meter CR-300 (Minolta Italia S.p.A., Milan, Italy). The left PM was removed, vacuum packaged, and stored frozen (−20°C) for analyses. Histological Evaluation For histological examination, samples of the pectoral muscle (pectoralis superficialis muscle) from all carcasses were taken at 45 min after slaughter. Samples of muscles were frozen in liquid nitrogen (−196°C) and placed in cryovials in which they were stored until analysis. Frozen muscle samples were mounted on a specimen disc with a few drops of water (or tissue freezing medium). Mounted muscle tissue was cut into sections (10 μm thick) at around –25°C using a cryostat (Thermo Shandon microtome, Thermo Fisher Scientific, Runcorn, UK) and then placed on the glass slides and stained. Hematoxylin and eosin reaction were used to evaluate the muscle fiber diameter (Dubovitz et al., 1973). A Carl Zeiss microscope (Jena, Germany) equipped with a Toup View camera was used to record microscopic images on a computer disk. MultiScan v. 18.03 microscope imaging software (Computer Scanning Systems II Ltd., Warsaw, Poland) was used to calculate diameter of muscle fibers per 1 mm2. Muscle fiber diameter was measured according to a method of smallest diameters described by Brook (1970). In brief, the mean diameter of muscle fibers value per each group was calculated. Measurement of Muscle Cholesterol Cholesterol was extracted using the method of Maraschiello et al. (1996) and then quantified by HPLC. A Kontron HPLC (Kontron Instruments, Milan, Italy) model 535, equipped with a Kinetex 5μ C18 reverse-phase column (150 × 4.6 mm x 5 μm; Phenomenex, Torrance, CA), was used. The HPLC mobile phase consisted of acetonitrile: 2-propanol (55:45, vol/vol) at a flow rate of 1.0 mL/min. The detection wavelength was 210 nm. The quantitation of muscle cholesterol content was based on the external standard method using a pure cholesterol standard (Sigma, St. Louis, MO). Fatty Acid Composition of Muscle Lipid extraction from breast muscle samples was performed by Folch et al. (1957) method. Fatty acids were quantified as methyl esters (FAME) using a gas chromatograph an HRGC 5300 Fisons (Rodano, Milan, Italy), equipped with a flame ionization detector and a fused silica capillary Column (CP-Sil RTX 2330), 30 m x 0.25 mm x 0.5 μm film thickness (Restek, Bellefonte, PA, USA). Helium was used as carrier gas. The oven temperature program was 120°C for 1 min then increasing at 5°C/min up to 230°C where it was maintained for 20 min. The individual FA peaks were identified by comparison of retention times with those of FAME authentic standards run under the same operating conditions. Results were expressed as percentage of the total FA identified. To assess the nutritional implications, the n-6 FA/n-3 FA and the PUFA/SFA (P/S) ratios were calculated. Moreover, in order to evaluate the risk of atherosclerosis and the potential aggregation of blood platelets, respectively the atherogenic index (AI) and the thrombogenic index (TI) were calculated, according to the formulas suggested by Ulbricht and Southgate (1991). Statistical Analyses All data were evaluated by ANOVA (SPSS, 2010). Differences among groups were determined by contrasts (prebiotic contrast 1: 3 × μC—μDN—μBI − μRFO = 0; contrast 2: 2 × μRFO—μDN—μBI = 0; contrast 3: μBI − μDN = 0. Mode of prebiotics’ administration contrast 1: 3 × μC − μT1 − μT2 − μT3 = 0; contrast 2: 2 × μT3 − μT1 − μT2 = 0; contrast 3: μT2 − μT1 = 0. μ = overall mean). RESULTS AND DISCUSSION In Vivo and Post Mortem Performance Effects of the prebiotics DN, BI and RFO and 3 different routes of delivery (T1, T2 and T3) were tested. The impact on slaughter performance and fiber diameter of PM in broiler chickens is presented in Table 2. Compared with the C group, chickens from prebiotic groups were significantly (P < 0.01) heavier (RFO = +4.6%; DN = +2.8%; BI = +2.2%) and among them RFO chickens were the heaviest (P < 0.01) compared to DN and BI groups. Route of prebiotic administration (Table 2) had an effect on final BW, results showing higher (P < 0.01) values for all the prebiotic-treated groups (T1, T2 and T3) compared with C group. No differences (P > 0.05) between T1, T2 or T3 were observed. Neither carcass weight nor carcass yield (ranging from 69.7% to 71.7%) was affected by prebiotic treatment (P > 0.05), while breast muscle weight and yield were higher (P < 0.01) in DN, BI and RFO treated groups compared to C group. The prebiotics injected in ovo only (T1) or by combining in ovo route and administration in-water (T2) resulted in a significantly higher carcass weight as compared with the chickens of T3 group (P > 0.01). There were no differences (P > 0.05) observed between C group and T1, T2 and T3 groups; whereas T3 group had reduced carcass yield compared to the other groups (C, T1 and T2; P > 0.05). The chickens treated with prebiotics (T1, T2 and T3) had heavier (P < 0.01) breast muscle compared to the ones from C group. The breast yield was higher (P < 0.01) for T1, T2 and T3 groups in comparison to C group. The higher breast muscle yield was more evident (P < 0.05) in chickens that received prebiotics in water (T3) than those injected in ovo (T1) or by combining both in ovo and in-water routes (T2); while, T1 and T2 did not differ (P > 0.05). In addition, prebiotic-treated groups showed a greater (P < 0.05) thickness of breast muscle fibers, compared to C group, irrespective the way of administration. These results are partially in agreement with those of our recent study (Maiorano et al., 2017) conducted on 275,000 broiler chickens (Ross 308) under commercial conditions. The authors reported enhanced growth performance in birds at 42 d which were injected in ovo with Bi2tos and DiNovo. Moreover, those animals showed higher carcass weight, carcass yield and breast muscle weight. Similar results were obtained by Bednarczyk et al. (2016), who reported a significant increase in body weight gain during the first 3 wk of life upon injection in ovo with different prebiotics (Bi2tos, DiNovo and RFO) as compared with the control group. On the contrary, Berrocoso et al. (2017) did not find any influence on growth performance or slaughter yield in Cobb 500 broilers, which were in ovo injected with different RFO doses and slaughtered at 21 d of age. In addition, these authors observed that RFO had the potential of enhancing ileum mucosa morphology and improving immunity in the small intestine, which are indicators of improved gut health. In another study conducted on Ross broiler chickens, Pruszynska-Oszmalek et al. (2015) found that the in ovo injection of prebiotics (inulin and Bi2tos) and synbiotics (inulin + Lactococcus lactis subsp. lactis and Bi2tos + Lactococcus lactis subsp. cremoris) caused an elevation of the activity of pancreatic enzymes, which can explain the observed higher BW of treated chickens. The positive effect on growth performance exerted by prebiotics injected in ovo seems to be related to the early stimulation of intestinal microbiota development in the chicken gut; this is due to the ability of prebiotics to enhance lactobacilli and bifidobacteria populations, and these beneficial bacteria compete with harmful bacteria for colonization (Biggs and Parsons, 2008; Depeint et al., 2008; Tzortzis, 2009). Results obtained using different types and doses of prebiotics administered in feed are variable and not always comparable (Gaggìa et al., 2010). Nevertheless, it is suggested that such bioactive substances might be used in broiler diets since they do not interfere or positively affect the yield of the most commercially valuable edible cuts (Pelicano et al., 2005). Table 2. Effect of different prebiotics and mode of their administration on slaughter performance and fiber diameter of pectoral muscle in broiler chickens.   Group1      Treatment2        C  DN  BI  RFO  SEM  P-value  C  T1  T2  T3  SEM  P-value  Final BW, g  2651.7C  2726.1B  2709.4B  2774.4A  0.01  0.001  2651.7B  2748.9A  2736.1A  2725.0A  0.03  0.008  Carcass weight, g  1900.0  1917.1  1910.3  1935.1  9.92  0.713  1900.0A,B  1958.0A  1936.0A  1871.9B  7.92  0.004  Carcass yield, g  71.67  70.32  70.45  69.66  0.32  0.362  71.67A  71.17A  70.65A  68.71B  0.12  0.005  Breast weight, g  579.8B  638.3A  618.9A  625.6A  5.34  0.024  579.8B  632.4A  619.8A  630.7A  6.34  0.040  Breast yield, %  30.7B  33.3A  32.6A  32.5A  0.24  0.023  30.7C  32.3B  32.3B  33.7A  0.27  0.002  Fiber diameter, μm  47.37b  54.02a  52.23a  54.69a  0.85  0.097  47.37b  54.56a  50.98a  55.40a  0.83  0.020    Group1      Treatment2        C  DN  BI  RFO  SEM  P-value  C  T1  T2  T3  SEM  P-value  Final BW, g  2651.7C  2726.1B  2709.4B  2774.4A  0.01  0.001  2651.7B  2748.9A  2736.1A  2725.0A  0.03  0.008  Carcass weight, g  1900.0  1917.1  1910.3  1935.1  9.92  0.713  1900.0A,B  1958.0A  1936.0A  1871.9B  7.92  0.004  Carcass yield, g  71.67  70.32  70.45  69.66  0.32  0.362  71.67A  71.17A  70.65A  68.71B  0.12  0.005  Breast weight, g  579.8B  638.3A  618.9A  625.6A  5.34  0.024  579.8B  632.4A  619.8A  630.7A  6.34  0.040  Breast yield, %  30.7B  33.3A  32.6A  32.5A  0.24  0.023  30.7C  32.3B  32.3B  33.7A  0.27  0.002  Fiber diameter, μm  47.37b  54.02a  52.23a  54.69a  0.85  0.097  47.37b  54.56a  50.98a  55.40a  0.83  0.020  1Group: C = Control, in ovo injection of physiological saline; DN = DiNovo; BI = Bi2tos; RFO = raffinose family oligosaccharides. 2Treatment: C = Control, in ovo injection of physiological saline; T1 = in ovo; T2 = in ovo + in-water; T3 = in-water. Means in a row with different letters are significantly different at:A-CP < 0.01;a-bP < 0.05. View Large Table 2. Effect of different prebiotics and mode of their administration on slaughter performance and fiber diameter of pectoral muscle in broiler chickens.   Group1      Treatment2        C  DN  BI  RFO  SEM  P-value  C  T1  T2  T3  SEM  P-value  Final BW, g  2651.7C  2726.1B  2709.4B  2774.4A  0.01  0.001  2651.7B  2748.9A  2736.1A  2725.0A  0.03  0.008  Carcass weight, g  1900.0  1917.1  1910.3  1935.1  9.92  0.713  1900.0A,B  1958.0A  1936.0A  1871.9B  7.92  0.004  Carcass yield, g  71.67  70.32  70.45  69.66  0.32  0.362  71.67A  71.17A  70.65A  68.71B  0.12  0.005  Breast weight, g  579.8B  638.3A  618.9A  625.6A  5.34  0.024  579.8B  632.4A  619.8A  630.7A  6.34  0.040  Breast yield, %  30.7B  33.3A  32.6A  32.5A  0.24  0.023  30.7C  32.3B  32.3B  33.7A  0.27  0.002  Fiber diameter, μm  47.37b  54.02a  52.23a  54.69a  0.85  0.097  47.37b  54.56a  50.98a  55.40a  0.83  0.020    Group1      Treatment2        C  DN  BI  RFO  SEM  P-value  C  T1  T2  T3  SEM  P-value  Final BW, g  2651.7C  2726.1B  2709.4B  2774.4A  0.01  0.001  2651.7B  2748.9A  2736.1A  2725.0A  0.03  0.008  Carcass weight, g  1900.0  1917.1  1910.3  1935.1  9.92  0.713  1900.0A,B  1958.0A  1936.0A  1871.9B  7.92  0.004  Carcass yield, g  71.67  70.32  70.45  69.66  0.32  0.362  71.67A  71.17A  70.65A  68.71B  0.12  0.005  Breast weight, g  579.8B  638.3A  618.9A  625.6A  5.34  0.024  579.8B  632.4A  619.8A  630.7A  6.34  0.040  Breast yield, %  30.7B  33.3A  32.6A  32.5A  0.24  0.023  30.7C  32.3B  32.3B  33.7A  0.27  0.002  Fiber diameter, μm  47.37b  54.02a  52.23a  54.69a  0.85  0.097  47.37b  54.56a  50.98a  55.40a  0.83  0.020  1Group: C = Control, in ovo injection of physiological saline; DN = DiNovo; BI = Bi2tos; RFO = raffinose family oligosaccharides. 2Treatment: C = Control, in ovo injection of physiological saline; T1 = in ovo; T2 = in ovo + in-water; T3 = in-water. Means in a row with different letters are significantly different at:A-CP < 0.01;a-bP < 0.05. View Large In this study, prebiotic administration had a positive impact on breast muscle weight which was also associated with a greater thickness (diameter) of breast muscle fibers as compared with the C group. In poultry meat production, muscle fiber properties play a key role in meat quantity and quality. In fact, it is reported that in chickens, selection for overall growth has been shown to induce greater muscle weight with simultaneous increases in fiber diameter and length (reviewed in Berri et al., 2007). Similar results were earlier obtained by Maiorano et al. (2012) who found a slightly greater thickness of muscle fiber in chickens, which were injected in ovo with RFO prebiotic. On the contrary, Maiorano et al. (2017) found that prebiotic treated groups (Bi2tos and DiNovo) were characterized by slightly thinner muscle fiber compared to control group. The reduced thickness of the fibers might be considered an indicator of fibrillarity and a delicate structure of the meat, being beneficial to meat quality from the consumer point of view (Maiorano et al., 2017). pH and Color Results of the effect of the 3 prebiotics and way of their administration on muscle pH and color are presented in Table 3. pH is one of the most important qualitative attribute of meat that has a central role in determining the protein behavior both in fresh and processed meat products (Lonergan, 2008) and it is also an important contributing factor to meat quality expressed as tenderness, color, and storage life (Van Laack and Lane, 2000). Type of prebiotics used did not influenced pH24, a finding in accord with the results of Maiorano et al. (2012). Whereas, Sang-Oh and Byung-Sung (2011) noted that dietary inuloprebiotic (250 g ton−1) reduced significantly pH of chicken meat. On the contrary, route of prebiotic administration affected ultimate meat pH, in fact it was higher (P < 0.01) in T2 group compared to T3, intermediate values were found for C and T1 groups (P > 0.05). The pH values observed in the present study (ranging from 5.76 and 5.87) fully fit within the pH range accepted for commercial poultry meat (Haščík et al., 2015). Table 3. Effect of different prebiotics and mode of their administration on meat quality traits of broiler chickens.   Group1      Treatment2        C  DN  BI  RFO  SEM  P-value  C  T1  T2  T3  SEM  P-value  pH24  5.86  5.81  5.84  5.79  0.01  0.350  5.86A,B  5.82A,B  5.87A  5.76B  0.02  0.001  Color3, 24 h post-mortem   L*  50.01  50.93  48.70  51.30  0.37  0.129  50.01  51.09  50.20  49.64  0.39  0.496   a*  3.90a  3.06b  3.35b  3.00b  0.10  0.047  3.90a  2.85b  3.23a,b  3.33a,b  0.12  0.036   b*  4.11  3.50  4.47  4.50  0.19  0.155  4.11  4.01  4.16  4.30  0.21  0.952    Group1      Treatment2        C  DN  BI  RFO  SEM  P-value  C  T1  T2  T3  SEM  P-value  pH24  5.86  5.81  5.84  5.79  0.01  0.350  5.86A,B  5.82A,B  5.87A  5.76B  0.02  0.001  Color3, 24 h post-mortem   L*  50.01  50.93  48.70  51.30  0.37  0.129  50.01  51.09  50.20  49.64  0.39  0.496   a*  3.90a  3.06b  3.35b  3.00b  0.10  0.047  3.90a  2.85b  3.23a,b  3.33a,b  0.12  0.036   b*  4.11  3.50  4.47  4.50  0.19  0.155  4.11  4.01  4.16  4.30  0.21  0.952  1Group: C = Control, in ovo injection of physiological saline; DN = DiNovo; BI = Bi2tos; RFO = raffinose family oligosaccharides. 2Treatment: C = Control, in ovo injection of physiological saline; T1 = in ovo; T2 = in ovo + in-water; T3 = in-water. 3Color: L* = lightness; a* = redness index; b* = yellow index. Means in a row with different letters are significantly different at:A-BP < 0.01;a-bP < 0.05. View Large Table 3. Effect of different prebiotics and mode of their administration on meat quality traits of broiler chickens.   Group1      Treatment2        C  DN  BI  RFO  SEM  P-value  C  T1  T2  T3  SEM  P-value  pH24  5.86  5.81  5.84  5.79  0.01  0.350  5.86A,B  5.82A,B  5.87A  5.76B  0.02  0.001  Color3, 24 h post-mortem   L*  50.01  50.93  48.70  51.30  0.37  0.129  50.01  51.09  50.20  49.64  0.39  0.496   a*  3.90a  3.06b  3.35b  3.00b  0.10  0.047  3.90a  2.85b  3.23a,b  3.33a,b  0.12  0.036   b*  4.11  3.50  4.47  4.50  0.19  0.155  4.11  4.01  4.16  4.30  0.21  0.952    Group1      Treatment2        C  DN  BI  RFO  SEM  P-value  C  T1  T2  T3  SEM  P-value  pH24  5.86  5.81  5.84  5.79  0.01  0.350  5.86A,B  5.82A,B  5.87A  5.76B  0.02  0.001  Color3, 24 h post-mortem   L*  50.01  50.93  48.70  51.30  0.37  0.129  50.01  51.09  50.20  49.64  0.39  0.496   a*  3.90a  3.06b  3.35b  3.00b  0.10  0.047  3.90a  2.85b  3.23a,b  3.33a,b  0.12  0.036   b*  4.11  3.50  4.47  4.50  0.19  0.155  4.11  4.01  4.16  4.30  0.21  0.952  1Group: C = Control, in ovo injection of physiological saline; DN = DiNovo; BI = Bi2tos; RFO = raffinose family oligosaccharides. 2Treatment: C = Control, in ovo injection of physiological saline; T1 = in ovo; T2 = in ovo + in-water; T3 = in-water. 3Color: L* = lightness; a* = redness index; b* = yellow index. Means in a row with different letters are significantly different at:A-BP < 0.01;a-bP < 0.05. View Large Here, all the tested prebiotics and different routes of prebiotic delivery significantly affected redness (a*) of the fillets. In fact, the redness index (a*) was reduced (P < 0.05) in meat from prebiotics groups (DN, BI an RFO) compared to that of C group and in T1 compared to C (P < 0.05). This is likely to be related to the muscle-fiber types (Zhao et al., 2012). The other color parameters (L* and b*) were similar (P > 0.05) between the experimental groups. However, the observed color coordinates fit within the range which is accepted for good chicken meat appearance. Therefore, different prebiotics might be used since they did not negatively affect meat color, which is an extremely important parameter influencing consumer decisions. Cholesterol Content and Fatty Acid Composition All prebiotics (DN, BI and RFO) did not affect cholesterol content (Table 4), irrespective of route of delivery (T1, T2 or T3), as compared with the C group (P < 0.05). Also Maiorano et al. (2012) found that prebiotic did not affect breast muscle cholesterol content. On the contrary, Pilarski et al. (2005) reported that in ovo application of fructooligosaccharides in Hybro G broiler breeder eggs caused a decrease of breast muscle cholesterol in comparison with the control group. The cholesterol values found in the present study (ranging from 46.74 to 49.57 mg/100 g) are similar than those reported by Pilarski et al. (2005) in breast muscle of 42-d-old broiler chickens (ranging from 49.3 to 54.7 mg/100 g) and lower (- 50%) than that reported by Maiorano et al. (2012). Cholesterol content in broiler meat can be altered by various factors (composition of diet, age, sex; Wang et al., 2006) as well as the use of different methodologies for cholesterol quantification or for sampling (Bragagnolo and Rodriguez-Amaya, 2002). The FA proportion of meat is considered an important index for meat quality. Broiler fat is characterized by a significant amount of monounsaturated fatty acids (MUFA), and, in comparison with red meat, substantial amounts of polyunsaturated fats, especially the n-6 linoleic acid and arachidonic acid. Moreover, it may represent an important source of long-chain n-3 fatty acids (Hibbeln et al., 2006; Attia et al., 2017). The FA composition of breast meat is shown in Table 4 for prebiotics and route of their administration. Taking into account the general FA profile, total MUFA were the most abundant FA (ranging from 32.83 to 36.86%), followed in descending order by saturated fatty acids (SFA) (ranging from 34.55 to 35.67%) and polyunsaturated fatty acids (PUFA) (ranging from 28.59 to 32.13%). To our knowledge, limited information is available in literature on the effect of prebiotics on modification in the FA pattern of lipids in chicken meat. Whereas, there is no available information regarding the potential effect of in ovo injection of different type of prebiotics using different routes of delivery (in ovo vs. in-water vs. in ovo and in-water combined) in chicken breast muscle; therefore, this subject should be considered in new investigations. Total SFA in breast muscle were higher (P < 0.01) in prebiotic groups than in C group, reflecting the trends of C18:0 (stearic) and C20:0 (arachidic). No statistically significant differences were found among the 3 prebiotic groups. Palmitic acid (C16:0), instead, was similar (P > 0.05) among the groups. The total MUFA content of the breast samples decreased (P < 0.01) in prebiotic groups compared to C group; it was mainly in the form of oleic acid (C18:1 n9) that was lower (P < 0.01) in prebiotic groups compared to C group. The same trend (P < 0.01) was found for palmitoleic acid (C16:1) and for C18:1 trans, even if this latter was present in very small amount. The total PUFA content was higher (P < 0.01) in all prebiotic groups compared to C group. The precursor of the n-6 family, the linoleic acid (C18:2), quantitatively the most concentrated n-6 PUFA, was not affected (P > 0.05) by the prebiotic treatment; while significant differences (P < 0.01) in the other n-6 LC-PUFA were observed among groups. The γ-linolenic acid (C18:3 n-6) was higher (P < 0.01) in all prebiotic groups compared to C group; while, meat from prebiotic groups displayed a lower (P < 0.01) content of arachidonic acid (C20:4 n-6). In general, the total content of n-6 PUFA was lower (P < 0.01) in prebiotic groups compared to C group. On the contrary, the total content of n-3 PUFA was about 2 fold higher (P < 0.01) in prebiotic groups compared to C group. The precursors of the n-3 family, α-linolenic acid (C18:3), significantly increased (P < 0.01) with prebiotic administration. All n-3 LC-PUFA were higher (P < 0.01) in all prebiotic groups compared to C group, except for the eicosapentaenoic acid (C20:5) that was lower (P < 0.01) in prebiotic groups compared to C. Very small amount (less than 1%) of conjugated linoleic acid (C18:2cis-9, trans-11 and C18:2 trans-10, cis-12) was detected in breast samples. The n-6/n-3 ratio was favourably lower (P < 0.01) in prebiotic-treated groups compared to C group. Route of prebiotic administration had also a marked effect on meat FA profile (Table 4). T2 and T3 increased (P < 0.05) the SFA content in breast muscle compared to C. No significant effect (P > 0.05) was found for the main SFA: palmitic (C16:0), stearic (C18:0) and myristic (C14:0) acids; only the content of arachidic acid (C20:0) was higher (P < 0.01) in all treated groups. The MUFA content was lower (P < 0.01) in prebiotic-treated groups compared to C group, reflecting the trends of oleic acid (P < 0.01), palmitoleic acid (P < 0.01) and C18:1 trans (P < 0.01), even if the latter was present in negligible amount in treated groups. The PUFA content was higher (P < 0.01) in all treated groups (T1, T2 and T3) compared to C group. Also for the route of prebiotic administration, the linoleic acid was not affected (P > 0.05) by the treatment; while all routes of prebiotic administration increased (P < 0.01) the γ-linolenic acid and decreased (P < 0.01) the arachidonic acid compared to C group. As a consequence, the total content of n-6 PUFA was lower (P < 0.01) in prebiotic-treated groups compared to C group, with T2 lower than T1 (P < 0.05). The total content of n-3 PUFA was significantly higher in T1, T2 and T3 groups compared to C group, reflecting the trend (P < 0.01) of α-linolenic, docosapentaenoic (C22:5) and docosahexaenoic (C22:6) acids. The eicosapentaenoic acid (C20:5) was higher (P < 0.01) in C group compared to the other groups; T1 and T2 showed similar (P > 0.05) values that were higher compared with the value of T3 group. Also for the route of prebiotic administration, the n-6/n-3 ratio was favourably lower (P < 0.01) in prebiotic-treated groups compared to C group. The P/S ratio was higher (P < 0.05) in prebiotic groups (DN, BI and RFO) compared to C group; in addition, T1 was higher than T2 (P < 0.05) and T3 similar (P > 0.05) to T1 and T2. Our results are inconsistent with those of Zhou et al. (2009), who found a greater concentration of total MUFA and lower amount of SFA in breast muscle of broiler chickens fed basal diet + 0.2% chitooligosaccharide (COS, oligosaccharide obtained by chemical and enzymatic hydrolysis of polychitosan) supplementation, but PUFA concentration was not affected by COS. The predominant SFA was palmitic acid (ranging from 22.29% to 22.90%). This acid is thought to increase cholesterol level together with lauric and myristic acid (Zock et al., 1994). Among the individual MUFA, the most abundant was the oleic acid (ranging from 32.33% to 34.17%). This finding is in agreement with levels found by other authors in chicken meat (Boschetti et al., 2016, fast growing birds; Alina et al., 2012). From a nutritional point of view, oleic acid plays a key role in human diet as involved in reducing lipaemia and consequently the risk of stroke (D’Alessandro et al., 2012). As general rule, poultry meat is characterized by the highest n-6/n-3 ratio compared to other meats, essentially due to the higher amount of n-6 FA than muscles of the other species (Rule et al., 2002; Wood et al., 2003). In fact, linoleic acid is the predominant essential FA in poultry and as a result the n-6 PUFA are the primary products found in tissue lipids. Consequently, the n-6/n-3 ratio in poultry meat is at a distance from the ideal value of 1 and above the recommended maximum of 4. The values found in the present study are within the ideal range of 1 to 4 and additionally birds of the prebiotic treated groups had improved values when compared with the control group, resulting in a positive impact on the dietetic value of meat. The P/S ratio has great nutritional implications also and it is taken as a measure of the propensity of the diet to influence the incidence of coronary disease (Wood et al., 2003). From a nutritional point of view, a higher P/S ratio is recommended, indeed it should be increased to above 0.4 (Wood et al., 2003). The P/S values observed in the present study are favourably high and in particular the prebiotic-treated groups showed a higher (P < 0.05) P/S ratio compared to C group. The AI and TI indexes represent criteria for evaluating the level and interrelation through which some FA may have atherogenic or thrombogenic properties, respectively. Only TI was significantly affected by prebiotic administration, being reduced in all prebiotic groups, compared to C group. The low AI and TI values found in the current study revealed good nutritional quality of the meat. Table 4. Effect of different prebiotics and mode of their administration on cholesterol (mg/100 g) and fatty acid composition (% of total fatty acids) of breast muscle from broiler chickens.   Group2      Treatment3      Item1  C  DN  BI  RFO  SEM  P-value  C  T1  T2  T3  SEM  P-value  Cholesterol  47.74  49.44  47.20  47.98  0.59  0.120  47.74  49.57  47.70  46.74  0.56  0.284  C14:0  1.19  1.07  1.16  1.16  0.03  0.608  1.19  1.11  1.15  1.12  0.04  0.890  C16:0  22.51  22.56  22.58  22.88  0.13  0.759  22.51  22.29  22.90  22.84  0.15  0.277  C16:1  1.43A  0.47B  0.47B  0.47B  0.05  0.001  1.43A  0.49B  0.47B  0.46B  0.04  0.001  C18:0  9.74b  9.83a,b  10.49a  10.37a  0.08  0.001  9.74  10.06  10.26  10.37  0.12  0.108  C18:1trans  1.26A  0.04B  0.03B  0.04B  0.04  0.001  1.26A  0.04B  0.03B  0.04B  0.05  0.001  C18:1cis-9  34.17A  32.47B  32.55B  32.54B  0.12  0.001  34.17A  32.64B  32.58B  32.33B  0.11  0.001  C18:2 n-6  19.11  19.52  19.16  18.84  0.12  0.224  19.11  19.38  18.92  19.22  0.14  0.542  C18:2cis-9, trans-11  0.22b  0.24a  0.26a  0.28a  0.01  0.046  0.22b  0.25c  0.23c,b  0.28a  0.01  0.037  C18:2trans-10, cis-12  0.21  0.23  0.26  0.27  0.01  0.225  0.21  0.27  0.24  0.26  0.01  0.468  C18:3 n-6  1.36B  1.90A  1.80A  1.82A  0.04  0.005  1.36B  1.90A  1.78A  1.84A  0.05  0.004  C18:3 n-3  1.77B  2.26A  2.25A  2.24A  0.03  0.001  1.77B  2.26A  2.26A  2.24A  0.02  0.001  C20:0  1.11B  1.43A,a  1.30A,b  1.26A,b  0.02  0.001  1.11B  1.38A  1.34A  1.28A  0.01  0.005  C20:4 n-6  2.33A  0.63B  0.60B  0.64B  0.05  0.001  2.33A  0.65B  0.58B  0.65B  0.06  0.001  C20:5 n-3  1.46A  1.43B  1.35B  1.26C  0.02  0.003  1.46A  1.42B  1.35B  1.27C  0.02  0.016  C22:5 n-3  0.75B  2.86A  2.90A  2.95A  0.07  0.001  0.75B  2.85A  2.92A  2.93A  0.08  0.001  C22:6 n-3  1.38B  3.06A  2.84A  2.98A  0.07  0.001  1.38B  3.01A  2.99A  2.87A  0.08  0.001  ΣSFA  34.55B  34.89A  35.53A  35.67A  0.15  0.068  34.55b  34.84a,b  35.65a  35.60a  0.13  0.046  ΣMUFA  36.86A  32.98B  33.05B  33.05B  0.17  0.001  36.86A  33.17B  33.08B  32.83B  0.18  0.001  ΣPUFA  28.59B  32.13A  31.42A  31.28A  0.19  0.001  28.59B  31.99A  31.27A  31.56A  0.120  0.001  Total n-6  22.81A  22.05B  21.56B  21.31B  0.12  0.005  22.81A  21.93B,a  21.28B,b  21.71B,a,b  0.14  0.009  Total n-3  5.36B  9.61A  9.34A  9.43A  0.16  0.001  5.36B  9.54A  9.52A  9.30A  0.14  0.001  n-6/n-3  4.25A  2.29B  2.31B  2.26B  0.06  0.001  4.25A  2.30B  2.24B  2.33B  0.08  0.001  P/S  0.83b  0.92a  0.88a  0.88a  0.01  0.006  0.83b  0.92a  0.88c  0.89a,c  0.01  0.012  AI  0.42  0.42  0.43  0.43  0.02  0.429  0.42  0.41  0.43  0.43  0.03  0.291  TI  0.73A  0.59B  0.62B  0.62B  0.01  0.001  0.73A  0.59B  0.61B  0.62B  0.01  0.001    Group2      Treatment3      Item1  C  DN  BI  RFO  SEM  P-value  C  T1  T2  T3  SEM  P-value  Cholesterol  47.74  49.44  47.20  47.98  0.59  0.120  47.74  49.57  47.70  46.74  0.56  0.284  C14:0  1.19  1.07  1.16  1.16  0.03  0.608  1.19  1.11  1.15  1.12  0.04  0.890  C16:0  22.51  22.56  22.58  22.88  0.13  0.759  22.51  22.29  22.90  22.84  0.15  0.277  C16:1  1.43A  0.47B  0.47B  0.47B  0.05  0.001  1.43A  0.49B  0.47B  0.46B  0.04  0.001  C18:0  9.74b  9.83a,b  10.49a  10.37a  0.08  0.001  9.74  10.06  10.26  10.37  0.12  0.108  C18:1trans  1.26A  0.04B  0.03B  0.04B  0.04  0.001  1.26A  0.04B  0.03B  0.04B  0.05  0.001  C18:1cis-9  34.17A  32.47B  32.55B  32.54B  0.12  0.001  34.17A  32.64B  32.58B  32.33B  0.11  0.001  C18:2 n-6  19.11  19.52  19.16  18.84  0.12  0.224  19.11  19.38  18.92  19.22  0.14  0.542  C18:2cis-9, trans-11  0.22b  0.24a  0.26a  0.28a  0.01  0.046  0.22b  0.25c  0.23c,b  0.28a  0.01  0.037  C18:2trans-10, cis-12  0.21  0.23  0.26  0.27  0.01  0.225  0.21  0.27  0.24  0.26  0.01  0.468  C18:3 n-6  1.36B  1.90A  1.80A  1.82A  0.04  0.005  1.36B  1.90A  1.78A  1.84A  0.05  0.004  C18:3 n-3  1.77B  2.26A  2.25A  2.24A  0.03  0.001  1.77B  2.26A  2.26A  2.24A  0.02  0.001  C20:0  1.11B  1.43A,a  1.30A,b  1.26A,b  0.02  0.001  1.11B  1.38A  1.34A  1.28A  0.01  0.005  C20:4 n-6  2.33A  0.63B  0.60B  0.64B  0.05  0.001  2.33A  0.65B  0.58B  0.65B  0.06  0.001  C20:5 n-3  1.46A  1.43B  1.35B  1.26C  0.02  0.003  1.46A  1.42B  1.35B  1.27C  0.02  0.016  C22:5 n-3  0.75B  2.86A  2.90A  2.95A  0.07  0.001  0.75B  2.85A  2.92A  2.93A  0.08  0.001  C22:6 n-3  1.38B  3.06A  2.84A  2.98A  0.07  0.001  1.38B  3.01A  2.99A  2.87A  0.08  0.001  ΣSFA  34.55B  34.89A  35.53A  35.67A  0.15  0.068  34.55b  34.84a,b  35.65a  35.60a  0.13  0.046  ΣMUFA  36.86A  32.98B  33.05B  33.05B  0.17  0.001  36.86A  33.17B  33.08B  32.83B  0.18  0.001  ΣPUFA  28.59B  32.13A  31.42A  31.28A  0.19  0.001  28.59B  31.99A  31.27A  31.56A  0.120  0.001  Total n-6  22.81A  22.05B  21.56B  21.31B  0.12  0.005  22.81A  21.93B,a  21.28B,b  21.71B,a,b  0.14  0.009  Total n-3  5.36B  9.61A  9.34A  9.43A  0.16  0.001  5.36B  9.54A  9.52A  9.30A  0.14  0.001  n-6/n-3  4.25A  2.29B  2.31B  2.26B  0.06  0.001  4.25A  2.30B  2.24B  2.33B  0.08  0.001  P/S  0.83b  0.92a  0.88a  0.88a  0.01  0.006  0.83b  0.92a  0.88c  0.89a,c  0.01  0.012  AI  0.42  0.42  0.43  0.43  0.02  0.429  0.42  0.41  0.43  0.43  0.03  0.291  TI  0.73A  0.59B  0.62B  0.62B  0.01  0.001  0.73A  0.59B  0.61B  0.62B  0.01  0.001  1SFA = saturated fatty acids; MUFA = monounsaturated fatty acids; PUFA = polyunsaturated fatty acids; P/S = PUFA/SFA ratio; AI = atherogenic index; TI = thrombogenic index. 2Group: C = Control, in ovo injection of physiological saline; DN = DiNovo; BI = Bi2tos; RFO = raffinose family oligosaccharides. 3Treatment: C = Control, in ovo injection of physiological saline; T1 = in ovo; T2 = in ovo + in-water; T3 = in-water. Means in a row with different letters are significantly different at:A-CP < 0.01;a-cP < 0.05. View Large Table 4. Effect of different prebiotics and mode of their administration on cholesterol (mg/100 g) and fatty acid composition (% of total fatty acids) of breast muscle from broiler chickens.   Group2      Treatment3      Item1  C  DN  BI  RFO  SEM  P-value  C  T1  T2  T3  SEM  P-value  Cholesterol  47.74  49.44  47.20  47.98  0.59  0.120  47.74  49.57  47.70  46.74  0.56  0.284  C14:0  1.19  1.07  1.16  1.16  0.03  0.608  1.19  1.11  1.15  1.12  0.04  0.890  C16:0  22.51  22.56  22.58  22.88  0.13  0.759  22.51  22.29  22.90  22.84  0.15  0.277  C16:1  1.43A  0.47B  0.47B  0.47B  0.05  0.001  1.43A  0.49B  0.47B  0.46B  0.04  0.001  C18:0  9.74b  9.83a,b  10.49a  10.37a  0.08  0.001  9.74  10.06  10.26  10.37  0.12  0.108  C18:1trans  1.26A  0.04B  0.03B  0.04B  0.04  0.001  1.26A  0.04B  0.03B  0.04B  0.05  0.001  C18:1cis-9  34.17A  32.47B  32.55B  32.54B  0.12  0.001  34.17A  32.64B  32.58B  32.33B  0.11  0.001  C18:2 n-6  19.11  19.52  19.16  18.84  0.12  0.224  19.11  19.38  18.92  19.22  0.14  0.542  C18:2cis-9, trans-11  0.22b  0.24a  0.26a  0.28a  0.01  0.046  0.22b  0.25c  0.23c,b  0.28a  0.01  0.037  C18:2trans-10, cis-12  0.21  0.23  0.26  0.27  0.01  0.225  0.21  0.27  0.24  0.26  0.01  0.468  C18:3 n-6  1.36B  1.90A  1.80A  1.82A  0.04  0.005  1.36B  1.90A  1.78A  1.84A  0.05  0.004  C18:3 n-3  1.77B  2.26A  2.25A  2.24A  0.03  0.001  1.77B  2.26A  2.26A  2.24A  0.02  0.001  C20:0  1.11B  1.43A,a  1.30A,b  1.26A,b  0.02  0.001  1.11B  1.38A  1.34A  1.28A  0.01  0.005  C20:4 n-6  2.33A  0.63B  0.60B  0.64B  0.05  0.001  2.33A  0.65B  0.58B  0.65B  0.06  0.001  C20:5 n-3  1.46A  1.43B  1.35B  1.26C  0.02  0.003  1.46A  1.42B  1.35B  1.27C  0.02  0.016  C22:5 n-3  0.75B  2.86A  2.90A  2.95A  0.07  0.001  0.75B  2.85A  2.92A  2.93A  0.08  0.001  C22:6 n-3  1.38B  3.06A  2.84A  2.98A  0.07  0.001  1.38B  3.01A  2.99A  2.87A  0.08  0.001  ΣSFA  34.55B  34.89A  35.53A  35.67A  0.15  0.068  34.55b  34.84a,b  35.65a  35.60a  0.13  0.046  ΣMUFA  36.86A  32.98B  33.05B  33.05B  0.17  0.001  36.86A  33.17B  33.08B  32.83B  0.18  0.001  ΣPUFA  28.59B  32.13A  31.42A  31.28A  0.19  0.001  28.59B  31.99A  31.27A  31.56A  0.120  0.001  Total n-6  22.81A  22.05B  21.56B  21.31B  0.12  0.005  22.81A  21.93B,a  21.28B,b  21.71B,a,b  0.14  0.009  Total n-3  5.36B  9.61A  9.34A  9.43A  0.16  0.001  5.36B  9.54A  9.52A  9.30A  0.14  0.001  n-6/n-3  4.25A  2.29B  2.31B  2.26B  0.06  0.001  4.25A  2.30B  2.24B  2.33B  0.08  0.001  P/S  0.83b  0.92a  0.88a  0.88a  0.01  0.006  0.83b  0.92a  0.88c  0.89a,c  0.01  0.012  AI  0.42  0.42  0.43  0.43  0.02  0.429  0.42  0.41  0.43  0.43  0.03  0.291  TI  0.73A  0.59B  0.62B  0.62B  0.01  0.001  0.73A  0.59B  0.61B  0.62B  0.01  0.001    Group2      Treatment3      Item1  C  DN  BI  RFO  SEM  P-value  C  T1  T2  T3  SEM  P-value  Cholesterol  47.74  49.44  47.20  47.98  0.59  0.120  47.74  49.57  47.70  46.74  0.56  0.284  C14:0  1.19  1.07  1.16  1.16  0.03  0.608  1.19  1.11  1.15  1.12  0.04  0.890  C16:0  22.51  22.56  22.58  22.88  0.13  0.759  22.51  22.29  22.90  22.84  0.15  0.277  C16:1  1.43A  0.47B  0.47B  0.47B  0.05  0.001  1.43A  0.49B  0.47B  0.46B  0.04  0.001  C18:0  9.74b  9.83a,b  10.49a  10.37a  0.08  0.001  9.74  10.06  10.26  10.37  0.12  0.108  C18:1trans  1.26A  0.04B  0.03B  0.04B  0.04  0.001  1.26A  0.04B  0.03B  0.04B  0.05  0.001  C18:1cis-9  34.17A  32.47B  32.55B  32.54B  0.12  0.001  34.17A  32.64B  32.58B  32.33B  0.11  0.001  C18:2 n-6  19.11  19.52  19.16  18.84  0.12  0.224  19.11  19.38  18.92  19.22  0.14  0.542  C18:2cis-9, trans-11  0.22b  0.24a  0.26a  0.28a  0.01  0.046  0.22b  0.25c  0.23c,b  0.28a  0.01  0.037  C18:2trans-10, cis-12  0.21  0.23  0.26  0.27  0.01  0.225  0.21  0.27  0.24  0.26  0.01  0.468  C18:3 n-6  1.36B  1.90A  1.80A  1.82A  0.04  0.005  1.36B  1.90A  1.78A  1.84A  0.05  0.004  C18:3 n-3  1.77B  2.26A  2.25A  2.24A  0.03  0.001  1.77B  2.26A  2.26A  2.24A  0.02  0.001  C20:0  1.11B  1.43A,a  1.30A,b  1.26A,b  0.02  0.001  1.11B  1.38A  1.34A  1.28A  0.01  0.005  C20:4 n-6  2.33A  0.63B  0.60B  0.64B  0.05  0.001  2.33A  0.65B  0.58B  0.65B  0.06  0.001  C20:5 n-3  1.46A  1.43B  1.35B  1.26C  0.02  0.003  1.46A  1.42B  1.35B  1.27C  0.02  0.016  C22:5 n-3  0.75B  2.86A  2.90A  2.95A  0.07  0.001  0.75B  2.85A  2.92A  2.93A  0.08  0.001  C22:6 n-3  1.38B  3.06A  2.84A  2.98A  0.07  0.001  1.38B  3.01A  2.99A  2.87A  0.08  0.001  ΣSFA  34.55B  34.89A  35.53A  35.67A  0.15  0.068  34.55b  34.84a,b  35.65a  35.60a  0.13  0.046  ΣMUFA  36.86A  32.98B  33.05B  33.05B  0.17  0.001  36.86A  33.17B  33.08B  32.83B  0.18  0.001  ΣPUFA  28.59B  32.13A  31.42A  31.28A  0.19  0.001  28.59B  31.99A  31.27A  31.56A  0.120  0.001  Total n-6  22.81A  22.05B  21.56B  21.31B  0.12  0.005  22.81A  21.93B,a  21.28B,b  21.71B,a,b  0.14  0.009  Total n-3  5.36B  9.61A  9.34A  9.43A  0.16  0.001  5.36B  9.54A  9.52A  9.30A  0.14  0.001  n-6/n-3  4.25A  2.29B  2.31B  2.26B  0.06  0.001  4.25A  2.30B  2.24B  2.33B  0.08  0.001  P/S  0.83b  0.92a  0.88a  0.88a  0.01  0.006  0.83b  0.92a  0.88c  0.89a,c  0.01  0.012  AI  0.42  0.42  0.43  0.43  0.02  0.429  0.42  0.41  0.43  0.43  0.03  0.291  TI  0.73A  0.59B  0.62B  0.62B  0.01  0.001  0.73A  0.59B  0.61B  0.62B  0.01  0.001  1SFA = saturated fatty acids; MUFA = monounsaturated fatty acids; PUFA = polyunsaturated fatty acids; P/S = PUFA/SFA ratio; AI = atherogenic index; TI = thrombogenic index. 2Group: C = Control, in ovo injection of physiological saline; DN = DiNovo; BI = Bi2tos; RFO = raffinose family oligosaccharides. 3Treatment: C = Control, in ovo injection of physiological saline; T1 = in ovo; T2 = in ovo + in-water; T3 = in-water. Means in a row with different letters are significantly different at:A-CP < 0.01;a-cP < 0.05. View Large In summary, the final BW of broiler chickens was increased upon delivery of prebiotics (DN, BI and RFO) irrespective of the method used. The prebiotic which showed the most significant improvement of performance traits was RFO. Each of the prebiotics which were injected in ovo (T1) or delivered by combining in ovo and in-water routes (T2) have resulted in an increase of carcass weight as compared with prebiotics given only in water (T3). Irrespective of delivery method used, prebiotics showed a positive impact on breast muscle weight and yield, which was also associated with a greater thickness (diameter) of breast muscle fibers. The redness (a*) of fillets was decreased upon delivery of prebiotics irrespective of the method used. As for FA composition, the intramuscular SFA were higher in the meat from prebiotic groups, while MUFA were lower. Moreover, PUFA and n-3 FA were higher in the meat from prebiotic groups, displaying more favorable indexes for human health (n-6/n-3, P/S, TI). Meat cholesterol content was not affected by any prebiotic treatment. In conclusion, even if the calculated increase of final body weight of the birds obtained with prebiotics seems minor in experimental scale, the economic impact could be relevant if one considers the high number of chickens reared under commercial conditions. In addition, according to these data, we can conclude that prebiotics support good performance results regardless of the mode of administration. It should be considered that the dose of a selected bioactive used for the in ovo injection is 10 times less than of dose which is added in water. This seems of an apparent economic benefit if one takes into account cost of routine prebiotic supplementation. Nevertheless, further research is required to increase the knowledge on prebiotic composition and elaborate the optimal bioactive formulation and route of their delivery, with proven strong effects on poultry meat quality; with a special emphasis placed on FA profile and other health promoting meat indices. Those further optimization trials must be performed in experimental and field conditions. ACKNOWLEDGMENTS The research leading to these results has received funding from the European Union's Seventh Framework Programme managed by REA Research Executive Agency http://ec.europa.eu/research/rea (FP7/2007–2013) under grant agreement number: 315198. This research was undertaken as part of a project entitled “Thrive-Rite: Natural Compounds to enhance Productivity, Quality and Health in Intensive Farming Systems”. Further details are provided on the consortium's website: www.thriverite.eu and the EU Commission's webpage: http://cordis.europa.eu/project/rcn/104395_en.html). 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Prebiotics offered to broiler chicken exert positive effect on meat quality traits irrespective of delivery route

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Oxford University Press
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© 2018 Poultry Science Association Inc.
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0032-5791
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10.3382/ps/pey149
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Abstract

ABSTRACT Elimination of antibiotic growth promoters from poultry production has encouraged intensive search for relevant alternatives. Prebiotics are proposed as efficient replacements to stimulate colonization/expansion of beneficial microflora in chickens. The aim of this study was to deepen the knowledge on the effect of prebiotic administration on slaughter performance and meat quality traits of broiler chickens by evaluating different routes of their delivery (in ovo vs. in-water vs. in ovo + in-water). At d 12 of incubation, 1,500 eggs (Ross 308) containing viable embryos were randomly allotted into 4 groups and injected in ovo with 0.2 mL solution containing: 3.5 mg/embryo BI (Bi2tos, trans-galactooligosaccharides); 0.88 mg/embryo DN (DiNovo, extract of Laminaria spp.); 1.9 mg/embryo RFO (raffinose family oligosaccharides) and 0.2 mL physiological saline (C). All prebiotics increased final BW compared to C group (P < 0.01), irrespective of delivery route. The prebiotics injected in ovo (T1) or in ovo combined with in-water supplementation (T2) increased carcass weight as compared with in-water group (T3), while T3 had the lowest carcass yield compared to the other groups. All prebiotics increased breast muscle weight and yield (P < 0.01), as well as fiber diameter (P < 0.05). Ultimate meat pH was lower (P < 0.01) in T3 than in T2 group. Meat from chickens treated with prebiotics showed a lower redness index, while lightness and yellowness were not affected by the treatments. Saturated fatty acid (SFA), polyunsaturated fatty acid (PUFA) and n-3 fatty acids contents were higher (P < 0.01), and monounsaturated fatty acid (MUFA) level was lower (P < 0.01) in prebiotic groups compared with C group. Nutritional indexes (n-6/n-3, PUFA/SFA ratio and thrombogenic index) displayed favorable human health-promoting values in the meat of chickens which were treated with prebiotics, irrespective of delivery route. Muscle cholesterol content was not affected by prebiotics. In conclusion, this study has shown that prebiotics can exert positive effects on growth of broiler chickens, carcass and meat quality traits, irrespective of delivery route. INTRODUCTION World production of poultry meat is expected to increase by over 181 million tonnes by 2050 (Alexandratos and Bruinsma, 2012). Such a raise in poultry production will likely be powered by continued genetic progress resulting from the work of primary breeders. However, a great number of scientific evidence suggests that the intense genetic selection is linked with the elevated incidence of histological and biochemical changes to muscle tissue that lead to different types of myopathies, which adversely affect the poultry meat industry (Petracci et al., 2015; Kuttappan et al., 2016; Baldi et al., 2018). In addition, new antibiotic-resistant bacteria are emerging and spreading globally, while known poultry pathogens like Campylobacter and Salmonella are still defined as a major food borne threat for consumers. In fact, European countries have implemented bans on the use of growth-promoting antibiotics, while in the USA the Center for Veterinary Medicine of the US FDA (Food and Drug Administration) recently issued a “Guidance for Industry”, which describes requirements for label claims and recommended restrictions on usage of antibiotics in food producing animals (FDA, 2012). Pre- and pro-biotics are proposed as one of the alternatives to antibiotic growth promoters, to prevent enteric diseases and improve performance in poultry. Prebiotics are non-digestible components of feed derived from sugars, including raffinose, galactooligosaccharides and β-glucans (Sobolewska et al., 2017). Recently, the prebiotic is defined as “a non-digestible compound that, through its metabolization by microorganisms in the gut, modulates composition and/or activity of the gut microbiota, thus conferring a beneficial physiological effect on the host” (Bindels et al., 2015). Numerous probiotic and prebiotic products are commercially available and are conventionally supplemented in-feed or in-water at first hours/days post hatching. There are substantial inconsistencies regarding efficacy of bioactives, which are found in the reported broiler chicken feeding trials (Waldroup et al., 2003; Bozkurt et al., 2009; Kim et al., 2011; Nabizadeh, 2012; Fowler et al., 2015). These discrepancies could be related to a number of conditions, which namely are: the physiological state of the bird, contaminated hatchery area, microbiota balance in the animal gut, disadvantageous interaction among various dietary additives (i.e., oligosaccharides, coccidiostatics, probiotics), different chemical structure and composition of the prebiotics used, mode of administration and duration of the treatment, doses of biological active substances applied as well as the amount of feed or water intake, including the water quality (Gaggìa et al., 2010; Huyghebaert et al., 2011; Nabil Alloui et al., 2013; Ducatelle et al., 2015; Bednarczyk et al., 2016; Sugiharto, 2016). Therefore, the major challenges are: (i) to observe the evident effect of prebiotics under fully controlled rearing condition, (ii) to optimize the method of prebiotic delivery. During the last years, we have tested different prebiotics (commercial type and in-house extracted) and their synergistic combinations with probiotics (synbiotics) in field and laboratory studies (Pilarski et al., 2005; Bednarczyk et al., 2011, 2016; Maiorano et al., 2012, 2017; Dunislawska et al., 2017). To be effective, these compounds have to be administered in fully controlled conditions, as early as possible. In our earlier study (Villaluenga et al., 2004) we have determined the 12th day of incubation for prebiotic injection into the air cell of the incubated egg, as the optimal time to provide stimulation of embryonic microflora with no harm done to the incubating eggs. At this time the chorioallantoic membrane is highly vascularized and allows transferring of the prebiotic from the air cell into the circulatory system and further to the developing intestine. Alike in the current trial, also in the previous one (Bednarczyk et al., 2016; Maiorano et al., 2017) we have offered to broiler chickens a broad range of bioactive formulations, including: i) commercially available prebiotics, such as DN (DiNovo, BioAtlantis Ltd., Tralee, Co. Kerry, Ireland), extract of Laminaria spp. containing laminarin and fucoidan and BI (Bi2tos, Clasado Ltd., Sliema, Malta), a non-digestive trans-galactooligosaccharides (GOS) from milk lactose digested with Bifidobacterium bifidum NCIMB 41,171; ii) in-house extracted prebiotic (RFO—raffinose family oligosaccharides) from lupin (Lupinus luteus) seeds (Ciesiolka et al., 2005). We hypothesized that the combination of different modes of prebiotic administration: injection in ovo (into the air chamber of the egg on the 12th d of incubation), followed by additional application of prebiotic in drinking water, during the first week of chicken rearing, may exhibit a synergistic effect not only on growth performance (Bednarczyk et al., 2016) but also on meat quality traits. Only few publications which report the effect of prebiotic administration on meat quality traits of broiler chickens can be found (Maiorano et al., 2012, 2017). None of them included the analysis of fatty acid (FA) composition. Some authors proved that prebiotics can alter lipid metabolism (Letexier et al., 2003) and enhance the ratio of polyunsaturated fatty acid (PUFA) to saturated fatty acid (SFA) in chicken meat (Zhou et al., 2009; Velasco et al., 2010), which is beneficial to human health. The aim of this study was to deepen the knowledge on the effect of prebiotic administration on slaughter performance and meat quality traits of broiler chicken by evaluating different routes of their delivery (in ovo vs. in-water vs. in ovo and in-water combined). MATERIALS AND METHODS Birds and Experimental Design Eggs (60 g average weight) were obtained from single 32-wk-old breeder flock (Ross 308) and incubated in a commercial broiler hatchery (Drobex-Agro Sp. z o.o., Solec Kujawski, Poland). Prior to the injection, the eggs were candled to select only the ones containing viable embryos. At d 12 of incubation, 1500 eggs containing viable embryos were randomly allotted into 4 experimental groups (375 eggs/group). Prebiotic solutions of each experimental group contained different doses of bioactives: 3.5 mg/embryo BI (Bi2tos, Clasado Ltd., Sliema, Malta); 0.88 mg/embryo DN (DiNovo, BioAtlantis Ltd., Tralee, Co. Kerry, Ireland); 1.9 mg/embryo RFO. The above mentioned prebiotic solutions were used to compare effects of different routes of delivery: (T1) in ovo injection, (T2) in ovo injection combined with in-water delivery, and (T3) in-water delivery. Control group (C) was injected in ovo with physiological saline only and did not receive any prebiotic in-water. As for in ovo treatments, the eggs were injected in ovo with 0.2 mL of selected prebiotic solution, which was deposited in the air cell followed by immediate sealing of the eggs. After hatching, the chicks were sexed and 600 males (42.0 ± 2 g average weight) were randomly assigned to 10 experimental groups (60 males/group): T1 (DN, BI and RFO), T2 (DN, BI and RFO), T3 (DN, BI and RFO) and C. Birds were grown to 42 d of age in collective cages (n = 6 replicate cages, 10 birds in each cage). Chicks from T1 group were raised without any additional supplementation with prebiotics in water. T2 and T3 groups were supplemented in-water with respective prebiotic (DN, BI or RFO) for the first 7 d of life. Those animals received 12 mL of the prebiotic dissolved in water per pen (20 mg of prebiotic/mL). Birds were reared according to the regulations and permission of the local Ethical Commission (decision No.22/2012 21.06.2012) and in accordance with the animal welfare recommendations of European Union directive 86/609/EEC, in an experimental poultry house Drobex-Agro (Solec Kujawski, Poland) that provided good husbandry conditions (e.g., stocking density, litter, ventilation). Animals were fed ad libitum the standard commercial feed mixtures (Table 1): starter (d 1–21), grower (d 22–35), finisher (d 35–42). In the same trial, along the rearing period, in vivo performance and mortality were recorded as described by Bednarczyk et al. (2016). Table 1. Composition and nutrient content of diets.   Starter  Grower  Finisher    (1–21 d)  (22–35 d)  (36–42 d)  Ingredient, g/Kg   Wheat  26.73  29.19  30.66   Maize  30.00  30.00  30.00   Soybean meal  32.50  28.20  25.33   Canola seeds  5.00  6.00  7.00   Soybean oil  2.10  1.33  1.80   Lard  -  2.00  2.50   NaCl  0.30  0.30  0.28   Mel stern  1.09  0.95  0.85   Phosphate 1-Calcium  1.15  0.94  0.63   DL-methionine  0.25  0.18  0.13   L-lysine  0.32  0.32  0.27   L-threonine  0.06  0.09  0.05   Vitamin—mineral premix1  0.50  0.50  0.50  Calculated composition   ME, MJ/kg  12.47  12.97  13.39   CP, % DM  22.00  20.50  19.50   Lysine, % DM  1.35  1.25  1.15   Methionine, % DM  0.57  0.49  0.43   Methionine + Cystine, % DM  0.95  0.85  0.78   Ca, % DM  0.90  0.80  0.70   P, % DM  0.40  0.35  0.28    Starter  Grower  Finisher    (1–21 d)  (22–35 d)  (36–42 d)  Ingredient, g/Kg   Wheat  26.73  29.19  30.66   Maize  30.00  30.00  30.00   Soybean meal  32.50  28.20  25.33   Canola seeds  5.00  6.00  7.00   Soybean oil  2.10  1.33  1.80   Lard  -  2.00  2.50   NaCl  0.30  0.30  0.28   Mel stern  1.09  0.95  0.85   Phosphate 1-Calcium  1.15  0.94  0.63   DL-methionine  0.25  0.18  0.13   L-lysine  0.32  0.32  0.27   L-threonine  0.06  0.09  0.05   Vitamin—mineral premix1  0.50  0.50  0.50  Calculated composition   ME, MJ/kg  12.47  12.97  13.39   CP, % DM  22.00  20.50  19.50   Lysine, % DM  1.35  1.25  1.15   Methionine, % DM  0.57  0.49  0.43   Methionine + Cystine, % DM  0.95  0.85  0.78   Ca, % DM  0.90  0.80  0.70   P, % DM  0.40  0.35  0.28  1Provided per kilogram of diets: vitamin A 12,500 IU, vitamin D3 - 4500 IU, vitamin E 45 mg, vitamin K3 3 mg, vitamin B1 3 mg, vitamin B2 6 mg, vitamin B6 4 mg, pantothenic acid 14 mg, nicotinic acid 50 mg, folic acid 1.75 mg, choline 1.6 g, vitamin B12 0.02 mg, biotin 0.2 mg, Fe 50 mg, Mn 120 mg, Zn 100 mg, Cu 15 mg, I 1.2 mg, Se 0.3 mg, fitase 500 FTU. View Large Table 1. Composition and nutrient content of diets.   Starter  Grower  Finisher    (1–21 d)  (22–35 d)  (36–42 d)  Ingredient, g/Kg   Wheat  26.73  29.19  30.66   Maize  30.00  30.00  30.00   Soybean meal  32.50  28.20  25.33   Canola seeds  5.00  6.00  7.00   Soybean oil  2.10  1.33  1.80   Lard  -  2.00  2.50   NaCl  0.30  0.30  0.28   Mel stern  1.09  0.95  0.85   Phosphate 1-Calcium  1.15  0.94  0.63   DL-methionine  0.25  0.18  0.13   L-lysine  0.32  0.32  0.27   L-threonine  0.06  0.09  0.05   Vitamin—mineral premix1  0.50  0.50  0.50  Calculated composition   ME, MJ/kg  12.47  12.97  13.39   CP, % DM  22.00  20.50  19.50   Lysine, % DM  1.35  1.25  1.15   Methionine, % DM  0.57  0.49  0.43   Methionine + Cystine, % DM  0.95  0.85  0.78   Ca, % DM  0.90  0.80  0.70   P, % DM  0.40  0.35  0.28    Starter  Grower  Finisher    (1–21 d)  (22–35 d)  (36–42 d)  Ingredient, g/Kg   Wheat  26.73  29.19  30.66   Maize  30.00  30.00  30.00   Soybean meal  32.50  28.20  25.33   Canola seeds  5.00  6.00  7.00   Soybean oil  2.10  1.33  1.80   Lard  -  2.00  2.50   NaCl  0.30  0.30  0.28   Mel stern  1.09  0.95  0.85   Phosphate 1-Calcium  1.15  0.94  0.63   DL-methionine  0.25  0.18  0.13   L-lysine  0.32  0.32  0.27   L-threonine  0.06  0.09  0.05   Vitamin—mineral premix1  0.50  0.50  0.50  Calculated composition   ME, MJ/kg  12.47  12.97  13.39   CP, % DM  22.00  20.50  19.50   Lysine, % DM  1.35  1.25  1.15   Methionine, % DM  0.57  0.49  0.43   Methionine + Cystine, % DM  0.95  0.85  0.78   Ca, % DM  0.90  0.80  0.70   P, % DM  0.40  0.35  0.28  1Provided per kilogram of diets: vitamin A 12,500 IU, vitamin D3 - 4500 IU, vitamin E 45 mg, vitamin K3 3 mg, vitamin B1 3 mg, vitamin B2 6 mg, vitamin B6 4 mg, pantothenic acid 14 mg, nicotinic acid 50 mg, folic acid 1.75 mg, choline 1.6 g, vitamin B12 0.02 mg, biotin 0.2 mg, Fe 50 mg, Mn 120 mg, Zn 100 mg, Cu 15 mg, I 1.2 mg, Se 0.3 mg, fitase 500 FTU. View Large Slaughter Surveys At 42 d of age, 7 birds per cage were randomly chosen (42 birds per treatment), individually weighed (after a fasting period of 12 h), and slaughtered. After evisceration, the hot carcass weight (without head and feet) was recorded, and carcass yield was calculated. In addition, the breast muscle was removed from all carcasses and its percentage based on hot carcass weight was calculated. After 24 h of refrigeration, the Pectoral muscle (PM) pH was measured using a portable pH meter (FiveGo, Mettler-Toledo AG, Schwerzenbach, Switzerland) equipped with a penetrating glass electrode. At the same time, tri-stimulus color coordinates (lightness, L*; redness, a*; yellowness, b*) were detected on the PM using a Chroma Meter CR-300 (Minolta Italia S.p.A., Milan, Italy). The left PM was removed, vacuum packaged, and stored frozen (−20°C) for analyses. Histological Evaluation For histological examination, samples of the pectoral muscle (pectoralis superficialis muscle) from all carcasses were taken at 45 min after slaughter. Samples of muscles were frozen in liquid nitrogen (−196°C) and placed in cryovials in which they were stored until analysis. Frozen muscle samples were mounted on a specimen disc with a few drops of water (or tissue freezing medium). Mounted muscle tissue was cut into sections (10 μm thick) at around –25°C using a cryostat (Thermo Shandon microtome, Thermo Fisher Scientific, Runcorn, UK) and then placed on the glass slides and stained. Hematoxylin and eosin reaction were used to evaluate the muscle fiber diameter (Dubovitz et al., 1973). A Carl Zeiss microscope (Jena, Germany) equipped with a Toup View camera was used to record microscopic images on a computer disk. MultiScan v. 18.03 microscope imaging software (Computer Scanning Systems II Ltd., Warsaw, Poland) was used to calculate diameter of muscle fibers per 1 mm2. Muscle fiber diameter was measured according to a method of smallest diameters described by Brook (1970). In brief, the mean diameter of muscle fibers value per each group was calculated. Measurement of Muscle Cholesterol Cholesterol was extracted using the method of Maraschiello et al. (1996) and then quantified by HPLC. A Kontron HPLC (Kontron Instruments, Milan, Italy) model 535, equipped with a Kinetex 5μ C18 reverse-phase column (150 × 4.6 mm x 5 μm; Phenomenex, Torrance, CA), was used. The HPLC mobile phase consisted of acetonitrile: 2-propanol (55:45, vol/vol) at a flow rate of 1.0 mL/min. The detection wavelength was 210 nm. The quantitation of muscle cholesterol content was based on the external standard method using a pure cholesterol standard (Sigma, St. Louis, MO). Fatty Acid Composition of Muscle Lipid extraction from breast muscle samples was performed by Folch et al. (1957) method. Fatty acids were quantified as methyl esters (FAME) using a gas chromatograph an HRGC 5300 Fisons (Rodano, Milan, Italy), equipped with a flame ionization detector and a fused silica capillary Column (CP-Sil RTX 2330), 30 m x 0.25 mm x 0.5 μm film thickness (Restek, Bellefonte, PA, USA). Helium was used as carrier gas. The oven temperature program was 120°C for 1 min then increasing at 5°C/min up to 230°C where it was maintained for 20 min. The individual FA peaks were identified by comparison of retention times with those of FAME authentic standards run under the same operating conditions. Results were expressed as percentage of the total FA identified. To assess the nutritional implications, the n-6 FA/n-3 FA and the PUFA/SFA (P/S) ratios were calculated. Moreover, in order to evaluate the risk of atherosclerosis and the potential aggregation of blood platelets, respectively the atherogenic index (AI) and the thrombogenic index (TI) were calculated, according to the formulas suggested by Ulbricht and Southgate (1991). Statistical Analyses All data were evaluated by ANOVA (SPSS, 2010). Differences among groups were determined by contrasts (prebiotic contrast 1: 3 × μC—μDN—μBI − μRFO = 0; contrast 2: 2 × μRFO—μDN—μBI = 0; contrast 3: μBI − μDN = 0. Mode of prebiotics’ administration contrast 1: 3 × μC − μT1 − μT2 − μT3 = 0; contrast 2: 2 × μT3 − μT1 − μT2 = 0; contrast 3: μT2 − μT1 = 0. μ = overall mean). RESULTS AND DISCUSSION In Vivo and Post Mortem Performance Effects of the prebiotics DN, BI and RFO and 3 different routes of delivery (T1, T2 and T3) were tested. The impact on slaughter performance and fiber diameter of PM in broiler chickens is presented in Table 2. Compared with the C group, chickens from prebiotic groups were significantly (P < 0.01) heavier (RFO = +4.6%; DN = +2.8%; BI = +2.2%) and among them RFO chickens were the heaviest (P < 0.01) compared to DN and BI groups. Route of prebiotic administration (Table 2) had an effect on final BW, results showing higher (P < 0.01) values for all the prebiotic-treated groups (T1, T2 and T3) compared with C group. No differences (P > 0.05) between T1, T2 or T3 were observed. Neither carcass weight nor carcass yield (ranging from 69.7% to 71.7%) was affected by prebiotic treatment (P > 0.05), while breast muscle weight and yield were higher (P < 0.01) in DN, BI and RFO treated groups compared to C group. The prebiotics injected in ovo only (T1) or by combining in ovo route and administration in-water (T2) resulted in a significantly higher carcass weight as compared with the chickens of T3 group (P > 0.01). There were no differences (P > 0.05) observed between C group and T1, T2 and T3 groups; whereas T3 group had reduced carcass yield compared to the other groups (C, T1 and T2; P > 0.05). The chickens treated with prebiotics (T1, T2 and T3) had heavier (P < 0.01) breast muscle compared to the ones from C group. The breast yield was higher (P < 0.01) for T1, T2 and T3 groups in comparison to C group. The higher breast muscle yield was more evident (P < 0.05) in chickens that received prebiotics in water (T3) than those injected in ovo (T1) or by combining both in ovo and in-water routes (T2); while, T1 and T2 did not differ (P > 0.05). In addition, prebiotic-treated groups showed a greater (P < 0.05) thickness of breast muscle fibers, compared to C group, irrespective the way of administration. These results are partially in agreement with those of our recent study (Maiorano et al., 2017) conducted on 275,000 broiler chickens (Ross 308) under commercial conditions. The authors reported enhanced growth performance in birds at 42 d which were injected in ovo with Bi2tos and DiNovo. Moreover, those animals showed higher carcass weight, carcass yield and breast muscle weight. Similar results were obtained by Bednarczyk et al. (2016), who reported a significant increase in body weight gain during the first 3 wk of life upon injection in ovo with different prebiotics (Bi2tos, DiNovo and RFO) as compared with the control group. On the contrary, Berrocoso et al. (2017) did not find any influence on growth performance or slaughter yield in Cobb 500 broilers, which were in ovo injected with different RFO doses and slaughtered at 21 d of age. In addition, these authors observed that RFO had the potential of enhancing ileum mucosa morphology and improving immunity in the small intestine, which are indicators of improved gut health. In another study conducted on Ross broiler chickens, Pruszynska-Oszmalek et al. (2015) found that the in ovo injection of prebiotics (inulin and Bi2tos) and synbiotics (inulin + Lactococcus lactis subsp. lactis and Bi2tos + Lactococcus lactis subsp. cremoris) caused an elevation of the activity of pancreatic enzymes, which can explain the observed higher BW of treated chickens. The positive effect on growth performance exerted by prebiotics injected in ovo seems to be related to the early stimulation of intestinal microbiota development in the chicken gut; this is due to the ability of prebiotics to enhance lactobacilli and bifidobacteria populations, and these beneficial bacteria compete with harmful bacteria for colonization (Biggs and Parsons, 2008; Depeint et al., 2008; Tzortzis, 2009). Results obtained using different types and doses of prebiotics administered in feed are variable and not always comparable (Gaggìa et al., 2010). Nevertheless, it is suggested that such bioactive substances might be used in broiler diets since they do not interfere or positively affect the yield of the most commercially valuable edible cuts (Pelicano et al., 2005). Table 2. Effect of different prebiotics and mode of their administration on slaughter performance and fiber diameter of pectoral muscle in broiler chickens.   Group1      Treatment2        C  DN  BI  RFO  SEM  P-value  C  T1  T2  T3  SEM  P-value  Final BW, g  2651.7C  2726.1B  2709.4B  2774.4A  0.01  0.001  2651.7B  2748.9A  2736.1A  2725.0A  0.03  0.008  Carcass weight, g  1900.0  1917.1  1910.3  1935.1  9.92  0.713  1900.0A,B  1958.0A  1936.0A  1871.9B  7.92  0.004  Carcass yield, g  71.67  70.32  70.45  69.66  0.32  0.362  71.67A  71.17A  70.65A  68.71B  0.12  0.005  Breast weight, g  579.8B  638.3A  618.9A  625.6A  5.34  0.024  579.8B  632.4A  619.8A  630.7A  6.34  0.040  Breast yield, %  30.7B  33.3A  32.6A  32.5A  0.24  0.023  30.7C  32.3B  32.3B  33.7A  0.27  0.002  Fiber diameter, μm  47.37b  54.02a  52.23a  54.69a  0.85  0.097  47.37b  54.56a  50.98a  55.40a  0.83  0.020    Group1      Treatment2        C  DN  BI  RFO  SEM  P-value  C  T1  T2  T3  SEM  P-value  Final BW, g  2651.7C  2726.1B  2709.4B  2774.4A  0.01  0.001  2651.7B  2748.9A  2736.1A  2725.0A  0.03  0.008  Carcass weight, g  1900.0  1917.1  1910.3  1935.1  9.92  0.713  1900.0A,B  1958.0A  1936.0A  1871.9B  7.92  0.004  Carcass yield, g  71.67  70.32  70.45  69.66  0.32  0.362  71.67A  71.17A  70.65A  68.71B  0.12  0.005  Breast weight, g  579.8B  638.3A  618.9A  625.6A  5.34  0.024  579.8B  632.4A  619.8A  630.7A  6.34  0.040  Breast yield, %  30.7B  33.3A  32.6A  32.5A  0.24  0.023  30.7C  32.3B  32.3B  33.7A  0.27  0.002  Fiber diameter, μm  47.37b  54.02a  52.23a  54.69a  0.85  0.097  47.37b  54.56a  50.98a  55.40a  0.83  0.020  1Group: C = Control, in ovo injection of physiological saline; DN = DiNovo; BI = Bi2tos; RFO = raffinose family oligosaccharides. 2Treatment: C = Control, in ovo injection of physiological saline; T1 = in ovo; T2 = in ovo + in-water; T3 = in-water. Means in a row with different letters are significantly different at:A-CP < 0.01;a-bP < 0.05. View Large Table 2. Effect of different prebiotics and mode of their administration on slaughter performance and fiber diameter of pectoral muscle in broiler chickens.   Group1      Treatment2        C  DN  BI  RFO  SEM  P-value  C  T1  T2  T3  SEM  P-value  Final BW, g  2651.7C  2726.1B  2709.4B  2774.4A  0.01  0.001  2651.7B  2748.9A  2736.1A  2725.0A  0.03  0.008  Carcass weight, g  1900.0  1917.1  1910.3  1935.1  9.92  0.713  1900.0A,B  1958.0A  1936.0A  1871.9B  7.92  0.004  Carcass yield, g  71.67  70.32  70.45  69.66  0.32  0.362  71.67A  71.17A  70.65A  68.71B  0.12  0.005  Breast weight, g  579.8B  638.3A  618.9A  625.6A  5.34  0.024  579.8B  632.4A  619.8A  630.7A  6.34  0.040  Breast yield, %  30.7B  33.3A  32.6A  32.5A  0.24  0.023  30.7C  32.3B  32.3B  33.7A  0.27  0.002  Fiber diameter, μm  47.37b  54.02a  52.23a  54.69a  0.85  0.097  47.37b  54.56a  50.98a  55.40a  0.83  0.020    Group1      Treatment2        C  DN  BI  RFO  SEM  P-value  C  T1  T2  T3  SEM  P-value  Final BW, g  2651.7C  2726.1B  2709.4B  2774.4A  0.01  0.001  2651.7B  2748.9A  2736.1A  2725.0A  0.03  0.008  Carcass weight, g  1900.0  1917.1  1910.3  1935.1  9.92  0.713  1900.0A,B  1958.0A  1936.0A  1871.9B  7.92  0.004  Carcass yield, g  71.67  70.32  70.45  69.66  0.32  0.362  71.67A  71.17A  70.65A  68.71B  0.12  0.005  Breast weight, g  579.8B  638.3A  618.9A  625.6A  5.34  0.024  579.8B  632.4A  619.8A  630.7A  6.34  0.040  Breast yield, %  30.7B  33.3A  32.6A  32.5A  0.24  0.023  30.7C  32.3B  32.3B  33.7A  0.27  0.002  Fiber diameter, μm  47.37b  54.02a  52.23a  54.69a  0.85  0.097  47.37b  54.56a  50.98a  55.40a  0.83  0.020  1Group: C = Control, in ovo injection of physiological saline; DN = DiNovo; BI = Bi2tos; RFO = raffinose family oligosaccharides. 2Treatment: C = Control, in ovo injection of physiological saline; T1 = in ovo; T2 = in ovo + in-water; T3 = in-water. Means in a row with different letters are significantly different at:A-CP < 0.01;a-bP < 0.05. View Large In this study, prebiotic administration had a positive impact on breast muscle weight which was also associated with a greater thickness (diameter) of breast muscle fibers as compared with the C group. In poultry meat production, muscle fiber properties play a key role in meat quantity and quality. In fact, it is reported that in chickens, selection for overall growth has been shown to induce greater muscle weight with simultaneous increases in fiber diameter and length (reviewed in Berri et al., 2007). Similar results were earlier obtained by Maiorano et al. (2012) who found a slightly greater thickness of muscle fiber in chickens, which were injected in ovo with RFO prebiotic. On the contrary, Maiorano et al. (2017) found that prebiotic treated groups (Bi2tos and DiNovo) were characterized by slightly thinner muscle fiber compared to control group. The reduced thickness of the fibers might be considered an indicator of fibrillarity and a delicate structure of the meat, being beneficial to meat quality from the consumer point of view (Maiorano et al., 2017). pH and Color Results of the effect of the 3 prebiotics and way of their administration on muscle pH and color are presented in Table 3. pH is one of the most important qualitative attribute of meat that has a central role in determining the protein behavior both in fresh and processed meat products (Lonergan, 2008) and it is also an important contributing factor to meat quality expressed as tenderness, color, and storage life (Van Laack and Lane, 2000). Type of prebiotics used did not influenced pH24, a finding in accord with the results of Maiorano et al. (2012). Whereas, Sang-Oh and Byung-Sung (2011) noted that dietary inuloprebiotic (250 g ton−1) reduced significantly pH of chicken meat. On the contrary, route of prebiotic administration affected ultimate meat pH, in fact it was higher (P < 0.01) in T2 group compared to T3, intermediate values were found for C and T1 groups (P > 0.05). The pH values observed in the present study (ranging from 5.76 and 5.87) fully fit within the pH range accepted for commercial poultry meat (Haščík et al., 2015). Table 3. Effect of different prebiotics and mode of their administration on meat quality traits of broiler chickens.   Group1      Treatment2        C  DN  BI  RFO  SEM  P-value  C  T1  T2  T3  SEM  P-value  pH24  5.86  5.81  5.84  5.79  0.01  0.350  5.86A,B  5.82A,B  5.87A  5.76B  0.02  0.001  Color3, 24 h post-mortem   L*  50.01  50.93  48.70  51.30  0.37  0.129  50.01  51.09  50.20  49.64  0.39  0.496   a*  3.90a  3.06b  3.35b  3.00b  0.10  0.047  3.90a  2.85b  3.23a,b  3.33a,b  0.12  0.036   b*  4.11  3.50  4.47  4.50  0.19  0.155  4.11  4.01  4.16  4.30  0.21  0.952    Group1      Treatment2        C  DN  BI  RFO  SEM  P-value  C  T1  T2  T3  SEM  P-value  pH24  5.86  5.81  5.84  5.79  0.01  0.350  5.86A,B  5.82A,B  5.87A  5.76B  0.02  0.001  Color3, 24 h post-mortem   L*  50.01  50.93  48.70  51.30  0.37  0.129  50.01  51.09  50.20  49.64  0.39  0.496   a*  3.90a  3.06b  3.35b  3.00b  0.10  0.047  3.90a  2.85b  3.23a,b  3.33a,b  0.12  0.036   b*  4.11  3.50  4.47  4.50  0.19  0.155  4.11  4.01  4.16  4.30  0.21  0.952  1Group: C = Control, in ovo injection of physiological saline; DN = DiNovo; BI = Bi2tos; RFO = raffinose family oligosaccharides. 2Treatment: C = Control, in ovo injection of physiological saline; T1 = in ovo; T2 = in ovo + in-water; T3 = in-water. 3Color: L* = lightness; a* = redness index; b* = yellow index. Means in a row with different letters are significantly different at:A-BP < 0.01;a-bP < 0.05. View Large Table 3. Effect of different prebiotics and mode of their administration on meat quality traits of broiler chickens.   Group1      Treatment2        C  DN  BI  RFO  SEM  P-value  C  T1  T2  T3  SEM  P-value  pH24  5.86  5.81  5.84  5.79  0.01  0.350  5.86A,B  5.82A,B  5.87A  5.76B  0.02  0.001  Color3, 24 h post-mortem   L*  50.01  50.93  48.70  51.30  0.37  0.129  50.01  51.09  50.20  49.64  0.39  0.496   a*  3.90a  3.06b  3.35b  3.00b  0.10  0.047  3.90a  2.85b  3.23a,b  3.33a,b  0.12  0.036   b*  4.11  3.50  4.47  4.50  0.19  0.155  4.11  4.01  4.16  4.30  0.21  0.952    Group1      Treatment2        C  DN  BI  RFO  SEM  P-value  C  T1  T2  T3  SEM  P-value  pH24  5.86  5.81  5.84  5.79  0.01  0.350  5.86A,B  5.82A,B  5.87A  5.76B  0.02  0.001  Color3, 24 h post-mortem   L*  50.01  50.93  48.70  51.30  0.37  0.129  50.01  51.09  50.20  49.64  0.39  0.496   a*  3.90a  3.06b  3.35b  3.00b  0.10  0.047  3.90a  2.85b  3.23a,b  3.33a,b  0.12  0.036   b*  4.11  3.50  4.47  4.50  0.19  0.155  4.11  4.01  4.16  4.30  0.21  0.952  1Group: C = Control, in ovo injection of physiological saline; DN = DiNovo; BI = Bi2tos; RFO = raffinose family oligosaccharides. 2Treatment: C = Control, in ovo injection of physiological saline; T1 = in ovo; T2 = in ovo + in-water; T3 = in-water. 3Color: L* = lightness; a* = redness index; b* = yellow index. Means in a row with different letters are significantly different at:A-BP < 0.01;a-bP < 0.05. View Large Here, all the tested prebiotics and different routes of prebiotic delivery significantly affected redness (a*) of the fillets. In fact, the redness index (a*) was reduced (P < 0.05) in meat from prebiotics groups (DN, BI an RFO) compared to that of C group and in T1 compared to C (P < 0.05). This is likely to be related to the muscle-fiber types (Zhao et al., 2012). The other color parameters (L* and b*) were similar (P > 0.05) between the experimental groups. However, the observed color coordinates fit within the range which is accepted for good chicken meat appearance. Therefore, different prebiotics might be used since they did not negatively affect meat color, which is an extremely important parameter influencing consumer decisions. Cholesterol Content and Fatty Acid Composition All prebiotics (DN, BI and RFO) did not affect cholesterol content (Table 4), irrespective of route of delivery (T1, T2 or T3), as compared with the C group (P < 0.05). Also Maiorano et al. (2012) found that prebiotic did not affect breast muscle cholesterol content. On the contrary, Pilarski et al. (2005) reported that in ovo application of fructooligosaccharides in Hybro G broiler breeder eggs caused a decrease of breast muscle cholesterol in comparison with the control group. The cholesterol values found in the present study (ranging from 46.74 to 49.57 mg/100 g) are similar than those reported by Pilarski et al. (2005) in breast muscle of 42-d-old broiler chickens (ranging from 49.3 to 54.7 mg/100 g) and lower (- 50%) than that reported by Maiorano et al. (2012). Cholesterol content in broiler meat can be altered by various factors (composition of diet, age, sex; Wang et al., 2006) as well as the use of different methodologies for cholesterol quantification or for sampling (Bragagnolo and Rodriguez-Amaya, 2002). The FA proportion of meat is considered an important index for meat quality. Broiler fat is characterized by a significant amount of monounsaturated fatty acids (MUFA), and, in comparison with red meat, substantial amounts of polyunsaturated fats, especially the n-6 linoleic acid and arachidonic acid. Moreover, it may represent an important source of long-chain n-3 fatty acids (Hibbeln et al., 2006; Attia et al., 2017). The FA composition of breast meat is shown in Table 4 for prebiotics and route of their administration. Taking into account the general FA profile, total MUFA were the most abundant FA (ranging from 32.83 to 36.86%), followed in descending order by saturated fatty acids (SFA) (ranging from 34.55 to 35.67%) and polyunsaturated fatty acids (PUFA) (ranging from 28.59 to 32.13%). To our knowledge, limited information is available in literature on the effect of prebiotics on modification in the FA pattern of lipids in chicken meat. Whereas, there is no available information regarding the potential effect of in ovo injection of different type of prebiotics using different routes of delivery (in ovo vs. in-water vs. in ovo and in-water combined) in chicken breast muscle; therefore, this subject should be considered in new investigations. Total SFA in breast muscle were higher (P < 0.01) in prebiotic groups than in C group, reflecting the trends of C18:0 (stearic) and C20:0 (arachidic). No statistically significant differences were found among the 3 prebiotic groups. Palmitic acid (C16:0), instead, was similar (P > 0.05) among the groups. The total MUFA content of the breast samples decreased (P < 0.01) in prebiotic groups compared to C group; it was mainly in the form of oleic acid (C18:1 n9) that was lower (P < 0.01) in prebiotic groups compared to C group. The same trend (P < 0.01) was found for palmitoleic acid (C16:1) and for C18:1 trans, even if this latter was present in very small amount. The total PUFA content was higher (P < 0.01) in all prebiotic groups compared to C group. The precursor of the n-6 family, the linoleic acid (C18:2), quantitatively the most concentrated n-6 PUFA, was not affected (P > 0.05) by the prebiotic treatment; while significant differences (P < 0.01) in the other n-6 LC-PUFA were observed among groups. The γ-linolenic acid (C18:3 n-6) was higher (P < 0.01) in all prebiotic groups compared to C group; while, meat from prebiotic groups displayed a lower (P < 0.01) content of arachidonic acid (C20:4 n-6). In general, the total content of n-6 PUFA was lower (P < 0.01) in prebiotic groups compared to C group. On the contrary, the total content of n-3 PUFA was about 2 fold higher (P < 0.01) in prebiotic groups compared to C group. The precursors of the n-3 family, α-linolenic acid (C18:3), significantly increased (P < 0.01) with prebiotic administration. All n-3 LC-PUFA were higher (P < 0.01) in all prebiotic groups compared to C group, except for the eicosapentaenoic acid (C20:5) that was lower (P < 0.01) in prebiotic groups compared to C. Very small amount (less than 1%) of conjugated linoleic acid (C18:2cis-9, trans-11 and C18:2 trans-10, cis-12) was detected in breast samples. The n-6/n-3 ratio was favourably lower (P < 0.01) in prebiotic-treated groups compared to C group. Route of prebiotic administration had also a marked effect on meat FA profile (Table 4). T2 and T3 increased (P < 0.05) the SFA content in breast muscle compared to C. No significant effect (P > 0.05) was found for the main SFA: palmitic (C16:0), stearic (C18:0) and myristic (C14:0) acids; only the content of arachidic acid (C20:0) was higher (P < 0.01) in all treated groups. The MUFA content was lower (P < 0.01) in prebiotic-treated groups compared to C group, reflecting the trends of oleic acid (P < 0.01), palmitoleic acid (P < 0.01) and C18:1 trans (P < 0.01), even if the latter was present in negligible amount in treated groups. The PUFA content was higher (P < 0.01) in all treated groups (T1, T2 and T3) compared to C group. Also for the route of prebiotic administration, the linoleic acid was not affected (P > 0.05) by the treatment; while all routes of prebiotic administration increased (P < 0.01) the γ-linolenic acid and decreased (P < 0.01) the arachidonic acid compared to C group. As a consequence, the total content of n-6 PUFA was lower (P < 0.01) in prebiotic-treated groups compared to C group, with T2 lower than T1 (P < 0.05). The total content of n-3 PUFA was significantly higher in T1, T2 and T3 groups compared to C group, reflecting the trend (P < 0.01) of α-linolenic, docosapentaenoic (C22:5) and docosahexaenoic (C22:6) acids. The eicosapentaenoic acid (C20:5) was higher (P < 0.01) in C group compared to the other groups; T1 and T2 showed similar (P > 0.05) values that were higher compared with the value of T3 group. Also for the route of prebiotic administration, the n-6/n-3 ratio was favourably lower (P < 0.01) in prebiotic-treated groups compared to C group. The P/S ratio was higher (P < 0.05) in prebiotic groups (DN, BI and RFO) compared to C group; in addition, T1 was higher than T2 (P < 0.05) and T3 similar (P > 0.05) to T1 and T2. Our results are inconsistent with those of Zhou et al. (2009), who found a greater concentration of total MUFA and lower amount of SFA in breast muscle of broiler chickens fed basal diet + 0.2% chitooligosaccharide (COS, oligosaccharide obtained by chemical and enzymatic hydrolysis of polychitosan) supplementation, but PUFA concentration was not affected by COS. The predominant SFA was palmitic acid (ranging from 22.29% to 22.90%). This acid is thought to increase cholesterol level together with lauric and myristic acid (Zock et al., 1994). Among the individual MUFA, the most abundant was the oleic acid (ranging from 32.33% to 34.17%). This finding is in agreement with levels found by other authors in chicken meat (Boschetti et al., 2016, fast growing birds; Alina et al., 2012). From a nutritional point of view, oleic acid plays a key role in human diet as involved in reducing lipaemia and consequently the risk of stroke (D’Alessandro et al., 2012). As general rule, poultry meat is characterized by the highest n-6/n-3 ratio compared to other meats, essentially due to the higher amount of n-6 FA than muscles of the other species (Rule et al., 2002; Wood et al., 2003). In fact, linoleic acid is the predominant essential FA in poultry and as a result the n-6 PUFA are the primary products found in tissue lipids. Consequently, the n-6/n-3 ratio in poultry meat is at a distance from the ideal value of 1 and above the recommended maximum of 4. The values found in the present study are within the ideal range of 1 to 4 and additionally birds of the prebiotic treated groups had improved values when compared with the control group, resulting in a positive impact on the dietetic value of meat. The P/S ratio has great nutritional implications also and it is taken as a measure of the propensity of the diet to influence the incidence of coronary disease (Wood et al., 2003). From a nutritional point of view, a higher P/S ratio is recommended, indeed it should be increased to above 0.4 (Wood et al., 2003). The P/S values observed in the present study are favourably high and in particular the prebiotic-treated groups showed a higher (P < 0.05) P/S ratio compared to C group. The AI and TI indexes represent criteria for evaluating the level and interrelation through which some FA may have atherogenic or thrombogenic properties, respectively. Only TI was significantly affected by prebiotic administration, being reduced in all prebiotic groups, compared to C group. The low AI and TI values found in the current study revealed good nutritional quality of the meat. Table 4. Effect of different prebiotics and mode of their administration on cholesterol (mg/100 g) and fatty acid composition (% of total fatty acids) of breast muscle from broiler chickens.   Group2      Treatment3      Item1  C  DN  BI  RFO  SEM  P-value  C  T1  T2  T3  SEM  P-value  Cholesterol  47.74  49.44  47.20  47.98  0.59  0.120  47.74  49.57  47.70  46.74  0.56  0.284  C14:0  1.19  1.07  1.16  1.16  0.03  0.608  1.19  1.11  1.15  1.12  0.04  0.890  C16:0  22.51  22.56  22.58  22.88  0.13  0.759  22.51  22.29  22.90  22.84  0.15  0.277  C16:1  1.43A  0.47B  0.47B  0.47B  0.05  0.001  1.43A  0.49B  0.47B  0.46B  0.04  0.001  C18:0  9.74b  9.83a,b  10.49a  10.37a  0.08  0.001  9.74  10.06  10.26  10.37  0.12  0.108  C18:1trans  1.26A  0.04B  0.03B  0.04B  0.04  0.001  1.26A  0.04B  0.03B  0.04B  0.05  0.001  C18:1cis-9  34.17A  32.47B  32.55B  32.54B  0.12  0.001  34.17A  32.64B  32.58B  32.33B  0.11  0.001  C18:2 n-6  19.11  19.52  19.16  18.84  0.12  0.224  19.11  19.38  18.92  19.22  0.14  0.542  C18:2cis-9, trans-11  0.22b  0.24a  0.26a  0.28a  0.01  0.046  0.22b  0.25c  0.23c,b  0.28a  0.01  0.037  C18:2trans-10, cis-12  0.21  0.23  0.26  0.27  0.01  0.225  0.21  0.27  0.24  0.26  0.01  0.468  C18:3 n-6  1.36B  1.90A  1.80A  1.82A  0.04  0.005  1.36B  1.90A  1.78A  1.84A  0.05  0.004  C18:3 n-3  1.77B  2.26A  2.25A  2.24A  0.03  0.001  1.77B  2.26A  2.26A  2.24A  0.02  0.001  C20:0  1.11B  1.43A,a  1.30A,b  1.26A,b  0.02  0.001  1.11B  1.38A  1.34A  1.28A  0.01  0.005  C20:4 n-6  2.33A  0.63B  0.60B  0.64B  0.05  0.001  2.33A  0.65B  0.58B  0.65B  0.06  0.001  C20:5 n-3  1.46A  1.43B  1.35B  1.26C  0.02  0.003  1.46A  1.42B  1.35B  1.27C  0.02  0.016  C22:5 n-3  0.75B  2.86A  2.90A  2.95A  0.07  0.001  0.75B  2.85A  2.92A  2.93A  0.08  0.001  C22:6 n-3  1.38B  3.06A  2.84A  2.98A  0.07  0.001  1.38B  3.01A  2.99A  2.87A  0.08  0.001  ΣSFA  34.55B  34.89A  35.53A  35.67A  0.15  0.068  34.55b  34.84a,b  35.65a  35.60a  0.13  0.046  ΣMUFA  36.86A  32.98B  33.05B  33.05B  0.17  0.001  36.86A  33.17B  33.08B  32.83B  0.18  0.001  ΣPUFA  28.59B  32.13A  31.42A  31.28A  0.19  0.001  28.59B  31.99A  31.27A  31.56A  0.120  0.001  Total n-6  22.81A  22.05B  21.56B  21.31B  0.12  0.005  22.81A  21.93B,a  21.28B,b  21.71B,a,b  0.14  0.009  Total n-3  5.36B  9.61A  9.34A  9.43A  0.16  0.001  5.36B  9.54A  9.52A  9.30A  0.14  0.001  n-6/n-3  4.25A  2.29B  2.31B  2.26B  0.06  0.001  4.25A  2.30B  2.24B  2.33B  0.08  0.001  P/S  0.83b  0.92a  0.88a  0.88a  0.01  0.006  0.83b  0.92a  0.88c  0.89a,c  0.01  0.012  AI  0.42  0.42  0.43  0.43  0.02  0.429  0.42  0.41  0.43  0.43  0.03  0.291  TI  0.73A  0.59B  0.62B  0.62B  0.01  0.001  0.73A  0.59B  0.61B  0.62B  0.01  0.001    Group2      Treatment3      Item1  C  DN  BI  RFO  SEM  P-value  C  T1  T2  T3  SEM  P-value  Cholesterol  47.74  49.44  47.20  47.98  0.59  0.120  47.74  49.57  47.70  46.74  0.56  0.284  C14:0  1.19  1.07  1.16  1.16  0.03  0.608  1.19  1.11  1.15  1.12  0.04  0.890  C16:0  22.51  22.56  22.58  22.88  0.13  0.759  22.51  22.29  22.90  22.84  0.15  0.277  C16:1  1.43A  0.47B  0.47B  0.47B  0.05  0.001  1.43A  0.49B  0.47B  0.46B  0.04  0.001  C18:0  9.74b  9.83a,b  10.49a  10.37a  0.08  0.001  9.74  10.06  10.26  10.37  0.12  0.108  C18:1trans  1.26A  0.04B  0.03B  0.04B  0.04  0.001  1.26A  0.04B  0.03B  0.04B  0.05  0.001  C18:1cis-9  34.17A  32.47B  32.55B  32.54B  0.12  0.001  34.17A  32.64B  32.58B  32.33B  0.11  0.001  C18:2 n-6  19.11  19.52  19.16  18.84  0.12  0.224  19.11  19.38  18.92  19.22  0.14  0.542  C18:2cis-9, trans-11  0.22b  0.24a  0.26a  0.28a  0.01  0.046  0.22b  0.25c  0.23c,b  0.28a  0.01  0.037  C18:2trans-10, cis-12  0.21  0.23  0.26  0.27  0.01  0.225  0.21  0.27  0.24  0.26  0.01  0.468  C18:3 n-6  1.36B  1.90A  1.80A  1.82A  0.04  0.005  1.36B  1.90A  1.78A  1.84A  0.05  0.004  C18:3 n-3  1.77B  2.26A  2.25A  2.24A  0.03  0.001  1.77B  2.26A  2.26A  2.24A  0.02  0.001  C20:0  1.11B  1.43A,a  1.30A,b  1.26A,b  0.02  0.001  1.11B  1.38A  1.34A  1.28A  0.01  0.005  C20:4 n-6  2.33A  0.63B  0.60B  0.64B  0.05  0.001  2.33A  0.65B  0.58B  0.65B  0.06  0.001  C20:5 n-3  1.46A  1.43B  1.35B  1.26C  0.02  0.003  1.46A  1.42B  1.35B  1.27C  0.02  0.016  C22:5 n-3  0.75B  2.86A  2.90A  2.95A  0.07  0.001  0.75B  2.85A  2.92A  2.93A  0.08  0.001  C22:6 n-3  1.38B  3.06A  2.84A  2.98A  0.07  0.001  1.38B  3.01A  2.99A  2.87A  0.08  0.001  ΣSFA  34.55B  34.89A  35.53A  35.67A  0.15  0.068  34.55b  34.84a,b  35.65a  35.60a  0.13  0.046  ΣMUFA  36.86A  32.98B  33.05B  33.05B  0.17  0.001  36.86A  33.17B  33.08B  32.83B  0.18  0.001  ΣPUFA  28.59B  32.13A  31.42A  31.28A  0.19  0.001  28.59B  31.99A  31.27A  31.56A  0.120  0.001  Total n-6  22.81A  22.05B  21.56B  21.31B  0.12  0.005  22.81A  21.93B,a  21.28B,b  21.71B,a,b  0.14  0.009  Total n-3  5.36B  9.61A  9.34A  9.43A  0.16  0.001  5.36B  9.54A  9.52A  9.30A  0.14  0.001  n-6/n-3  4.25A  2.29B  2.31B  2.26B  0.06  0.001  4.25A  2.30B  2.24B  2.33B  0.08  0.001  P/S  0.83b  0.92a  0.88a  0.88a  0.01  0.006  0.83b  0.92a  0.88c  0.89a,c  0.01  0.012  AI  0.42  0.42  0.43  0.43  0.02  0.429  0.42  0.41  0.43  0.43  0.03  0.291  TI  0.73A  0.59B  0.62B  0.62B  0.01  0.001  0.73A  0.59B  0.61B  0.62B  0.01  0.001  1SFA = saturated fatty acids; MUFA = monounsaturated fatty acids; PUFA = polyunsaturated fatty acids; P/S = PUFA/SFA ratio; AI = atherogenic index; TI = thrombogenic index. 2Group: C = Control, in ovo injection of physiological saline; DN = DiNovo; BI = Bi2tos; RFO = raffinose family oligosaccharides. 3Treatment: C = Control, in ovo injection of physiological saline; T1 = in ovo; T2 = in ovo + in-water; T3 = in-water. Means in a row with different letters are significantly different at:A-CP < 0.01;a-cP < 0.05. View Large Table 4. Effect of different prebiotics and mode of their administration on cholesterol (mg/100 g) and fatty acid composition (% of total fatty acids) of breast muscle from broiler chickens.   Group2      Treatment3      Item1  C  DN  BI  RFO  SEM  P-value  C  T1  T2  T3  SEM  P-value  Cholesterol  47.74  49.44  47.20  47.98  0.59  0.120  47.74  49.57  47.70  46.74  0.56  0.284  C14:0  1.19  1.07  1.16  1.16  0.03  0.608  1.19  1.11  1.15  1.12  0.04  0.890  C16:0  22.51  22.56  22.58  22.88  0.13  0.759  22.51  22.29  22.90  22.84  0.15  0.277  C16:1  1.43A  0.47B  0.47B  0.47B  0.05  0.001  1.43A  0.49B  0.47B  0.46B  0.04  0.001  C18:0  9.74b  9.83a,b  10.49a  10.37a  0.08  0.001  9.74  10.06  10.26  10.37  0.12  0.108  C18:1trans  1.26A  0.04B  0.03B  0.04B  0.04  0.001  1.26A  0.04B  0.03B  0.04B  0.05  0.001  C18:1cis-9  34.17A  32.47B  32.55B  32.54B  0.12  0.001  34.17A  32.64B  32.58B  32.33B  0.11  0.001  C18:2 n-6  19.11  19.52  19.16  18.84  0.12  0.224  19.11  19.38  18.92  19.22  0.14  0.542  C18:2cis-9, trans-11  0.22b  0.24a  0.26a  0.28a  0.01  0.046  0.22b  0.25c  0.23c,b  0.28a  0.01  0.037  C18:2trans-10, cis-12  0.21  0.23  0.26  0.27  0.01  0.225  0.21  0.27  0.24  0.26  0.01  0.468  C18:3 n-6  1.36B  1.90A  1.80A  1.82A  0.04  0.005  1.36B  1.90A  1.78A  1.84A  0.05  0.004  C18:3 n-3  1.77B  2.26A  2.25A  2.24A  0.03  0.001  1.77B  2.26A  2.26A  2.24A  0.02  0.001  C20:0  1.11B  1.43A,a  1.30A,b  1.26A,b  0.02  0.001  1.11B  1.38A  1.34A  1.28A  0.01  0.005  C20:4 n-6  2.33A  0.63B  0.60B  0.64B  0.05  0.001  2.33A  0.65B  0.58B  0.65B  0.06  0.001  C20:5 n-3  1.46A  1.43B  1.35B  1.26C  0.02  0.003  1.46A  1.42B  1.35B  1.27C  0.02  0.016  C22:5 n-3  0.75B  2.86A  2.90A  2.95A  0.07  0.001  0.75B  2.85A  2.92A  2.93A  0.08  0.001  C22:6 n-3  1.38B  3.06A  2.84A  2.98A  0.07  0.001  1.38B  3.01A  2.99A  2.87A  0.08  0.001  ΣSFA  34.55B  34.89A  35.53A  35.67A  0.15  0.068  34.55b  34.84a,b  35.65a  35.60a  0.13  0.046  ΣMUFA  36.86A  32.98B  33.05B  33.05B  0.17  0.001  36.86A  33.17B  33.08B  32.83B  0.18  0.001  ΣPUFA  28.59B  32.13A  31.42A  31.28A  0.19  0.001  28.59B  31.99A  31.27A  31.56A  0.120  0.001  Total n-6  22.81A  22.05B  21.56B  21.31B  0.12  0.005  22.81A  21.93B,a  21.28B,b  21.71B,a,b  0.14  0.009  Total n-3  5.36B  9.61A  9.34A  9.43A  0.16  0.001  5.36B  9.54A  9.52A  9.30A  0.14  0.001  n-6/n-3  4.25A  2.29B  2.31B  2.26B  0.06  0.001  4.25A  2.30B  2.24B  2.33B  0.08  0.001  P/S  0.83b  0.92a  0.88a  0.88a  0.01  0.006  0.83b  0.92a  0.88c  0.89a,c  0.01  0.012  AI  0.42  0.42  0.43  0.43  0.02  0.429  0.42  0.41  0.43  0.43  0.03  0.291  TI  0.73A  0.59B  0.62B  0.62B  0.01  0.001  0.73A  0.59B  0.61B  0.62B  0.01  0.001    Group2      Treatment3      Item1  C  DN  BI  RFO  SEM  P-value  C  T1  T2  T3  SEM  P-value  Cholesterol  47.74  49.44  47.20  47.98  0.59  0.120  47.74  49.57  47.70  46.74  0.56  0.284  C14:0  1.19  1.07  1.16  1.16  0.03  0.608  1.19  1.11  1.15  1.12  0.04  0.890  C16:0  22.51  22.56  22.58  22.88  0.13  0.759  22.51  22.29  22.90  22.84  0.15  0.277  C16:1  1.43A  0.47B  0.47B  0.47B  0.05  0.001  1.43A  0.49B  0.47B  0.46B  0.04  0.001  C18:0  9.74b  9.83a,b  10.49a  10.37a  0.08  0.001  9.74  10.06  10.26  10.37  0.12  0.108  C18:1trans  1.26A  0.04B  0.03B  0.04B  0.04  0.001  1.26A  0.04B  0.03B  0.04B  0.05  0.001  C18:1cis-9  34.17A  32.47B  32.55B  32.54B  0.12  0.001  34.17A  32.64B  32.58B  32.33B  0.11  0.001  C18:2 n-6  19.11  19.52  19.16  18.84  0.12  0.224  19.11  19.38  18.92  19.22  0.14  0.542  C18:2cis-9, trans-11  0.22b  0.24a  0.26a  0.28a  0.01  0.046  0.22b  0.25c  0.23c,b  0.28a  0.01  0.037  C18:2trans-10, cis-12  0.21  0.23  0.26  0.27  0.01  0.225  0.21  0.27  0.24  0.26  0.01  0.468  C18:3 n-6  1.36B  1.90A  1.80A  1.82A  0.04  0.005  1.36B  1.90A  1.78A  1.84A  0.05  0.004  C18:3 n-3  1.77B  2.26A  2.25A  2.24A  0.03  0.001  1.77B  2.26A  2.26A  2.24A  0.02  0.001  C20:0  1.11B  1.43A,a  1.30A,b  1.26A,b  0.02  0.001  1.11B  1.38A  1.34A  1.28A  0.01  0.005  C20:4 n-6  2.33A  0.63B  0.60B  0.64B  0.05  0.001  2.33A  0.65B  0.58B  0.65B  0.06  0.001  C20:5 n-3  1.46A  1.43B  1.35B  1.26C  0.02  0.003  1.46A  1.42B  1.35B  1.27C  0.02  0.016  C22:5 n-3  0.75B  2.86A  2.90A  2.95A  0.07  0.001  0.75B  2.85A  2.92A  2.93A  0.08  0.001  C22:6 n-3  1.38B  3.06A  2.84A  2.98A  0.07  0.001  1.38B  3.01A  2.99A  2.87A  0.08  0.001  ΣSFA  34.55B  34.89A  35.53A  35.67A  0.15  0.068  34.55b  34.84a,b  35.65a  35.60a  0.13  0.046  ΣMUFA  36.86A  32.98B  33.05B  33.05B  0.17  0.001  36.86A  33.17B  33.08B  32.83B  0.18  0.001  ΣPUFA  28.59B  32.13A  31.42A  31.28A  0.19  0.001  28.59B  31.99A  31.27A  31.56A  0.120  0.001  Total n-6  22.81A  22.05B  21.56B  21.31B  0.12  0.005  22.81A  21.93B,a  21.28B,b  21.71B,a,b  0.14  0.009  Total n-3  5.36B  9.61A  9.34A  9.43A  0.16  0.001  5.36B  9.54A  9.52A  9.30A  0.14  0.001  n-6/n-3  4.25A  2.29B  2.31B  2.26B  0.06  0.001  4.25A  2.30B  2.24B  2.33B  0.08  0.001  P/S  0.83b  0.92a  0.88a  0.88a  0.01  0.006  0.83b  0.92a  0.88c  0.89a,c  0.01  0.012  AI  0.42  0.42  0.43  0.43  0.02  0.429  0.42  0.41  0.43  0.43  0.03  0.291  TI  0.73A  0.59B  0.62B  0.62B  0.01  0.001  0.73A  0.59B  0.61B  0.62B  0.01  0.001  1SFA = saturated fatty acids; MUFA = monounsaturated fatty acids; PUFA = polyunsaturated fatty acids; P/S = PUFA/SFA ratio; AI = atherogenic index; TI = thrombogenic index. 2Group: C = Control, in ovo injection of physiological saline; DN = DiNovo; BI = Bi2tos; RFO = raffinose family oligosaccharides. 3Treatment: C = Control, in ovo injection of physiological saline; T1 = in ovo; T2 = in ovo + in-water; T3 = in-water. Means in a row with different letters are significantly different at:A-CP < 0.01;a-cP < 0.05. View Large In summary, the final BW of broiler chickens was increased upon delivery of prebiotics (DN, BI and RFO) irrespective of the method used. The prebiotic which showed the most significant improvement of performance traits was RFO. Each of the prebiotics which were injected in ovo (T1) or delivered by combining in ovo and in-water routes (T2) have resulted in an increase of carcass weight as compared with prebiotics given only in water (T3). Irrespective of delivery method used, prebiotics showed a positive impact on breast muscle weight and yield, which was also associated with a greater thickness (diameter) of breast muscle fibers. The redness (a*) of fillets was decreased upon delivery of prebiotics irrespective of the method used. As for FA composition, the intramuscular SFA were higher in the meat from prebiotic groups, while MUFA were lower. Moreover, PUFA and n-3 FA were higher in the meat from prebiotic groups, displaying more favorable indexes for human health (n-6/n-3, P/S, TI). Meat cholesterol content was not affected by any prebiotic treatment. In conclusion, even if the calculated increase of final body weight of the birds obtained with prebiotics seems minor in experimental scale, the economic impact could be relevant if one considers the high number of chickens reared under commercial conditions. In addition, according to these data, we can conclude that prebiotics support good performance results regardless of the mode of administration. It should be considered that the dose of a selected bioactive used for the in ovo injection is 10 times less than of dose which is added in water. This seems of an apparent economic benefit if one takes into account cost of routine prebiotic supplementation. Nevertheless, further research is required to increase the knowledge on prebiotic composition and elaborate the optimal bioactive formulation and route of their delivery, with proven strong effects on poultry meat quality; with a special emphasis placed on FA profile and other health promoting meat indices. Those further optimization trials must be performed in experimental and field conditions. ACKNOWLEDGMENTS The research leading to these results has received funding from the European Union's Seventh Framework Programme managed by REA Research Executive Agency http://ec.europa.eu/research/rea (FP7/2007–2013) under grant agreement number: 315198. This research was undertaken as part of a project entitled “Thrive-Rite: Natural Compounds to enhance Productivity, Quality and Health in Intensive Farming Systems”. Further details are provided on the consortium's website: www.thriverite.eu and the EU Commission's webpage: http://cordis.europa.eu/project/rcn/104395_en.html). 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Journal

Poultry ScienceOxford University Press

Published: Apr 20, 2018

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