Effects of free-fatty-acid content and saturation degree of the dietary oil sources on lipid-class content and fatty-acid digestibility along the gastrointestinal tract in broilers from 22 to 37 days of age R. Rodriguez-Sanchez,*,1,2 A. Tres,y R. Sala,* M. D. Soler,z F. Guardiola,y and A. C. Barroeta* * Animal Nutrition and Welfare Service, Department of Animal and Food Science, Facultat de Veterin a ria, Universitat Autonoma de Barcelona, Bellaterra, Barcelona, E-08193 Spain; yNutrition, Food Science and Gastronomy Department − XIA-INSA, Facultat de Farm acia i Ciencies de l’Alimentaci o, Universitat de Barcelona, Av Prat de la Riba, 171, Santa Coloma de Gramenet, E-08921 Spain; and zAviFeed Science, Department of Animal Production and Health Public Veterinary Health and Food Science and Technology, Facultad de Veterinaria, Universidad Cardenal Herrera-CEU, CEU Universities, Calle Tirant lo Blanch, 7, E-46115 Alfara del Patriarca, Valencia, Spain ABSTRACT The aim of the present study is to assess the lower FFA content and the higher TFA and poly- the effect of the free-fatty-acid (FFA) content and satu- unsaturated FA (PUFA) digestibility coefficients in ration degree of dietary fat (added at 6%) on the fatty- the ileum (P < 0.001) observed in the former group. acid (FA) digestibility and lipid-class content along the The dietary FFA level did not affect the FA absorp- gastrointestinal tract and excreta in broilers from 22 to tion process as much as the dietary fat source did. 37 d of age. This is essential to determine the potential This was supported by the lack of statistical differen- use of acid oils (refining by-products rich in FFA) in ces among the diets with a similar saturation degree broiler diets as an alternative to crude oils. The study but rather different levels of FFA, for TFA, saturated consisted of a 2 £ 4 factorial arrangement, which FA, and PUFA digestibility coefficients both in the included 2 fat sources (soybean oils − unsaturated, or jejunum and ileum. However, the interactions palm oils − saturated) and 4 levels of FFA (5, 15, 35, reported in the ileum for triacylglycerol and diacyl- and 50%). Samples of digestive content of the gizzard, glycerol contents (P < 0.001), as well as for monoun- duodenum, jejunum, ileum and of the excreta were saturated FA digestibility coefficients (P < 0.05) obtained at 37 d of age. Irrespective of the dietary fat show that the dietary FFA content affects the FA source, more than 80% of total FA (TFA) was absorbed absorption process. The present results show that the in the jejunum. Broilers fed with unsaturated diets had inclusion of acid oils in grower-finisher broiler diets a higher absorption efficiency of FA than did those fed with FFA levels up to 35% does not have a negative with saturated diets. This conclusion is supported by impact on the FA absorption process. Key words: acid oil, broiler, fatty-acid digestibility, free-fatty-acid, lipid class 2021 Poultry Science 100:101261 https://doi.org/10.1016/j.psj.2021.101261 INTRODUCTION of an economic alternative, in comparison to conven- tional fats, that can be repurposed as a feed fat ingredi- The use of supplemental fats is a common practice for ent. These fat by-products come from the chemical meeting both energy and essential fatty-acid (FA) (called acid oils from chemical refining; e.g., soybean requirements in poultry diets. Acid oils are an example acid oil) or physical (called FA distillates from physical refining; e.g., palm FA distillate) refining processes of edible oils. In the text, they will be generically called Ó 2021 The Authors. Published by Elsevier Inc. on behalf of Poultry acid oils unless otherwise stated. Acid oils are rich in free Science Association Inc. This is an open access article under the CC BY-NC-ND license (http://creativecommons.org/licenses/by-nc-nd/ fatty acids (FFA; 32−94%, Varona et al., 2021), and 4.0/). their FA composition is related to the botanical origin of Received February 5, 2021. the crude oil. Nevertheless, the molecular structure of Accepted May 12, 2021. acylglcyerols, and the presence of energy-diluting com- 1 Present address: AgResearch Ltd., Ruakura Research Centre, 10 Bisley Road, Private Bag 3123, 19 Hamilton 3214, NZ. pounds, can affect their energy value (Roll et al., 2018; 2 Corresponding author: rodriguezsan.raquel@gmail.com Varona et al., 2021). 1 2 RODRIGUEZ-SANCHEZ ET AL. It is well documented that the saturation degree of Table 1. Ingredient composition, and energy and nutrient con- dietary fat has an important effect on fat utilization tent of the experimental basal diet. because unsaturated fatty acids (UFA) are easier to Grower-finisher diet digest and absorb than are saturated fatty acids (SFA; Item (from 22 to 37 d) Renner and Hill, 1961; Sklan, 1979; Tancharoenrat Ingredients (%) et al., 2014; Vilarrasa et al., 2015a). It has been Wheat 44.00 described that the saturation degree of the diet has a Soybean meal 48% CP 27.25 Barley 18.58 greater effect than does its FFA content on the fat Experimental fats1 6.00 absorption process (Vilarrasa et al., 2015b; Rodriguez- Calcium carbonate 1.39 Sanchez et al., 2019b; Jimenez-Moya et al 2021). How- Sodium chloride 0.35 Monocalcium phosphate 1.20 ever, there is a lack of consensus regarding the effect of Vitamin and mineral premix2 0.40 dietary FFA on fat digestion and absorption processes. DL-Methionine 0.17 Thus, while some authors have reported that dietary L-Lysine 0.12 L-Threonine 0.02 FFA content negatively affects dietary fat digestibility Titanium oxide 0.50 and feed AME values (Shannon, 1971; Wiseman and Ethoxyquin 66% 0.02 Salvador, 1991; Wiseman et al., 1992; Blanch et al., Calculated energy and nutrient contents (%) AMEn3 (kcal/kg) 3,075 1995, 1996; Vil a and Esteve-Garcia, 1996), others have Crude protein 20.30 reported no effects on dietary AME (Vilarrasa et al., Calcium 0.90 2015b; Roll et al., 2018) and growth performance Sodium 0.15 Available phosphorus 0.40 (Young, 1961; Bornstein and Lipstein, 1961, 1963; Art- Digestible Lys 1.10 man, 1964; Zumbado et al., 1994). The age of the chick- Digestible Met 0.47 ens could be a determining factor in the outcome of Digestible Met + Cys 0.83 Digestible Thr 0.74 these studies, as the ability of chickens to digest and Digestible Trp 0.25 absorb dietary fat improves with the age (Renner and 1 Crude soybean oil, crude palm oil, acid soybean oil from chemical Hill, 1960; Batal and Parsons, 2002; Tancharoenrat refining or palm fatty acid distillate from physical refining. et al., 2013; Rodriguez-Sanchez et al., 2019a). Under- 2 Provides per kg of feed: vitamin A (from retinol), 10,000 IU; vitamin standing how FFA affect the fat absorption process is D3 (from cholecalciferol), 4,800 IU; vitamin E (from alfa-tocopherol), 49.5 IU; vitamin B1, 3 mg; vitamin B2, 9 mg; vitamin B6, 4.5 mg; vitamin B12, essential to determine the potential use of acid oils in 16.5 mg; vitamin K3, 3 mg; calcium pantothenate, 16.5 mg; nicotinic acid, broiler diets. 51 mg; folic acid, 1.8 mg; biotin, 30 mg; Fe (from FeSO4¢7H2O), 54 mg; I The global aim of this study is to assess the effect of (from Ca(I2O3)2), 1.2 mg; Cu (from CuSO4¢5H2O), 12 mg; Mn (from MnO), 90 mg; Zn (from ZnO), 66 mg; Se (from Na2SeO3), 0.18 mg; Mo the FFA content and dietary fat saturation degree on (from (NH4)6Mo7O24), 1.2 mg. FA digestibility and lipid-class composition along the gastrointestinal tract, and also on excreta in broilers from 22 to 37 d of age. The same effects in broilers from the 8 dietary treatments, with 11 chicks per cage and 6 11 to 14 d of age are reported in Rodriguez-Sanchez cages per treatment. Birds were housed in wire-floor et al. (2019b). A completely random design with a 2 £ 4 cages. Throughout the study, feed and water were sup- factorial arrangement was applied, which included 2 die- plied for ad libitum consumption, and animals were tary fat sources (soybean oils − unsaturated, or palm raised under controlled conditions of light and tempera- oils − saturated), and 4 levels of FFA (5, 15, 35 and ture, as recommended by the breeder. 50%). Thus, there were 8 dietary treatments with 6% The feeding program consisted of 2 phases (0−21 d and added fat, which resulted from replacing crude soybean 22−37 d of age). All diets were based on wheat-soybean oil with soybean acid oil, or crude palm oil with palm meal, fed in mash form, formulated to meet or exceed FA distillate in different proportions. The results from FEDNA requirements (2008) and to minimize the basal this study give new insights, regarding the use of acid level of fat (Table 1). Titanium dioxide (TiO2) was added oils as a feed fat ingredient, and support the importance (5 g/kg) as an inert marker. The level of inclusion of fat in of continuing work towards finding the best way to use the 8 experimental diets was 6%. The diets differed in the them as alternative fat sources in broiler diets. type of added fat according to a 2 £ 4 factorial arrange- ment: 2 dietary fat sources (soybean oils − unsaturated, or palm oils − saturated), and 4 different levels of dietary MATERIAL AND METHODS FFA (Table 2). The lowest FFA level assayed for each fat Animals and Diets source corresponded to the addition of a 6% of crude oil (either crude soybean [SO] or crude palm oil [PO]) to the The experimental procedure was approved by the feed. For the unsaturated diets (S), the highest level of Animal Protocol Review Committee of the Universitat FFA corresponded to the use of acid soybean oil from Autonoma de Barcelona (Bellaterra, Barcelona, Spain), chemical refining (SA) instead of SO (Table 2). Then, a and conformed to the European Union Guidelines similar highest FFA level was designed for the saturated (2010/63/EU). diets (P) by blending palm fatty-acid distillate from phys- A total of 528 one-day-old female Ross 308 broilers ical refining (PA) with PO (Table 2). Two intermediate were obtained from a commercial hatchery (Pondex treatments were achieved by adding SO and SA at 70:30 SAU; Lleida, Spain), and randomly assigned to one of or 30:70, or PO and PA in the proportions detailed in FAT BY-PRODUCTS IN BROILER DIETS 3 Table 2. Oil blends used in the experimental diets.1 Unsaturated - S Saturated - P Dietary fat Dietary FFA% 5 15 35 50 5 15 35 50 Proportion in oil blends (%) Dietary treatments S5 S15 S35 S50 P5 P15 P35 P50 Crude soybean oil (SO) 100 70 30 - - - - - Acid soybean oil from chemical refining (SA) - 30 70 100 - - - - Crude palm oil (PO) - - - - 100 80 53 33 Palm fatty acid distillate from physical refining (PA) - - - - - 20 47 66 Abbreviation: FFA, free fatty acids. 1 All oils or oil blends were added at 6% into the basal diet. Table 2 to obtain dietary FFA levels as similar as possible Chemical Analysis and Calculations to those of the unsaturated groups. The lipid-class compo- sition, quality parameters, and gross energy of the 4 The experimental oils were analyzed in triplicate for experimental oils are presented in Table 3. moisture, unsaponifiable matter (AOAC International (2005) methods: 926.12 and 933.08, respectively), impu- rities (ISO 663:2007), and lipid-class composition Data Collection (Rodriguez-Sanchez et al. 2019a; Table 3). Analytical determinations of the diets were performed following the Feed consumption and BW were measured weekly to AOAC International (2005) methods: DM (934.01), ash calculate the ADFI, the ADG, and the feed conversion (942.05), CP (968.06), crude fat (2003.05), and crude ratio (FCR) throughout the experiment. These were fiber (962.09). Gross energy was determined by an adia- corrected for mortalities. At 37 d of age, 3 birds/cage batic calorimeter (IKA C-4000, Janke-Kunkel; Staufen, were euthanized and the following samples were col- Germany). The FA content of the feed, digestive con- lected: content of the gizzard, duodenum (from the pylo- tent, and excreta was determined following the ric junction to the distal-most point of insertion of the Sukhija and Palmquist (1988) method, and the digest- duodenal mesentery), jejunum (from the distal-most ibility coefficients of FA were determined using the TiO2 point of insertion of the duodenal mesentery to the junc- ratio (Rodriguez-Sanchez et al., 2019b). The TiO2 in the tion with Meckel’s diverticulum), ileum (from the junc- feed, digestive content and excreta was analyzed follow- tion with Meckel’s diverticulum to 1 cm proximal to the ing the procedures of Short et al. (1996) and determined ileocecal junction), and a representative sample of by ICP-OES (Optima 3200 RL, Perkin Elmer; Wal- excreta of each cage. Each sample from all birds within tham, MA). The AME was determined from the product each cage was pooled, homogenized, frozen at -20°C, of the energy utilization ratio and its corresponding lyophilized, and ground to pass through a 0.5-mm sieve. gross energy of feed. The lipid-class composition (triacyl- Then, the samples were kept at 4°C until further analy- glycerols [TAG], diacylglycerols [DAG], monoacylgly- ses. Another sampling took place at 14 d of age, and cerols [MAG], and FFA) of the extracted fat from the these results are presented and discussed in Rodriguez- feed, digestive content, and excreta samples was ana- Sanchez et al. (2019b). lyzed as described by Rodriguez-Sanchez et al. (2019b). Lipid classes are a way to assess the hydrolysis of TAG, through their disappearance and the parallel appearance of the lipolysis products (DAG, MAG, and FFA), as Table 3. Lipid-class composition, quality parameters and energy well as the absorption of the latter through their disap- content of the experimental oils. pearance from the intestinal lumen. The following for- Experimental oils mula was applied to determine the lipid-class content in Item SO 1 SA2 PO3 PA4 the different GIT segments, and excreta: Lipid-class composition (%) Lipid  class content ¼ ½LCdig=½TiO2 dig; TAG 94.40 20.40 84.08 2.10 DAG 3.30 10.63 12.22 1.70 where [LC]dig is the concentration of the lipid classes in MAG 0.60 1.72 < 0.10 0.80 the digesta of a GIT segment or excreta (mg/g DM) and FFA 1.60 67.24 3.0 95.40 Quality (%) [TiO2]dig is the concentration of TiO2 in the digesta of Moisture 0.08 0.69 0.06 0.04 the same GIT segment or excreta (mg/g DM). The mac- Impurities 0.40 0.85 0.77 1.27 ronutrient, FA and lipid-class composition of the experi- Unsaponifiable 0.52 2.78 0.31 0.42 Energy content mental diets are presented in Table 4. Gross energy (kcal/kg) 9479 9461 9257 9417 Abbreviations: DAG, diacylglycerols; FFA, free fatty acids; MAG, Statistical Analysis monoacylglycerols; TAG, triacylglycerols. 1 SO = crude soybean oil. 2 SA = acid soybean oil from chemical refining. Productive parameters, AME, lipid-class content and 3 PO = crude palm oil digestibility of FA were subjected to a univariate analy- 4 PA = palm fatty acid distillate from physical refining. sis using the GLM procedure (SPSS statistics 25.0.0.0, 4 RODRIGUEZ-SANCHEZ ET AL. Table 4. Analyzed1 macronutrient content, fatty-acid and lipid-class composition of the experimental diets.2 Grower-finisher diets Unsaturated - S diets Saturated - P diets Item S5 S15 S35 S50 P5 P15 P35 P50 Macronutrient content Dry matter % 90.80 90.84 90.70 90.76 90.92 90.87 90.84 90.76 Crude protein % 19.29 19.89 20.16 19.93 19.83 20.09 19.49 19.75 Crude fat % 7.29 7.46 7.26 7.28 7.47 7.42 7.22 7.35 Crude fiber % 5.59 5.24 5.27 5.08 5.60 5.61 6.02 6.81 Ash % 6.12 6.15 6.01 6.28 5.76 6.04 6.04 6.14 Gross energy, kcal/kg 4,134 4,198 4,175 4,118 4,130 4,184 4,172 4,151 Fatty acid composition, % C14:0 - - - - 1.1 1.1 1.0 1.0 C16:0 14.39 14.73 16.21 17.21 37.80 37.64 38.38 39.94 C18:0 4.02 4.18 4.62 4.71 3.85 3.83 3.78 3.88 C18:1 n-9 20.50 20.56 20.96 20.92 31.26 30.67 30.19 30.32 C18:1 n-7 1.35 1.36 1.34 1.34 0.85 0.85 0.82 0.80 C18:2 n-6 52.37 51.94 50.20 49.52 22.38 23.13 23.09 21.45 C18:3 n-3 6.12 5.90 5.26 4.79 1.71 1.78 1.79 1.71 Minor fatty acids 1.26 1.34 1.42 1.51 1.06 1.04 0.94 0.88 SFA 19.39 19.97 21.98 23.18 43.39 43.12 43.69 45.33 MUFA 22.12 22.19 22.56 22.51 32.51 31.97 31.43 31.52 PUFA 58.49 57.84 55.46 54.31 24.10 24.91 24.88 23.16 UFA:SFA ratio 4.16 4.01 3.55 3.31 1.30 1.32 1.29 1.21 Lipid-class composition, % TAG 96.03 79.67 56.50 35.42 83.23 73.05 56.72 44.99 DAG 2.59 3.84 6.06 8.01 8.83 7.63 5.76 4.29 MAG 0.49 0.66 1.48 2.28 0.70 0.92 0.90 1.06 FFA 0.89 15.83 35.96 54.28 7.23 18.39 36.63 49.66 Abbreviations: DAG, diacylglycerols; FFA, free fatty acids; MAG, monoacylglycerols; MUFA, monounsaturated fatty acids; PUFA, polyunsaturated fatty acids; SFA, saturated fatty acids; TAG, triacylglycerols; UFA, unsaturated fatty acids. 1 All samples were analyzed at least in duplicate. 2 Dietary treatments supplemented with 6% of an unsaturated (S) or saturated fat source (P): S5 - 100% crude soybean oil, S15 - oil blend with 70% crude soybean oil and 30% acid soybean oil from chemical refining (SA), S35 - oil blend with 30% crude soybean oil and 70% SA, S50 - 100% SA, P5 - 100% crude palm oil, P15 - oil blend with 80% crude palm oil and 20% palm fatty acid distillate from physical refining (PA), S35 - oil blend with 53% crude palm oil and 47% PA, S50 - oil blend with 33% crude palm oil and 66% PA. IBM 2017). The model included the dietary fat type increased. The percentage of DAG increased in S diets, (soybean oils − unsaturated, or palm oils − saturated), but decreased in P diets, as the acid oil replaced the cor- and the FFA level (5, 15, 35, and 50%) as the main fac- responding crude oil. tors, as well as their interaction. Differences among treatment means were tested using Tukeys test for mul- tiple comparisons. In the case of the lipid-class content Growth Performance and digestibility of FA, this analysis was performed for Growth performance results are presented in Table 5. each GIT segment. A regression analysis was carried out BW at 37 d of age was higher and the FCR was lower for for each GIT segment and fat type (unsaturated or satu- the broilers fed S diets than for those fed P diets (P < rated), in order to find the best-fit regression equations 0.01). BW at 37 d was higher for the broilers fed the considering the SFA digestibility as the dependent vari- diets with 15% of FFA than for those fed the diets with able and the content of dietary FFA as the independent 5 and 35% of FFA, but no differences were observed for variable. The cage served as the experimental unit, so the AME intake among the broilers fed the different there were 6 replicates per dietary treatment. Results in diets. tables are reported as least-squares means, and differen- ces were considered significant at P < 0.05. Lipid-Class Content Along the RESULTS Gastrointestinal Tract Characterization of Experimental Diets The lipid-class content in the jejunum, ileum and excreta for the different dietary treatments is presented The chemical analysis and lipid-class characterization in Table 6. The broilers fed S diets showed a lower con- of the experimental diets (Table 4) reflected the compo- tent of FFA in the jejunum, ileum, and excreta (P < sition of the added oils. The unsaturated-to-saturated 0.001) and a higher content of TAG in the jejunum and FA ratio (UFA:SFA) of S diets was higher than in P ileum than did those fed P diets. The broilers fed the diets. In both S and P diets, as the acid oil replaced the diets with 5% of FFA showed a lower content of FFA in corresponding crude oil, the percentage of TAG the jejunum, in comparison to those fed the diets with decreased, and the percentage of MAG and FFA 15% FFA (P < 0.05), but no differences were observed FAT BY-PRODUCTS IN BROILER DIETS 5 Diets supplemented with 6% of an unsaturated (S) or saturated fat source (P): S5 - 100% crude soybean oil, S15 - oil blend with 70% crude soybean oil and 30% acid soybean oil from chemical refining (SA), S35 - oil blend with 30% crude soybean oil and 70% SA, S50 - 100% SA, P5 - 100% crude palm oil, P15 - oil blend with 80% crude palm oil and 20% palm fatty acid distillate from physical refining (PA), S35 - oil blend Fat FFA Interaction P-values were obtained from the univariate GLM procedure of SPSS conducted to study whether the dietary treatments, the dietary fat saturation degree and the dietary FFA percentage affected the growth for TAG. A significant interaction between the dietary NS NS NS NS NS P values fat saturation degree and FFA percentage was observed for DAG and MAG content in the jejunum (P < 0.05); the broilers fed the S50 diet had higher DAG and MAG ** NS NS NS NS content than did those fed the P50 diet, but no differen- ces were observed among the other diets. Interactions ** ** NS NS NS were also reported in the ileum for TAG, DAG and FFA 11.994 content. While the TAG content was higher for the 420.6 21.530 0.622 0.985 1.68 0.008 SEM broilers fed S35 and S50 diets than for those fed the S5 diet, no differences were observed among the ones fed P diets (P < 0.01). Similarly, broilers fed the S50 diet 147.9 87.9 2,244ab 50 showed higher DAG content than did those fed S5, S15, and all P diets (P < 0.001). Regarding FFA, while broilers fed S diets had similar FFA content in the ileum, 1.70 148.5 424.5 88.0 2,192b those fed the P15 diet had higher FFA content, in com- 35 parison to those fed P5 and P50 diets (P < 0.001). In the FFA%4 excreta, the broilers fed the diets with 5% of FFA had 1.68 149.9 442.9 90.7 the lowest FFA content (P < 0.001). Interactions were 2,309a 15 reported for DAG (P < 0.05) and MAG (P < 0.01) con- tent; the broilers fed the S35 diet showed higher DAG 1.70 85.7 144.5 432.2 content than did the ones fed S5, P5 and P50 diets, and 2,152b 5 the MAG content was higher for the broilers fed the P50 diet than for the ones fed the other P diets. 1.72 148.9 426.5 87.6 2,193 P Abbreviations: ADG, average daily gain; ADFI, average daily feed intake; BW, body weight; FCR, feed conversion ratio. Apparent Fatty-Acid Digestibility Along the Fat3 Table 5. Growth performance of broiler chickens (22 to 37d) according to different fat sources in the diet.1 1.66 Gastrointestinal Tract and Apparent 88.5 146.9 431.3 2,256 S Metabolizable Energy 1.74 The FA digestibility coefficients reported in the jeju- 85.5 149.0 430.8 P50 2,172 num, ileum, and excreta and the AME values for the dif- ferent dietary treatments are presented in Table 7. means in a row not sharing a common superscript letter are significantly different (P < 0.05). Saturated − P diets 1.71 Broilers fed S diets showed higher polyunsaturated FA 88.5 148.9 410.5 P35 2,194 (PUFA) digestibility coefficients than did those fed P with 53% crude palm oil and 47% PA, S50 - oil blend with 33% crude palm oil and 66% PA. diets, both in the jejunum and ileum. The digestibility of 1.69 total FA (TFA) in the ileum and excreta, and the 90.4 151.0 435.8 P15 2,280 digestibility of SFA in the excreta were also higher for the broilers fed S diets (P < 0.001). The digestibility Dietary treatments2 1.73 coefficients for TFA and SFA in the excreta were signifi- 146.8 435.9 86.1 P5 2,156 cantly the highest for the broilers fed the diets with 5% of FFA (P < 0.01). Significant interactions between the dietary fat saturation degree and the FFA percentage 1.63 146.8 410.2 90.4 S50 2,290 were reported in the jejunum for TFA, SFA and mono- unsaturated FA (MUFA; (P < 0.05)), in the ileum for Unsaturated − S diets SFA and MUFA (P < 0.05), and in the excreta for Values are means of 24 cages with 3 chickens/cage. Values are means of 12 cages with 3 chickens/cage. 1.70 Values are means of 6 cages with 3 chickens/cage. 148.2 440.7 87.4 S35 MUFA and PUFA (P < 0.05) digestibility coefficients. 2,190 In the ileum, a quadratic regression equation could be fit for the SFA digestibility coefficients in the broilers fed S 1.67 149.0 448.7 90.9 S15 diets (Figure 1; P < 0.01); the coefficients observed for 2,337 *P < 0.05; **P < 0.01; ***P < 0.001. the broilers fed S5, S15, and S35 diets were similar, while more variable and lower values were reported for those 1.67 142.2 429.4 85.3 S5 2,181 fed S50 diet. The coefficients reported for the broilers fed P diets did not fit any model; they were similar among them and in general lower than the coefficients AME intake, kcal/b/d performance values. observed for S5, S15, and S35. In the ileum and excreta, broilers fed S5 diet showed higher MUFA digestibility BW at 37 d, g ADFI, g/b/d ADG, g/b/d coefficients than those fed S50 diet (and also than those FCR, g/g fed S35 diet in the excreta), while the coefficients for the a-b broilers fed P5, P15, P35, and P50 diets were similar. Item 1 2 3 4 The digestibility of PUFA in the excreta followed the 6 Table 6. Lipid-class content1 along the gastrointestinal tract and excreta according to different fat sources in the diet in 37-day-old broiler chickens.2 Dietary treatments3 Fat4 FFA%5 P values Unsaturated − S diets Saturated − P diets Item S5 S15 S35 S50 P5 P15 P35 P50 S P 5 15 35 50 SEM Fat FFA Interaction Jejunum TAG 0.74 0.89 0.91 1.00 0.40 0.39 0.40 0.37 0.88 0.39 0.57 0.64 0.65 0.68 0.027 *** NS NS DAG 1.02ab 1.33ab 0.94ab 1.55a 0.96ab 1.14ab 1.20ab 0.88b 1.21 1.05 0.99 1.24 1.07 1.21 0.048 NS NS * MAG 0.60ab 0.76ab 0.71ab 0.88a 0.66ab 0.56ab 0.69ab 0.45b 0.74 0.58 0.62 0.66 0.70 0.66 0.027 ** NS * FFA 3.33 4.42 3.78 4.31 5.18 6.52 6.49 4.88 3.96 5.77 4.26b 5.47a 5.13ab 4.6ab 0.153 *** * NS RODRIGUEZ-SANCHEZ ET AL. Ileum TAG 0.30b 0.38ab 0.53a 0.53a 0.30b 0.33b 0.28b 0.30b 0.44 0.30 0.30 0.36 0.41 0.41 0.013 *** * ** DAG 0.45b 0.58b 0.71ab 0.90a 0.58b 0.57b 0.55b 0.51b 0.66 0.55 0.52 0.57 0.63 0.7 0.022 * * *** MAG 0.65 0.72 0.73 0.75 0.73 0.66 0.75 0.67 0.71 0.71 0.69 0.69 0.74 0.71 0.030 NS NS NS FFA 1.27d 1.36d 1.56d 1.91d 2.88c 4.17a 3.70ab 3.27bc 1.52 3.51 2.07 2.76 2.63 2.59 0.061 *** ** *** Excreta TAG 0.31 0.44 0.48 0.49 0.43 0.40 0.36 0.40 0.43 0.40 0.37 0.42 0.42 0.44 0.019 NS NS NS DAG 0.61b 0.88ab 1.10a 0.89ab 0.66b 0.87ab 0.74ab 0.67b 0.87 0.74 0.64 0.89 0.93 0.78 0.028 * ** * MAG 0.40bc 0.61ab 0.49abc 0.52abc 0.40bc 0.37c 0.41bc 0.63a 0.51 0.45 0.40 0.49 0.45 0.58 0.016 NS ** ** FFA 3.13 4.19 4.18 4.30 4.48 5.40 6.40 7.20 3.95 5.87 3.8b 4.80a 5.29a 5.75a 0.129 *** *** NS Abbreviations: DAG, diacylglycerols; FFA, free fatty acids; MAG, monoacylglycerols; TAG, triacylglycerols. P-values were obtained from the univariate GLM procedure of SPSS conducted to study whether the dietary treatments, the dietary fat saturation degree and the dietary FFA percentage affected the lipid-class content. P < 0.05 was considered significant. *P < 0.05; **P < 0.01; ***P < 0.001. a-d means in a row not sharing a common superscript letter are significantly different (P < 0.05). 1 Lipid-class concentration (mg/g) / Ti concentration (mg/g) in each gastrointestinal segment and excreta. 2 Diets supplemented with 6% of an unsaturated (S) or saturated fat source (P): S5 - 100% crude soybean oil, S15 - oil blend with 70% crude soybean oil and 30% acid soybean oil from chemical refining (SA), S35 - oil blend with 30% crude soybean oil and 70% SA, S50 - 100% SA, P5 - 100% crude palm oil, P15 - oil blend with 80% crude palm oil and 20% palm fatty acid distillate from physical refining (PA), S35 - oil blend with 53% crude palm oil and 47% PA, S50 - oil blend with 33% crude palm oil and 66% PA. 3 Values are means of 6 cages with 3 chickens/cage. 4 Values are means of 24 cages with 3 chickens/cage. 5 Values are means of 12 cages with 3 chickens/cage. Table 7. Apparent fatty-acid digestibility coefficients along the gastrointestinal tract and excreta, and apparent metabolizable energy according to different fat sources in the diet in 37- day-old broiler chickens.1 Dietary treatments2 Fat3 FFA%4 P values Unsaturated − S diets Saturated − P diets Item S5 S15 S35 S50 P5 P15 P35 P50 S P 5 15 35 50 SEM Fat FFA Interaction FAT BY-PRODUCTS IN BROILER DIETS Jejunum TFA 0.84a 0.84a 0.84a 0.79a 0.71b 0.66b 0.68b 0.70b 0.83 0.69 0.77 0.75 0.76 0.75 0.005 *** NS * SFA 0.64 0.64 0.64 0.53 0.60 0.56 0.57 0.64 0.61 0.59 0.62 0.60 0.60 0.59 0.012 NS NS * MUFA 0.83 0.83 0.82 0.78 0.81 0.78 0.78 0.80 0.81 0.79 0.82 0.81 0.80 0.79 0.005 * NS * PUFA 0.91 0.92 0.93 0.90 0.77 0.69 0.73 0.77 0.91 0.74 0.84 0.81 0.83 0.84 0.006 *** NS NS Ileum TFA 0.92 0.90 0.90 0.86 0.79 0.75 0.77 0.79 0.89 0.78 0.85 0.82 0.83 0.83 0.006 *** NS NS SFA 0.81a 0.76ab 0.77ab 0.69abc 0.68bc 0.60c 0.66bc 0.69abc 0.76 0.66 0.74 0.68 0.72 0.69 0.010 *** NS * MUFA 0.91a 0.89ab 0.90ab 0.86b 0.88ab 0.86b 0.88ab 0.89ab 0.89 0.88 0.90 0.88 0.89 0.87 0.004 NS NS * PUFA 0.95 0.95 0.95 0.93 0.86 0.86 0.88 0.87 0.95 0.87 0.91 0.91 0.91 0.90 0.003 *** NS NS Excreta TFA 0.86 0.83 0.82 0.79 0.74 0.69 0.69 0.70 0.82 0.71 0.80a 0.76b 0.75b 0.75b 0.005 *** ** NS SFA 0.78 0.72 0.72 0.67 0.65 0.55 0.54 0.57 0.72 0.58 0.71a 0.63b 0.63b 0.62b 0.008 *** *** NS MUFA 0.87a 0.84abc 0.82bc 0.80c 0.85ab 0.84abc 0.84abc 0.85ab 0.83 0.84 0.86 0.84 0.83 0.82 0.004 NS ** * PUFA 0.88a 0.86ab 0.85ab 0.82bc 0.75d 0.75d 0.78cd 0.77d 0.85 0.76 0.82 0.80 0.82 0.79 0.004 *** NS ** AME5 3,019a 3,013a 2,987a 2,840cd 2,985ab 2,896bc 2,758d 2,870c 2,965 2,877 3002 2955 2873 2855 6.955 *** *** *** Abbreviations: MUFA, monounsaturated fatty acids; PUFA, polyunsaturated fatty acids; SFA, saturated fatty acids; TFA, total fatty acids. P-values were obtained from the univariate GLM procedure of SPSS conducted to study whether the dietary treatments, the dietary fat saturation degree and the dietary FFA percentage affected the fatty-acid digestibility results. *P < 0.05; **P < 0.01; ***P < 0.001. a-d means in a row not sharing a common superscript letter are significantly different (P < 0.05). 1 Diets supplemented with 6% of an unsaturated (S) or saturated fat source (P): S5 - 100% crude soybean oil, S15 - oil blend with 70% crude soybean oil and 30% acid soybean oil from chemical refining (SA), S35 - oil blend with 30% crude soybean oil and 70% SA, S50 - 100% SA, P5 - 100% crude palm oil, P15 - oil blend with 80% crude palm oil and 20% palm fatty acid distillate from physical refining (PA), S35 - oil blend with 53% crude palm oil and 47% PA, S50 - oil blend with 33% crude palm oil and 66% PA. 2 Values are means of 6 cages with 3 chickens/cage. 3 Values are means of 24 cages with 3 chickens/cage. 4 Values are means of 12 cages with 3 chickens/cage. 5 Apparent metabolizable energy (kcal/kg). Values are pooled means of 6 replicates with 3 chickens/replicate. 7 8 RODRIGUEZ-SANCHEZ ET AL. same trend. Regarding the AME values, there was a sig- nificant interaction between the dietary fat saturation degree and FFA percentage; the values for S5, S15, S35, and P5 diets were similar to each other and higher in comparison to the other dietary treatments, and the AME value for S50 was similar to the values reported for P15, P35, and P50. DISCUSSION Lipid classes, together with FA digestibility coeffi- cients, help in understanding the dynamics of fat diges- tion and absorption processes along the GIT in broilers (Rodriguez-Sanchez et al., 2019b). It has been docu- mented that fat digestion is initiated in the gizzard and continues in the duodenum (Ravindran et al., 2016). Figure 1. Relation between the saturated fatty-acid digestibility cal- culated in the ileum, and the four dietary free-fatty-acid percentages1 for The results from the gizzard and duodenum have not two different fat sources ( soybean oil -unsaturated diets, and  palm oil been presented, as they are likely to be affected by the − saturated diets) in 37-day-old broilers. 1Diets with an average of 5, 15, gastro-duodenal retrograde movement of digesta, and 35, and 50% of FFA. Each point represents each replicate value (with 3 chickens/replicate). The black line illustrates the quadratic model the secretion of endogenous fat into the duodenum observed for the unsaturated diets: y = -3-5 £ 2 − 0.006x, where lumen (Rodriguez-Sanchez et al., 2019a,b). As the R2 = 0.39 and P = 0.007. The saturated diets did not fit any model. absorption process of FA takes place in the jejunum Abbreviations: FFA, free fatty acids; SFA, saturated fatty acids. and, to a lesser extent, in the ileum (Tancharoenrat et al., 2014; Rodriguez-Sanchez et al., 2019a,b), the which is in agreement with Vilarrasa et al. (2015b) and results in these 2 GIT segments will be the focus of the Rodriguez-Sanchez et al. (2019b). The same pattern was Discussion. observed for the individual FA digestibility coefficients The lower FFA content observed in the jejunum, the in the jejunum and ileum (Figure 2). However, the inter- higher digestibility coefficients for TFA in the ileum and actions reported in the ileum for TAG and DAG for PUFA, both in the jejunum and ileum, the higher BW, and the lower FCR for the broilers fed S diets, con- firm the more efficient absorption of the unsaturated diets in 37-day-old broilers. All of this corroborates that the higher the degree of unsaturation of a fat, the greater the ability of the FA to be absorbed. This is in accor- dance with Tancharoenrat et al. (2014), and Rodriguez- Sanchez et al. (2019b), who described the better ability of UFA to be absorbed in 21-, and 14-day-old broilers, respectively, and with Rodriguez-Sanchez et al. (2019a), who reported that unsaturated diets are more efficiently used than are saturated diets regardless of age. The natural emulsifying properties of UFA could explain their higher absorption in the GIT, as they assist in the formation of mixed micelles and, therefore, in the FA absorption process (Krogdahl, 1985; Smulikowska and Mieczkowska, 1996). It is well known that the ability to absorb dietary fat in broilers increases with age (Noy and Sklan, 1995; Batal and Parsons, 2002; Tancharoenrat et al., 2013). In agreement with this, the ileal TFA digestibility coeffi- cients reported in the present study in 37-day-old broilers were higher than those reported in the starter period of the study in 14-day-old broilers (Rodriguez- Sanchez et al., 2019b). The lack of differences among the four S diets, and Figure 2. Individual fatty-acid apparent digestibility coefficients in among the four P diets with different dietary FFA per- the (A) jejunum, and (B) ileum for the four different dietary free-fatty- centages, both in the jejunum and ileum, for TFA, SFA, acid percentages (5%, 15%, 35%, and 50%) in 37-day-old broiler chickens. Values are means § SE of 12 replicates per each dietary FFA percentage and PUFA digestibility coefficients, showed that the die- (with 3 chickens/replicate). Palmitic (C16:0), stearic (C18:0), oleic tary fat saturation degree affected the absorption of FA (C18:1n9), and linoleic (C18:2n6) acids. Abbreviation: FFA, free fatty to a greater extent than the dietary FFA percentage, acids. FAT BY-PRODUCTS IN BROILER DIETS 9 content, as well as for MUFA digestibility coefficients, (Tancharoenrat et al., 2014), where 68% and 75% of and the regression analysis of the ileal SFA digestibility TFA, respectively, were absorbed in this GIT seg- coefficients (Figure 1), suggest that the dietary FFA ment. This suggests that the improvement in the content affects the FA absorption process, and that this absorption process of FA from starter to grower-fin- effect could be different between the broilers fed the isher broilers is mainly due to the higher contribution unsaturated (S), and saturated (P) diets. While dif- of the jejunum to this process. The contribution of ferences were observed between the broilers fed S5 the ileum to the FA absorption process was lower, as and S50 diets (the results for S5, S15 and S35 being there were less FA reaching this GIT segment, but it similar), the results reported for those fed P5 and was especially important for the absorption of SFA, P50 diets were equivalent. Thus, according to the being higher for the broilers fed S diets (19%) than present results, the replacement of crude oils with for those fed P diets (10%). This difference has also acid oils (fat by-products rich in FFA) in grower-fin- been reported in 14-day-old broilers (Rodriguez- isher broiler diets do not have a negative impact on Sanchez et al. (2019b), where the contribution of the the FA absorption process, at least when the dietary ileum to SFA absorption was 46% and 27% for the FFA percentage does not exceed 35%. This finding broilers fed S and P diets, respectively, and explains also supports the greater efficiency of the FA absorp- the higher absorption of unsaturated diets as sug- tion process in broilers of 37 d of age. Rodriguez- gested by these authors. Sanchez et al. (2019b) reported that in broilers of 14 The present results show that, despite the fact soy- d of age, unsaturated diets with more than 15% of bean oil sources are, in general, associated with higher FFA decreased the absorption of FA, and that palm FA digestibility coefficients, palm oil sources can also be oil sources were not suitable. Thus, the results show used as a fat source for grower-finisher broiler diets. The that 37-day-old broilers are less affected by both the results give information regarding the sampling process dietary fat saturation degree and FFA content than for fat digestibility studies; we do not recommend taking are 14-day-old broilers. It is important to mention digesta samples from either the gizzard or the duode- the differences in the composition of the experimental num, as these results are greatly affected by gastroduo- oils (SA had more moisture and unsaponifiable than denal reverse peristalsis and the endogenous secretion of SO and PA), and experimental diets (FFA increase lipids into the duodenal lumen. Similarly, we do not rec- was 98% in S diets vs. 85% in P diets). There were ommend using excreta samples, as the results are likely also differences in the FA composition among the die- to be affected by the presence of lipids from bacterial tary treatments, which were reflected in the UFA: activity. Jejunum and ileum digesta are the most SFA ratios (Table 4); while in S diets there was a representative samples to consider for FA digestibility greater content of dietary SFA, as the dietary FFA studies. percentage was higher, in P diets the SFA content In conclusion, the dietary fat type (saturation degree) remained constant. affected the FA absorption process to a greater extent The results reported in the excreta suggest that than did the dietary FFA content, and the absorption of diets with more than 5% of dietary FFA have a nega- the unsaturated diets was more efficient than was the tive impact on FA absorption. However, it is likely absorption of the saturated diets, irrespective of their that these results are affected by the presence of lip- dietary FFA content. The results from the present study ids from bacterial activity (Rodriguez-Sanchez et al., are promising as they demonstrate that including acid 2019b). In fact, FA of possible bacterial origin were oils (fat by-products rich in FFA) in grower-finisher detected in the excreta (capric, margaric, elaidic acid, broiler diets does not have a negative impact on the FA and vaccenic acids), their total concentration (mg/g) absorption process, at least when the dietary FFA per- being affected by the dietary FFA percentage (data centage does not exceed 35%, which means that chickens not shown in table; 5% FFA: 1.40b, 15% FFA: can successfully use these dietary fat sources. The use of 1.58ab, 35% FFA: 1.71a, and 50% FFA: 1.51ab). acid oils in broiler diets is an interesting strategy to This finding altogether with the noneffective absorp- reduce the cost of feed formulation while also reducing tion of FA by the animal after the ileum (Ren- their environmental impact. ner, 1965) is the reason why the results of the jejunum and ileum are more representative of the FA absorption process. Taking this into account, the con- ACKNOWLEDGMENTS tribution of the jejunum and ileum on FA absorption was calculated considering the ileal digestibility coef- This work was carried out thanks to the financial sup- ficients as the maximum (100%) and expressing the port provided by the Ministerio de Economía y Competi- digestibility coefficients reported in the jejunum and tividad of the Spanish Government (Project AGL2015- ileum towards these values. It was observed that irre- 64431-C2-1-R), a pre-doctoral research grant from the spective of the dietary fat source, more than 80% of Ministerio de Educacion y Formacion Profesional of the FA were absorbed in the jejunum. This has been pre- Spanish Government (Ref. FPU/06063), and a post- viously reported in 14-day-old broilers (Rodriguez- doctorate contract within the Juan de la Cierva program Sanchez et al., 2019b) and 21-day-old broilers from the Ministerio de Ciencia e Innovaci on of the 10 RODRIGUEZ-SANCHEZ ET AL. Spanish Government (Ref. JCI-2012-13412). The digestibility along the gastrointestinal tract of broiler chickens fed English of this manuscript has been proofread by Mr. different fat sources at different ages. Poult. Sci. 98:1341–1353. Rodriguez-Sanchez, R., A. Tres, R. Sala, C. Garces-Narro, Chuck Simmons, a native English-speaking, retired uni- F. Guardiola, J. Gasa, and A. C. Barroeta. 2019b. Effects of die- versity Instructor of English. tary free fatty-acid content and saturation degree on lipid-class composition and fatty-acid digestibility along the gastrointestinal tract in broiler starter chickens. Poult. Sci. 98:4929–4941. DISCLOSURES Roll, A. P., E. Vilarrasa, A. Tres, and A. C. Barroeta. 2018. The dif- ferent molecular structure and glycerol-to-fatty acid ratio of palm The authors declare no conflict of interest. All authors oils affect their nutritive value in broiler chicken diets. 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