Evolution of lipid classes and fatty acid digestibility along the gastrointestinal tract of broiler chickens fed different fat sources at different ages R. Rodriguez-Sanchez ,∗,1 A. Tres,† R. Sala,∗ F. Guardiola,† and A. C. Barroeta∗ ∗ Animal Nutrition and Welfare Service, Department of Animal and Food Science, Facultat de Veterinària, Universitat Autònoma de Barcelona, E-08193 Bellaterra, Barcelona, Spain; and † Nutrition, Food Science and Gastronomy Department – XaRTA-INSA, Facultat de Farmàcia i Ciències de l’Alimentació, Universitat de Barcelona, Joan XXIII, 27-31, E-08028 Barcelona, Spain ABSTRACT The aim of the present study is to eval- ability of adult chickens to absorb fat was higher than uate the effect of the dietary fat saturation degree and for young chickens. The results show that the duode- age on the lipid class (TAG, DAG, MAG, and FFA) num is the main place of fat digestion (hydrolysis), and composition and fatty acid digestibility along the gas- the jejunum the main place of fat absorption. The role trointestinal tract (GIT) and excreta in broiler chick- of the ileum on fat absorption is very important, as it ens. A total of 120 one-day-old female broiler chickens is the last segment of the GIT where the absorption of were randomly distributed in 2 dietary treatments (6 fatty acids has been described. Thus, it was the contri- cages/treatment), which resulted from the supplemen- bution of the ileum that was responsible for the higher tation of a basal diet with 6% of soybean oil or palm oil. fat utilization observed for animals fed the unsaturated Two digestibility balances were carried out at 14 and 35 diet than for those fed the saturated diet at 14 d, and d and fatty acid digestibility and lipid class composition it was also responsible for the improvement on the uti- were determined in the gizzard, duodenum, jejunum, lization of the saturated diet between 14 and 35 d. All ileum, and excreta. Along de GIT, both fatty acid di- the results suggest that the absorption of fatty acids is gestibility and lipid class composition were influenced more limiting than is hydrolysis, because the main dif- by the dietary fat source and the age of the chickens. ferences were observed in the jejunum and ileum, where The absorption of the unsaturated fat was more effi- the absorption of fatty acids takes place. cient and faster than it was for the saturated fat. The Key words: fat saturation degree, fatty acid digestibility, fatty acid absorption, lipid class, broiler 2019 Poultry Science 98:1341–1353 http://dx.doi.org/10.3382/ps/pey458 INTRODUCTION tion and are absorbed passively from the intestinal lu- men while others, like long-chain saturated FA (SFA) Among the ingredients used in the formulation of and diacylglycerols (DAG), need the solubilization in animal feeds, fats and oils are the most concentrated the hydrophobic cores of mixed micelles to be trans- sources of energy, and their use in poultry diets is a ported to the intestinal cells and absorbed. widespread practice. It is well known that factors re- It is accepted that the small intestine is where most lated to the diet like the saturation degree of fat or the of the digestion and absorption processes take place. chain length of fatty acids (FA), and factors related to However, in broiler chickens, the digestibility of fat is the animal like the age, strain, or gender affect the di- usually determined in the ileum or excreta and, conse- gestion and absorption of lipids (Renner and Hill 1961; quently, the information regarding the relative impor- Sklan et al., 1973; Ravindran et al., 2016). tance of the different segments of the gastrointestinal Briefly, fat digestion starts with the hydrolysis of tri- tract (GIT) is scarce. There are also other factors like acylglycerols (TAG), which are the main component the gastro-duodenal reverse peristalsis (Sacranie et al., of fat in the diet, by pancreatic lipase, mainly result- 2012) and the fat endogenous secretion of lipids into ing in a sn-2-monoacylgycerol (MAG) and two free the intestine that make these processes more complex. FA (FFA) molecules from the sn-1 and sn-3 positions. Tancharoenrat et al. (2014) considered the impor- Some of the resulting products do not need emulsifica- tance of studying the digestibility of FA in different segments of the GIT to understand the dynamics of  fat digestion; however, there is no available informa- C 2018 Poultry Science Association Inc. Received May 24, 2018. tion about the composition of lipid classes (TAG, DAG, Accepted September 6, 2018. MAG, and FFA) in the different GIT segments. The 1 Corresponding author: Raquel.Rodriguez@uab.cat determination of lipid classes is a way to assess the 1341 1342 RODRIGUEZ-SANCHEZ ET AL. hydrolysis of TAG, through their disappearance and the Table 1. Ingredient composition of the experimental diets. parallel appearance of lipolysis products (DAG, MAG, Starter diet Grower–finisher diet and FFA), as well as the absorption of lipolysis products Ingredients, % (from 0 to 21 d) (from 22 to 35 d) through their disappearance from the intestinal lumen. Wheat 54.46 44.00 The present study was conducted in order to see the Soybean meal 48% 35.40 27.25 effect of the dietary fat saturation degree and the age of Barley – 18.58 Experimental fats1 6.00 6.00 the chickens (14 and 35 d) on FA digestibility and lipid Calcium carbonate 1.44 1.39 class composition along the GIT (gizzard, duodenum, Sodium chloride 0.40 0.35 jejunum, and ileum) and excreta. This will allow for a Vitamin and mineral premix2 0.40 0.40 DL-Methionine 0.23 0.17 better understanding of the relative importance of the L-Lysine 0.15 0.12 different GIT segments in the fat digestion and absorp- L-Threonine – 0.02 tion processes, as well as to see if there are some changes Titanium dioxide 0.50 0.50 Ethoxyquin 66% 0.02 0.02 according to the dietary fat source and the age of the chickens, as it was hypothesized that the fat digestion 1 Soybean or palm oil. 2 and absorption dynamics will be affected by these two Provides per kg of feed: vitamin A (from retinol), 13,500 IU; vitamin D3 (from cholecalciferol), 4800 IU; vitamin E (from alfa-tocopherol), 49.5 factors. This information is essential to know the ideal IU; vitamin B1 , 3 mg; vitamin B2 , 9 mg; vitamin B6 , 4.5 mg; vitamin B12 , composition of dietary fats in order to optimize their 16.5 μ g; vitamin K3 , 3 mg; calcium pantothenate, 16.5 mg; nicotinic acid, utilization. 51 mg; folic acid, 1.8 mg; biotin, 30 μ g; Fe (from FeSO4 ·7H2 O), 54 mg; I (from Ca(I2 O3 )2 ), 1.2 mg; Co (from 2CoCO3 ·3Co(OH)2 ·H2 O), 0.6 mg; Cu (from CuSO4 ·5H2 O), 12 mg; Mn (from MnO), 90 mg; Zn (from ZnO), 66 mg; Se (from Na2 SeO3 ), 0.18 mg; Mo (from (NH4 )6 Mo7 O24 ), 1.2 mg. MATERIALS AND METHODS Animals and Diets Two digestibility balances were carried out from 11 The study was performed at the animal experimen- to 14 d and from 33 to 35 d. At 14 d of age, 7 birds per tal facilities of the Servei de Granges i Camps Ex- cage were killed by cervical dislocation and samples of perimentals (Universitat Autònoma de Barcelona; Bel- the content of the gizzard, duodenum (from the pyloric laterra, Barcelona, Spain). The experimental procedure junction to the distal-most point of insertion of the duo- received prior approval from the Animal Protocol Re- denal mesentery), jejunum (from the distal-most point view Committee of the same institution. All animal of insertion of the duodenal mesentery to the junction housing and husbandry conformed to the European with Meckel’s diverticulum), ileum (from the junction Union Guidelines (2010/63/EU). with Meckel’s diverticulum to a point 1 cm proximal to A total of 120 one-day-old female broiler chickens the ileocecal junction), and a representative sample of of the Ross 308 strain were obtained from a commer- excreta of each cage were taken. The digestive content cial hatchery (Pondex SAU; Lleida, Spain). On arrival, of each segment of the GIT from all birds within each chicks were wing-banded, weighed (initial BW, 40.54 g cage was pooled, homogenized, and frozen at −80◦ C un- ± 2.18 g) and randomly assigned to one of the two di- til further analysis. At 35 d of age, the remaining birds etary treatments, with 10 chicks per cage and 6 cages were euthanized and the same procedure was repeated. per treatment. Birds were housed in wire-floor cages. Throughout the study, feed and water were supplied for ad libitum consumption, and animals were raised Chemical Analysis under controlled conditions of light and temperature, Analytical determinations of the diets were per- as recommended by the breeder. formed according to the methods of the AOAC In- Birds received a starter feed (in mash form) until ternational (2005): dry matter (Method 934.01), ash day 21 and a grower–finisher feed (in mash form) from (Method 942.05), crude protein (Method 968.06), crude day 22 to day 35. Wheat- and soybean-meal-based di- fat (Method 2003.05), and crude fiber (Method 962.09). ets were formulated to meet or exceed FEDNA require- Gross energy was determined by an adiabatic calorime- ments (2008). The two dietary treatments were ob- ter (IKA C-4000, Janke-Kunkel; Staufen, Germany). tained including 6% of crude soybean oil (S) or crude TiO2 was analyzed following the procedures of Short palm oil (P) in the basal diet; the ingredient composi- et al. (1996) with small changes. Briefly, 0.2 g of fresh tion of the experimental diets is presented in Table 1. sample was ashed and dissolved in 2 mL of 7.4 M sul- Titanium dioxide (TiO2 ) was added (5 g/kg) as an in- phuric acid. A 1/10 dilution was prepared, using 0.5 mL ert marker for the determination of the digestibility of of the sample and 4.5 mL of a 1% HNO3 solution. Ti FA. was determined by ICP-OES (Optima 3200 RL, Perkin Elmer; Waltham, MA, USA) under standard condi- Controls and Sampling tions. For the determination of the FA composition of the Feed consumption and BW were measured weekly to feed, excreta, and digestive content, lipids were ex- calculate the ADFI, ADG, and the feed conversion ratio tracted as follows: 0.5 g of the fresh sample was weighed (FCR) throughout the experiment. in a test tube and 3 mL of HCl 1 N solution and FAT DIGESTION ALONG THE DIGESTIVE TRACT 1343 7.5 mL of diethyl ether were added; the content of columns (Styragel HR 1 and Styragel HR 0.5) of 30 cm each tube was homogenized for 30 s at 20,000 rpm, us- × 0.78 cm i.d., filled with a spherical styrenedivinyl- ing a high-speed homogenizer (Polytron R System PT benzene copolymer of 5-μm particle size and 100 Å 3100; Kinematica AG; Switzerland). After the homog- and 50 Å, respectively (Water Associates; Milford, MA, enization, the content of each tube was poured into USA), connected in series. The mobile phase consisted a centrifuge test tube, and the procedure of adding of tetrahydrofuran (HPLC quality grade) at 1 mL/min. 7.5 mL of diethyl ether and homogenizing was repeated Lipid classes were identified by matching their reten- and the mixtures were then pooled in the centrifuge tion times with those of standards (trioleoylglycerol tube. 1 mL of a saturated NaCl solution was added, for TAG, dioleoylglycerol for DAG, oleoylglycerol for and the tubes were centrifuged for 15 min at 618.5 g. MAG, and oleic acid for FFA; Sigma-Aldrich Co.; St. After the centrifugation, the upper layer was trans- Louis, MO, USA) and quantified by internal normaliza- ferred into a round-bottom flask. Then, 15 mL of di- tion (area percent). ethyl ether were added and the centrifugation process The macronutrient, FA, and lipid class composition was repeated as described above. The upper layer was of the experimental diets are presented in Table 2. transferred into the round-bottom flask. The content in the round-bottom flask was evaporated completely in a rotatory evaporator set at 35◦ C, the extracted fat Formulas used for Digestibility was transferred by dissolving it in diethyl ether in a Determination glass test tube and the content was evaporated using The digestibility coefficients of FA in each segment N2 at 35◦ C. The tubes with the extracted fat were kept of the GIT were determined using the TiO2 ratio in the at −20◦ C until FA determination. FA methyl esters feed and digestive content or excreta according to the were obtained as described by Guardiola et al. (1994). following formula: Briefly, the fat extracted was methylated with sodium methoxide (0.5 N), followed by boron trifluoride (20 wt % in methanol), and FA methyl esters were extracted Digestibility of FA = 1 − (([Ti]diet/ with n-hexane. Subsequently, FA methyl esters were an- [FA]diet)/([Ti]dig/[FA]dig)), alyzed using an Agilent 4890D gas chromatograph (Ag- ilent Technologies; Santa Clara, CA, USA) equipped with a flame ionization detector and a capillary col- where [Ti]diet is the concentration of TiO2 in the diet umn (SP-2380, 60 m × 0.25 mm i.d., 0.2 μm from (g/g DM), [FA]diet is the FA content in the diet (g/g Supelco; Bellefonte, PA, USA). Hydrogen (25 p.s.i.) DM), [Ti]dig is the concentration of TiO2 in the diges- was used as the carrier gas. Injector and detector were tive content or excreta (g/g DM), and [FA]dig is the FA set at 270 and 300◦ C, respectively. Oven temperature content in the digestive content or excreta (g/g DM). was set at 150◦ C for 1 min, increased by 3◦ C/min to Regarding the apparent metabolizable energy 180◦ C; after 0.5 min at 180◦ C, it was increased by (AME), it was determined from the product of the 14.5◦ C/min to 220◦ C, maintained at 220◦ C for 3 min, energy utilization ratio and its corresponding gross and finally increased by 9.9C◦ /min to 250◦ C, and main- energy of feed. tained for 3 min at this temperature. FA methyl esters were identified by matching their retention times with Statistical Analysis those of standards (Supelco 37 component FAME Mix, Sigma-Aldrich Co.; St. Louis, MO, USA). Quantifica- Productive parameters were subjected to Student’s tion was performed by means of calibration curves of t-test using the type of fat in feed as a factor (SPSS single FA methyl esters (C14:0, C16:0, C18:0, C16:1n- statistics 25.0.0.0, IBM 2017). For the comparison of 7, C18:1n-9, C18:2n-6, C18:3n-3, and C24:0 methyl es- the lipid class composition and FA digestibility between ters), using nonadecanoic acid methyl ester as an inter- both dietary treatments a Student’s t-test was also per- nal standard (all purchased from Sigma-Aldrich Co.; St. formed for each one of the GIT segments. A univariate Louis, MO, USA). analysis using the GLM procedure was also performed Lipid class composition (TAG, DAG, MAG, and for FA digestibility to study whether FA composition FFA) of the extracted fat from the feed, excreta, and di- of the intestinal content depended on the added fat, gestive content was determined by size-exclusion chro- and also of the GIT segment. For the comparison of matography on an Agilent 1100 series HPLC chromato- the results observed at different ages, both for the lipid graph equipped with an isocratic pump, with the oven classes and FA digestibility, a univariate analysis using and a Refractive Index Detector (RID) both set at the GLM procedure was carried out for each one of the 35◦ C. Previously, the lipids of the feed, excreta, and GIT segments in order to study whether the composi- digestive content were extracted as explained above. tion of intestinal content depended on the added fat, After lipid extraction, lipids were dissolved in 2 mL and on the age of the chickens. Pairwise comparisons of tetrahydrofuran and filtered through a Nylon filter were used to see the differences in every parameter be- (0.45 μm), and 100 μL were injected (20 μL loop) into tween both fat sources, and ages. The cage served as the HPLC. Separation was conducted using 2 Styragel the experimental unit, so that there were 6 replicates 1344 RODRIGUEZ-SANCHEZ ET AL. Table 2. Analyzed1 macronutrient content and fatty acid and lipid-class composition of the experimental diets2 for the starter. Starter diets Grower–finisher diets (from 0 to 21 d) (from 22 to 35 d) Item S P S P Macronutrient content, Dry matter % 92.41 89.72 92.95 90.48 Crude protein % 21.20 20.88 17.64 18.05 Crude fat % 7.39 7.68 6.88 6.76 Crude fiber % 3.18 3.37 2.94 3.09 Ash % 5.64 6.30 5.15 5.93 Gross energy, kcal/kg 4259 4207 4190 4161 AME3 3284 3119 3327 3199 Fatty acid composition, % C14:0 – 0.81 – 0.79 C16:0 12.43 36.79 13.83 35.96 C18:0 3.76 4.11 3.75 4.12 C18:1 n-9 19.16 32.45 19.45 31.44 C18:1 n-7 0.28 0.31 0.58 0.33 C18:2 n-6 54.39 21.78 52.83 23.36 C18:3 n-3 7.44 2.15 7.28 2.36 Minor fatty acids 2.54 1.59 2.28 1.62 SFA 17.07 42.06 18.24 41.22 MUFA 21.02 33.99 21.65 33.05 PUFA 61.83 23.93 60.11 25.72 UFA:SFA 4.86 1.38 4.29 1.62 Lipid-class composition, % TAG 89.28 80.84 94.91 81.40 DAG 3.51 9.75 2.93 9.45 MAG 2.79 2.59 0.71 0.94 FFA 4.41 6.81 1.45 8.21 1 All samples were analyzed at minimum in duplicate. 2 Diets with 6% of soybean oil (S) or palm oil (P). 3 Values are pooled means of 12 replicates: with 10 chickens/replicate at 14 days, and 3 chickens/ replicate at 35 days. SFA = saturated fatty acids; MUFA = monounsaturated fatty acids; PUFA = polyunsaturated fatty acids; UFA = unsaturated fatty acids; TAG = triacylglycerols; DAG = diacylglycerols; MAG = monoacylglycerols; FFA = free fatty acids. per treatment. Differences were considered significant in the starter (from 0 to 21 d), the grower–finisher (from at P < 0.05. 22 to 35 d) and the global (from 0 to 35 d) periods are presented in Table 3. No significant differences were RESULTS observed in any feeding period (P > 0.05). Characterization of Experimental Diets Determinations in 14-day-old Broiler The chemical analysis of the experimental diets re- Chickens flected the composition of the added oils (P or S) and is presented in Table 2. The main FA in S di- Lipid Class Composition along the Gastrointesti- ets were linoleic and oleic acids, while the main FA in nal Tract. The lipid class composition (TAG, DAG, P diets were palmitic, oleic, and linoleic acids, both MAG, and FFA) in different GIT segments (gizzard, in the starter and the grower–finisher period. The duodenum, jejunum, and ileum) and excreta was de- unsaturated-to-saturated FA ratios also reflected the termined in 14-day-old broiler chickens fed diets with composition of the added oils. All of the diets had high two different fat sources (S and P), and is shown in proportions of TAG (>80%), and low proportions of Figure 1. DAG, MAG, and FFA (Table 2). However, there were Considering the average results of both fat sources some differences between the S and P diets. S diets had together at 14 d, TAG was the major lipid class in the higher proportions of TAG, and lower proportions of gizzard (65.17%), while in the duodenum it only rep- DAG and FFA, in comparison to P diets, for both pe- resented 13.52% of all lipid classes. The percentage of riods. The proportion of MAG between S and P diets DAG was also lower in the duodenum (9.81%) than was similar in each period. in the gizzard (15.02%). In parallel to the TAG and DAG disappearance (hydrolysis), the percentage of the Growth Performance end lipolysis products (MAG and FFA) increased, espe- cially the FFA percentage, which was the most predom- The broiler chickens completed the trial successfully. inant lipid class in the duodenum (70.55%), jejunum The effect of dietary fat sources on growth performance (78.06%), ileum (75.19%), and excreta (61.38%). From FAT DIGESTION ALONG THE DIGESTIVE TRACT 1345 Table 3. Growth performance of broiler chickens according to in the excreta since the results are expressed in area different fat sources in the diet.1 normalization (%). Dietary treatments Statistics Considering the added fat source (Figure 1), TAG was higher for S than for P in the gizzard (P = 0.018), Item S P SEM P-value while both DAG (P = 0.034) and FFA (P = 0.024) From 0 to 21 d were lower for S than for P. There were no differences ADFI, g/d per bird 48.4 49.1 1.089 0.76 ADG, g/d per bird 36.9 37.1 0.705 0.89 in the duodenum for any of the lipid classes, but the FCR, g/g 1.31 1.33 0.016 0.72 reduction in the TAG percentage from the gizzard to BW at 21 d, g 815 819 14.815 0.89 the duodenum, as well as the percentage of MAG and From 22 to 35 d FFA in the duodenum, was numerically higher for S ADFI, g/d per bird 146.1 150.2 1.180 0.11 ADG, g/d per bird 88.1 89.6 1.072 0.52 than for P. In the jejunum, the percentage of TAG was FCR, g/g 1.66 1.68 0.014 0.45 significantly higher for S than for P (P = 0.046). Re- From 0 to 35 d garding the lipolysis products, the percentage of MAG ADFI, g/d per bird 87.5 89.6 0.878 0.26 was higher for S than for P in the jejunum and ileum ADG, g/d per bird 57.4 58.1 0.657 0.61 FCR, g/g 1.52 1.54 0.007 0.27 (P = 0.004 and P = 0.041, respectively), while the per- BW at 35 d, g 2049 2073 23.0 0.61 centage of FFA was lower for S than for P in the je- 1 junum (P = 0.011), ileum (P < 0.001), and excreta Diets with 6% of soybean oil (S) or palm oil (P). ADFI = average daily feed intake; ADG = average daily gain; FCR (P < 0.001). In the ileum and excreta, the percent- = feed conversion ratio; BW = body weight. age of TAG and DAG was also significantly higher Values are means of 6 replicates. SEM = standard error of the mean. for S than for P (P < 0.001). This finding is related P-values were obtained from Student’s t-test conducted to study whether the type of fat added to feed affected growth performance values. to the absorption (disappearance) of the other lipid P < 0.05 was considered significant. classes (MAG and FFA). Fatty Acid Digestibility Coefficients along the Gastrointestinal Tract. FA digestibility coefficients were also studied in different segments of the GIT, the duodenum to the ileum, the percentage of MAG was and in the excreta, for the 2 different fat sources (S lower in each segment (6.12% in the duodenum, 4.50% and P) and are shown in Table 4. SFA digestibil- in the jejunum, and 4.14% in the ileum). The increase ity was mainly represented by palmitic (C16:0) and in TAG, DAG, and MAG percentage in the excreta in stearic (C18:0) acids, monounsaturated FA (MUFA) comparison to the results observed in the ileum, were digestibility by oleic acid (C18:1n-9), and polyunsatu- due to the high absorption of FFA in the ileum, which rated FA (PUFA) digestibility by linoleic (C18:2n-6) implies a relative increase of the rest of lipid classes acid. Figure 1. Lipid class composition (%) of the feed and along the gastrointestinal tract (each value represents the mean of 6 replicates (7 chickens/replicate)) according to different fat sources (diets with 6% of soybean oil or palm oil) in the diet in 14-day-old broiler chickens. G = gizzard; D = duodenum; J = jejunum; I = ileum; E = excreta; TAG = triacylglycerols; DAG = diacylglycerols; MAG = monoacylglycerols; FFA = free fatty acids. 1346 RODRIGUEZ-SANCHEZ ET AL. Table 4. Digestibility coefficients along the gastrointestinal tract according to different fat sources in the diet in 14- and 35-day-old broiler chickens. Dietary treatments1 GIT segment2 S P P-values G D J I E G D J I E G D J I E SEM Fat GIT Interaction Digestibility coefficients at 14 d TFA 0.40 –3.00 0.59 0.81 0.84 0.11 –2.34 0.56 0.60 0.68 0.26 –2.67 0.57 0.70 0.76 0.266 0.954 < 0.001 0.17 SFA 0.36 –4.94 0.55 0.77 0.79 0.23 –1.67 0.47 0.48 0.53 0.29 –3.30 0.51 0.62 0.66 0.444 0.005 < 0.001 < 0.001 C16:0 0.32 –3.55 0.58 0.79 0.81 0.22 -1.17 0.48 0.49 0.54 0.27 –2.36 0.53 0.64 0.67 0.199 0.007 < 0.001 < 0.001 C18:0 0.47 –10.30 0.47 0.72 0.76 0.31 –6.15 0.39 0.40 0.45 0.39 –8.22 0.43 0.56 0.61 3.853 0.201 < 0.001 0.028 MUFA 0.42 –1.54 0.61 0.81 0.84 0.25 –1.56 0.64 0.68 0.78 0.33 –1.55 0.63 0.75 0.83 0.117 0.464 < 0.001 0.95 C18:1 n-9 0.42 –1.37 0.62 0.81 0.85 0.23 –1.55 0.63 0.68 0.81 0.33 –1.46 0.62 0.74 0.83 0.110 0.219 < 0.001 0.925 PUFA 0.41 –2.95 0.59 0.83 0.86 –0.29 –4.64 0.58 0.68 0.78 0.06 –3.80 0.59 0.76 0.82 0.621 0.013 < 0.001 0.06 C18:2 n-6 0.40 –3.15 0.58 0.82 0.85 –0.29 –4.85 0.57 0.68 0.78 0.06 –4.00 0.58 0.75 0.82 0.678 0.017 < 0.001 0.077 C18:3 n-3 0.42 –1.49 0.67 0.86 0.88 –0.33 –2.52 0.69 0.74 0.80 0.04 –2.01 0.68 0.80 0.84 0.216 0.002 < 0.001 0.024 Digestibility coefficients at 35 d TFA 0.46 –7.42 0.79 0.89 0.91 0.45 –4.35 0.66 0.77 0.84 0.46 –5.88 0.72 0.83 0.88 2.412 0.179 < 0.001 0.056 SFA 0.44 –11.10 0.77 0.83 0.88 0.52 –3.16 0.58 0.66 0.75 0.48 –7.13 0.67 0.75 0.82 5.920 0.020 < 0.001 < 0.001 C16:0 0.41 –8.44 0.77 0.88 0.88 0.51 –2.59 0.59 0.66 0.75 0.46 –5.51 0.68 0.77 0.81 3.204 0.023 < 0.001 < 0.001 C18:0 0.58 –22.01 0.77 0.72 0.92 0.59 –8.28 0.49 0.68 0.72 0.58 –15.14 0.63 0.70 0.82 27.143 0.055 < 0.001 0.005 MUFA 0.47 –4.60 0.84 0.91 0.93 0.54 –3.28 0.79 0.89 0.93 0.50 –3.94 0.81 0.90 0.93 1.052 0.322 < 0.001 0.412 C18:1 n-9 0.45 –4.49 0.83 0.91 0.93 0.53 –3.23 0.79 0.89 0.93 0.49 –3.86 0.81 0.90 0.93 1.014 0.326 < 0.001 0.449 PUFA 0.47 –7.31 0.78 0.89 0.92 0.22 –7.62 0.63 0.78 0.88 0.34 –7.47 0.70 0.84 0.90 2.876 0.696 < 0.001 1.000 C18:2 n-6 0.46 –7.79 0.77 0.89 0.92 0.22 –8.09 0.62 0.78 0.88 0.34 –7.94 0.70 0.83 0.90 3.241 0.720 < 0.001 1.000 C18:3 n-3 0.50 –3.82 0.84 0.92 0.94 0.21 –2.99 0.70 0.81 0.89 0.35 –3.40 0.77 0.87 0.91 0.746 0.826 < 0.001 0.532 1 Two dietary treatments: diets with soybean oil (S) or palm oil (P). Values are pooled means of 6 replicates (with 8 chickens/replicate at 14 d; and 3 chickens/replicate at 35 d). 2 Values are pooled means of 12 replicates (with 8 chickens/replicate at 14 d; and 3 chickens/replicate at 35 d). G = gizzard; D = duodenum; J = jejunum; I = ileum; E = excreta; GIT = gastrointestinal tract; TFA = total fatty acids; SFA = saturated fatty acids; MUFA = monounsaturated fatty acids; PUFA = polyunsaturated fatty acids; SEM = standard error of the mean. Values correspond to pooled means obtained from two-way ANOVA conducted to the effect of the type of fat added and the GIT segment. P < 0.05 was considered significant. Considering the average results of both fat sources together at 14 d, digestibility coefficients of FA close to 0.3 were reported in the gizzard for total FA (TFA), SFA, and MUFA, while the digestibility of PUFA in this GIT segment was lower in comparison with the other FA groups. In the duodenum, all of the di- gestibility coefficients were highly negative, especially for SFA and PUFA. At the jejunum level, FA di- gestibility coefficients close to 0.6 were observed, ex- cept for SFA, which had lower digestibility coeffi- cients than did the others. All FA groups had an in- crease in their digestibility from the jejunum to the Figure 2. Saturated fatty acid digestibility coefficients along the ileum, reaching digestibility coefficients over 0.7 for gastrointestinal tract for two different fat sources in the diet (diets with 6% of soybean oil or palm oil) in 14-day-old broiler chickens. G = TFA, MUFA, and PUFA, and close to 0.6 for SFA. gizzard; D = duodenum; J = jejunum; I = ileum; E = excreta. Values An increase in FA digestibility was also observed from are the means of 6 replicates (with 7 chickens/replicate). P values were the ileum to the excreta, observing digestibility coef- obtained from two-way ANOVA conducted to see the effect of the type ficients close to 0.8 for TFA, MUFA, and PUFA, and of the added fat and the GIT segment. ∗∗∗ P < 0.001. close to 0.65 for SFA. A significant interaction between the fat source and detailed explanation about this effect can be found in the GIT segment was observed for SFA (P < 0.001; the next paragraph. Figure 2); this interaction being explained by palmitic The digestibility coefficients observed for both fat (C16:0) and stearic (C18:0) acids, the major SFA in the sources were compared separately in the gizzard, duo- diet. This interaction was due to the change observed denum, jejunum, ileum, and excreta. In the gizzard, in the digestibility coefficients between the duodenum PUFA digestibility was significantly higher in those an- and jejunum. While the SFA digestibility coefficient in imals fed S (S: 0.41 and P: −0.29; P = 0.027). In the the duodenum was considerably lower for S than for P, duodenum, differences were found for SFA digestibility S had a higher digestibility coefficient in the jejunum, (S: −4.94 and P: −1.67; P = 0.003). As absorption is ileum, and excreta. Regarding PUFA, a significant ef- expected to happen from the jejunum on, the results fect was observed for the added fat to the diet; a more observed from the jejunum to the excreta are shown FAT DIGESTION ALONG THE DIGESTIVE TRACT 1347 Figure 3. Fatty acid digestibility coefficients for two different dietary fat sources (diets with 6% of soybean oil or palm oil) in the jejunum, ileum and excreta in 14-day-old broiler chickens. TFA = total fatty acids; SFA = saturated fatty acids; MUFA = monounsaturated fatty acids; PUFA = polyunsaturated; J = jejunum; I = ileum; E = excreta. Values are the mean of 6 replicates (7 chickens/replicate). P values were obtained from Student’s t-test conducted to see the effect of the type of the added fat in each gastrointestinal segment. ∗ P < 0.05; ∗∗ P < 0.01; ∗∗∗ P < 0.001. in Figure 3. Those chickens fed S had higher TFA di- of DAG in the duodenum was also lower in compari- gestibility coefficients than did those fed P, both in the son to the gizzard (9.81 and 15.02%, respectively). The ileum and excreta (P < 0.001). The same was observed highest percentage of MAG and FFA was found in the for PUFA in the ileum (P = 0.010) and excreta (P = duodenum (6.06 and 67.35%, respectively). From the 0.002). In the case of SFA, the digestibility coefficients duodenum on, the percentage of both MAG and FFA observed for S were higher than were the ones observed decreased, with FFA being the most predominant lipid for P in the jejunum (P = 0.036), ileum and excreta class in the jejunum (66.33%), ileum (55.51%), and exc- (P < 0.001). Regarding MUFA, differences were only reta (52.27%). observed in the ileum (P = 0.019), where S had higher Considering the added fat source (Figure 4), the per- digestibility coefficient than did P. centage of TAG was higher for S than for P in the giz- With reference to the AME (Table 2), those animals zard (P = 0.021), while the percentage of FFA was lower fed S showed a higher value than did those fed P (P = for S than for P (P = 0.002). No differences were found 0.002). in the duodenum between both fat sources for any of the lipid classes. In the jejunum, the percentage of DAG was higher for S than for P (P < 0.001). The percent- Determinations in 35-day-old Broiler age of FFA was lower for S than for P in the ileum and Chickens excreta (P = 0.003 and P < 0.001, respectively), and the same tendency was observed in the jejunum (P = Lipid Class Composition along the Gastrointesti- 0.055). The percentage of TAG and DAG was higher for nal Tract. The lipid class composition (TAG, DAG, S than for P, both in the ileum and excreta, and also MAG, and FFA) in different GIT segments (gizzard, the MAG percentage in the ileum. As explained before duodenum, jejunum, and ileum) and excreta was de- this finding is related to the disappearance (absorption) termined in 35-day-old broiler chickens fed diets with of FFA. two different fat sources (S and P), and is shown in Fatty Acid Digestibility Coefficients along the Figure 4. Gastrointestinal Tract. The FA digestibility coeffi- Regarding the general evolution of lipid classes along cients at 35 d depending on the GIT segment or excreta the GIT at 35 d, and considering the average results and the added fat (S and P) are shown in Table 4. of both fat sources together, TAG was the main lipid All of the FA groups showed a similar evolution of class in the gizzard (62.67%), while in the duodenum it their digestibility coefficients along the GIT. Consid- represented 16.15% of all lipid classes. The percentage ering the average results of both fat sources together, 1348 RODRIGUEZ-SANCHEZ ET AL. Figure 4. Lipid class composition (%) of the feed and along the gastrointestinal tract (each value represents the mean of 6 replicates (3 chickens/replicate)) according to different fat sources (diets with 6% of soybean oil or palm oil) in the diet in 35-day-old broiler chickens. G = gizzard; D = duodenum; J = jejunum; I = ileum; E = excreta; TAG = triacylglycerols; DAG = diacylglycerols; MAG = monoacylglycerols; FFA = free fatty acids. digestibility coefficients close to 0.5, were observed in than for P (Figure 5). Regarding MUFA, S had a higher the gizzard for TFA, SFA, and MUFA; the digestibility digestibility coefficient than did P in the jejunum (P = coefficients for PUFA were lower in comparison to the 0.042), and no differences were observed in the ileum other FA groups. In the duodenum, all the digestibility and excreta. In the case of PUFA, S had higher di- coefficients were highly negative, especially for SFA gestibility coefficients in the jejunum (P < 0.001) and and PUFA. The utilization of FA continued in the ileum (P = 0.001), while no differences were observed jejunum, where digestibility coefficients close to 0.70 in the excreta. were reported for TFA, SFA, and PUFA, and digestibil- Regarding AME (Table 2), those animals fed S had ity coefficients close to 0.80 were reported for MUFA. higher values than those fed P (P = 0.011). In the ileum, the digestibility increased, in general, the digestibility of SFA being the lowest (0.75), and higher than 0.80 for TFA, MUFA, and PUFA. In the excreta, Comparison Between 14- and 35-day-old all the digestibility coefficients were higher than in the Broiler Chickens ileum, being higher than 0.80 for SFA and close to 0.90 Lipid Class Composition along the Gastrointesti- for MUFA, PUFA, and TFA. nal Tract. Regarding the general evolution of lipid A significant interaction between the fat source and classes along the GIT (Figures 1 and 4) some differ- the GIT segment was observed for SFA. As found at 14 ences were observed between 14 and 35 d, despite the d, this interaction was explained by C16:0 and C18:0 (P fact the percentage of TAG in the diets was similar be- < 0.001 and P = 0.005, respectively), and it was due to tween both ages for each one of the fat sources. When the change in the digestibility coefficients from the je- the effect of the age of the chickens on the lipid class junum on. In the duodenum, the digestibility coefficient composition was studied in each GIT segment, higher was lower for S than for P (P < 0.001), while in the je- percentage of MAG (P = 0.005) and percentage of FFA junum, ileum, and excreta, the digestibility coefficient (P = 0.030) were observed in the gizzard at 35 d in com- for S was higher than for P. parison to the results observed at 14 d. No differences The results observed for both fat sources were com- were observed in the duodenum. From the duodenum pared in the gizzard, duodenum, jejunum, ileum, and on, a general increase of TAG and MAG percentage, excreta separately; the results from the jejunum to the and a decrease of FFA percentage, were observed at 35 excreta are shown in Figure 5. A statistically signifi- d. In the ileum, the DAG percentage was also higher (P cant difference was observed in the gizzard for PUFA < 0.001), while in the excreta it was lower (P = 0.036), (S: 0.47 vs. P: 0.22; P = 0.032). The TFA and SFA di- at 35 d than at 14 d. gestibility coefficients in the jejunum (P < 0.001), ileum However, there was a common pattern for these lipid (P ≤ 0.001), and excreta (P ≤ 0.007) were higher for S classes, both in S and P, between 14 and 35 d, a FAT DIGESTION ALONG THE DIGESTIVE TRACT 1349 Figure 5. Fatty acid digestibility coefficients for two different dietary fat sources (diets with 6% of soybean oil or palm oil) in the jejunum, ileum and excreta in 35-day-old broiler chickens. TFA = total fatty acids; SFA = saturated fatty acids; MUFA = monounsaturated fatty acids; PUFA = polyunsaturated; J = jejunum; I = ileum; E = excreta. Values are the mean of 6 replicates (3 chickens/replicate). P values were obtained from Student’s t-test conducted to see the effect of the type of the added fat in each gastrointestinal segment. ∗ P < 0.05; ∗∗ P < 0.01; ∗∗∗ P < 0.001. significant interaction between the fat source and the (P = 0.054) for TFA. This was explained because, de- age of the chickens was observed for DAG in the je- spite the fact the digestibility values were higher at 35 d junum (P = 0.004), and also for TAG (P < 0.001) and than at 14 d, the increase between both ages was higher FFA (P = 0.019) in the excreta. The interactions were for P than for S. explained by the more pronounced changes observed in Regarding AME, a tendency was observed taking into S than in P. account the age of the chickens (P = 0.071). The AME Fatty Acid Digestibility along the Gastrointestinal was numerically higher at 35 d than at 14 d both for Tract. The age of the chickens had a positive effect on S and P; however, no differences were observed for S FA digestibility (Table 4). In the gizzard, the digestibil- (P > 0.05), while a tendency was observed for P (P = ity of SFA was significantly higher at 35 d than at 14 0.095). d (P = 0.048), and a tendency was observed for the di- gestibility of TFA (P = 0.055), MUFA (P = 0.058) and DISCUSSION PUFA (P = 0.059). In the duodenum, the digestibility of TFA and all of the FA groups was lower (more nega- Taking into account the results of the percentage of tive) at 35 d than at 14 d (P ≤ 0.029). In the jejunum, the lipid classes and FA digestibility along the GIT, it MUFA digestibility was higher at 35 d than at 14 d (P is possible to better understand fat digestion and ab- < 0.001), and a significant interaction between the fat sorption in broiler chickens in order to improve fat uti- source and the age of the chickens (Figures 3 and 5) was lization by optimizing the fat formulation in diets. The observed for TFA (P = 0.022), SFA (P = 0.016), and digestion or hydrolysis is mainly related to the ante- PUFA (P = 0.004); this was explained because, despite rior part of the GIT (in this study represented by the the fact the values were higher at 35 d, for both dietary gizzard and duodenum), while the absorption of fat is treatments, the increase between both ages was higher related to the posterior part of the GIT (in this study for S than for P. In the ileum, there was an age effect for represented by the jejunum and ileum). TFA digestibility, which was higher at 35 d than at 14 Considering the first step of fat digestion (hydroly- d (P = 0.001); the same was observed in the excreta for sis), the decrease of TAG and the increase of the lipol- MUFA and PUFA (P < 0.001). A significant interac- ysis products (DAG, MAG, and FFA) from the feed tion between the fat source and the age of the chickens to the duodenum agreed with the hydrolysis of TAG, was observed for SFA, both in the ileum (P = 0.04) which requires pancreatic lipase, colipase, and bile salts. and excreta (P = 0.008), and also for TFA in the exc- Thus, as the lipid classes show, the digestion of fat reta (P = 0.01); a tendency was observed in the ileum could start before the duodenum; however, according 1350 RODRIGUEZ-SANCHEZ ET AL. to the literature, no FA absorption in this anterior part that 48% of endogenous fat losses are accounted for of the GIT is expected. Hence, the FA digestibility co- FA (Tancharoenrat et al., 2014). All FA digestibility efficients observed in the gizzard might be related to coefficients calculated in the duodenum were highly the gastro-duodenal contraction cycle (also known as negative, especially for stearic (C18:0) and linoleic reverse peristalsis), described in birds (Sacranie et al., (C18:2n-6) acids (Table 4). This partially agreed with 2005), which allows for the passage of the content be- Hurwitz et al. (1973), who reported that the main FA tween the gizzard and the duodenum. According to this, secreted into the duodenum were linoleic (C18:2n-6) products such as bile salts, MAG, DAG, and pancre- and palmitic (C16:0) acids, followed by stearic (C18:0) atic lipase that come from the duodenum are expected and oleic (C18:1n-9) acids. A little secretion of linolenic to be found in the gizzard (Sklan et al., 1978). Bile acid (C18:3n-3) was also reported. Regarding the lipid salts and MAG in the gizzard might start the emulsi- classes in the duodenum, the high percentage of fication of fat, which is the first step of fat digestion, the intermediate lipolysis products (DAG) and the but it is not expected that pancreatic lipase hydrolyze end-lipolysis products (MAG and FFA) suggest that TAG in this GIT segment because of its acidic condi- the hydrolysis of lipids starts and mainly takes place in tion (Sklan et al., 1978). Lipases from vegetable origin this GIT segment. The presence of fat in the duodenum in the ingredients of the feed, such as wheat lipases has been reported as a stimulus for the secretion of (Barros et al., 2010), could hydrolyze part of the di- cholecystokinin, and consequently, the secretion of etary TAG in the crop, where the enzyme could work, pancreatic enzymes and bile (Lindsay et al., 1969; as the pH in that GIT segment is neutral-alkaline, ac- Krogdahl, 1985; Tuchweber et al., 1996). Thus, the fat cording to Kapranchikov et al. (2004). All of this ex- source of the diet could have an effect on endogenous plains the results found in this GIT segment, and it is fat losses, as the SFA digestibility coefficients observed in accordance with Ravindran et al. (2016). In addi- for the animals fed the unsaturated diet in the duode- tion, reverse peristalsis provides more time for digesta num were more negative than for those animals fed the to be exposed to enzymatic activity and to mechanical saturated diet. It has been described that certain FA systems, and according to Sacranie et al. (2005) this are able to affect pancreatic lipase; oleic and linoleic can lead to an increase in the digestion and absorption acids stimulate lipase activity, while SFA with 8 or 10 time in the upper intestine. Sacranie et al. (2005) also carbons have little effect, and the long-chain saturated suggest that dietary factors like fiber can influence re- ones inhibit the activity of the enzyme (Van Kuiken verse peristalsis. Nevertheless, the effect of fat and age et al., 1994). This effect is slightly reflected in the on gastro-duodenal reverse peristalsis has not been pre- higher TAG percentage decrease from the feed to the viously studied. Regarding the effect of the added fat, duodenum in S than in P, at both 14 and 35 d. FA digestibility results reported in the gizzard were nu- Regarding age, the more negative digestibility coef- merically higher for those chickens fed the unsaturated ficients observed for TFA, SFA, MUFA, and PUFA in diet than for those fed the saturated diet, which could the duodenum at 35 d, in contrast to 14 d, could be suggest the fat source has an effect on reverse peri- related to higher endogenous fat losses into the duo- stalsis. The difference was statistically significant for denum lumen, and to an enhanced gastro-duodenal re- PUFA, both at 14 and 35 d. Thus, PUFA disappear- verse peristalsis. This suggests that could be an im- ance in those animals fed the saturated diet was de- provement on bile secretion as age increases, as bile is layed and was lower in comparison to the chickens fed the main source of endogenous fat losses. The higher the unsaturated diet; this could be related to the dif- bile secretion could also explain part of the improve- ferent PUFA dietary level, which was much lower for ment on the FA digestibility coefficients in the jejunum, the palm-oil-supplemented diet than for the soybean- ileum, and excreta, as they are essential both for the oil-supplemented diet (Table 2). Regarding the age of emulsification of the fat in the first step of fat di- the chickens, reverse peristalsis between the gizzard and gestion and the formation of mixed micelles, which the duodenum could be enhanced at 35 d, which is in are crucial for the absorption of FA (especially SFA). agreement with the higher digestibility coefficients re- During the first weeks of the age of chicks, the bil- ported in the gizzard at 35 d in contrast to the ones iary secretion is low, and it has been described as the reported at 14 d for SFA. most limiting factor for lipid digestion (Krogdahl, 1985; Moving on to the duodenum, the high amounts Noy and Sklan, 1995), and its effect on fat digestion- of all of the lipolysis products and the negative FA absorption has been well-documented (Hurwitz et al., digestibility coefficients reported in this GIT segment 1973). Pancreatic lipase could be another limitation could be explained both by the hydrolysis of TAG in young birds. However, there is a lack of agreement and endogenous fat losses; the gastro-duodenal reverse among different authors. Nitsan et al. (1991) and Uni peristalsis could also have influenced. Regarding the et al. (1995) reported that the activity of pancreatic endogenous fat, it comes from the bile in its majority lipase increases with age, and Noy and Sklan (1995) (Tancharoenrat et al., 2014), but there can be other reported that the secretion of lipase increased 20- to possible components, like mucin-associated lipids, and 100-fold between days 4 and 21 post-hatch. On the desquamated epithelial cells (Hurwitz et al., 1973; other hand, Meng et al. (2004) described a lack of re- Clément, 1980; Gong et al., 1990). It has been described sponse on fat digestibility after dietary supplementation FAT DIGESTION ALONG THE DIGESTIVE TRACT 1351 with lipase, and Sklan (2001) suggested that lipase se- tion of SFA in those animals fed the unsaturated diet cretion in young chickens may not be as inadequate (S) could be due to the natural emulsifying properties as expected when their feed intake is considered. This of unsaturated FA, which assist in the mixed micelle finding is in agreement with the results observed in formation and absorption. While unsaturated FA can the present study, which may lead to the assumption form micelles spontaneously, SFA are non-polar and that fat hydrolysis is not the most limiting factor in fat need the presence of the right amount of conjugated digestibility in young chickens. bile salts and unsaturated FA for the emulsification and All of the above suggest that the fat saturation degree incorporation into the micelles (Krogdahl, 1985; Smu- might have an effect on both reverse peristalsis and fat likowska and Mieczkowaka, 1996). The length of the endogenous losses, being the diets rich in unsaturated carbon chain of FA also has an effect on the FA uti- fat beneficial for these processes; this means more bile lization, the longer the carbon chain, the more difficult salts and enzymes in the intestinal lumen and a higher the incorporation of FA into micelles. As can be seen exposure time of the digestive content for the digestive in Table 4, the utilization of stearic acid (C18:0) from enzymes. The age of the chickens also seems to have an the jejunum on was lower than it was for palmitic acid effect on these two processes. (C16:0). The hydrophobic cores of the mixed micelles Moving on to the second step of fat digestion (the ab- also have the ability to solubilize the long-chain SFA, sorption of fat), FA digestibility, lipid classes, and AME among other components, and facilitate their absorp- results showed a difference between both fat sources. tion. The results of FA digestibility and lipid classes also Regarding the contribution of the jejunum and ileum showed the contribution of each GIT segment involved in the FA absorption process, a different pattern was in fat absorption. observed depending on the age of the chickens. This Starting with the AME results, they supported the fact shows that the absorption of FA is more limit- higher utilization of the unsaturated fat (S) in com- ing than their digestion. Thus, even though adult birds parison to the saturated one (P) at both 14 and 35 d. have greater amounts of bile salts and a higher activ- This was also evident with the FA digestibility coef- ity of pancreatic lipase, the higher digestibility coeffi- ficients; those animals fed S had higher apparent di- cients observed at 35 d were mainly due to the enhanced gestibility coefficients of TFA in the ileum, at both 14 FA absorption through the intestinal epithelial surface, and 35 d, and in the jejunum at 35 d, than did those which is also related to the lower percentage of FFA re- fed P. These results also agree with the evolution of ported in the jejunum and ileum at 35 d in contrast to lipid classes from the duodenum on. The evolution of 14 d. Another factor that has been previously described FFA percentage from the jejunum to the excreta re- and could have influenced the improvement of fat uti- flected a higher FFA absorption in those animals fed S lization at 35 d is the FA-binding protein (FABP) con- in comparison to the ones fed P (sometimes even lead- centration in the intestinal mucosa, which is also age- ing to a relative increase of TAG percentage). While in dependent and has been reported to be insufficient in those animals fed S the FFA% decreased at 14 d in the very young chicks, increasing its level after 4 wk of age ileum, in those fed P, the FFA percentage continued in- (Katongole et al., 1980). creasing in the ileum, indicating that even though TAG FA digestibility and lipid class results also showed are hydrolyzed, FFA are not as efficiently absorbed in that, independently of the fat source of the diet and the animals fed the saturated diet (P). Lipid classes at the age of the chickens, the jejunum was the major site 35 d showed an improvement in the FFA absorption of lipid absorption. Considering the digestibility of the that led not only to a decrease of FFA percentage, but excreta as the highest (100%), and expressing the di- also to a relative increase of TAG percentage, but still, gestibility in the jejunum and ileum in relation to that, the FFA absorption was higher for those animals fed it was seen that regardless of the dietary fat source, 76% S than for the ones fed P. This suggests that the ab- of TFA at 14 d, and 82% of TFA at 35 d, were absorbed sorption of unsaturated FFA throughout the intestinal in the jejunum; this is in accordance with Hurwitz et al. epithelial surface is easier than is the absorption of the (1973) and Tancharoenrat et al. (2014). It is important saturated ones. Being both the generation of the lipoly- to mention that the hydrolysis and absorption of fat sis products and their disappearance (absorption) from are dynamic processes, and although hydrolysis mainly the intestinal lumen faster and more efficient in those takes place in the duodenum, it probably continues in chickens fed the unsaturated diet. This higher utiliza- the jejunum where the generation and disappearance of tion of unsaturated fat could be explained by the higher the lipolysis products is likely to happen at the same apparent digestibility coefficients observed for SFA in time. In the ileum, some FA absorption was reported, as the jejunum and ileum, and for PUFA in the ileum. In the digestibility coefficients in this GIT segment were fact, SFA were the ones that differed the most between higher than the ones reported in the jejunum, and the the animals fed the unsaturated diet and the ones fed FFA decreased, being the second most important place the saturated diet, suggesting that SFA contributed the of fat absorption. The contribution of the ileum to the most to the lower digestibility coefficients reported for absorption of FA was found to be different according to the animals fed the saturated diet. The better utiliza- the fat source of the diet and the age of the chickens. 1352 RODRIGUEZ-SANCHEZ ET AL. Thus, considering TFA digestibility, it was seen that the the GIT and, together with the determination of contribution of the ileum in those young chickens fed the digestibility of FA, leads to a better un- the unsaturated diet (S) was higher (26%) than it was derstanding of the fat digestion–absorption process for those fed the saturated diet (6%). The contribution in broiler chickens. of the ileum in the absorption of SFA in those chick- The lipid classes and FA digestibility results con- ens fed P was even lower (<1%), which is in agreement firmed broiler chickens can use the unsaturated diet (S) with Whitehead and Fisher (1975), who questioned the better than the saturated one (P). This improvement role of the ileum in the absorption of SFA. In contrast, was mainly due to a more efficient and faster absorp- those adult chickens fed the unsaturated diet (S) had a tion of the unsaturated fat. Regarding the age of the higher utilization of FA due to the higher contribution chickens on fat utilization, the present data confirmed of the jejunum in its absorption (87%), leaving less re- that as age increases, the ability of the chickens to maining FA to be absorbed in the ileum (11%). On the digest and absorb fat is better. This was reflected other hand, the absorption of TFA in those adult chick- in the higher FA digestibility coefficients observed ens fed the saturated diet (P) also mainly took place in the adult chickens in comparison to the young in the jejunum (79%), however, the contribution of the ones in the jejunum, ileum, and excreta, the jejunum ileum (12.5%) at 35 d was also important, especially for and ileum being the main ones responsible for the the SFA, which improved from less than 1% at 14 d to improvement observed at 35 d. However, the previous 11.5% at 35 d; nevertheless, the FA absorption was still GIT segments could also have been positively influ- higher for S than for P. The increase in the contribution enced, as the hydrolysis process also improves with the of the ileum at 35 d in those animals fed P was also seen age of the chickens. by the lower FFA percentage observed in the ileum at Fat digestion starts before the duodenum, with emul- 35 d (66%) in comparison to the percentage observed sification, but it is in the duodenum where the hydroly- at 14 d (82%). This is in accordance with Hurwitz et sis of TAG starts, being the main place of lipid hydrol- al. (1973), who reported some lipid absorption in the ysis. The presence of high percentage of intermediate ileum, especially for linoleic, stearic, and palmitic acid. and end lipolysis products (DAG, MAG, and FFA) in All of this suggests that the contribution of the ileum the duodenum is in agreement with this. The jejunum is to fat digestion is really important, as the last chance of the most important place of FA absorption, contribut- those FA that leave the jejunum to be absorbed takes ing from 70 to 87% in FA apparent absorption. The place in the ileum. The dietary fat and the age of the ileum also plays an important role in FA absorption chickens also had an impact in the contribution of the (contributing from 6 to 26%), allowing the remaining jejunum and ileum to FA absorption. FA in the intestinal lumen to be absorbed after the Thus, those animals fed the unsaturated diet im- jejunum and explaining, in a great degree, the differ- proved fat utilization at 35 d mainly due to a higher ences observed in FA utilization among the 2 studied contribution of the jejunum in the absorption of FA, ages and dietary fats. The ileum was responsible for the while those animals fed the saturated diet improved fat higher digestibility values observed for the unsaturated utilization at 35 d for both a higher contribution of the fat in young broiler chickens and was also the responsi- jejunum and especially for a higher contribution of the ble of the improvement in the saturated fat utilization ileum in FA absorption in comparison to the results in adult broiler chickens. The role of the ileum is es- observed at 14 d. Thus, the higher contribution of the sential as there is no effective utilization by the animal ileum on fat absorption at 14 d could explain the bet- after this GIT segment. All of this suggests the absorp- ter utilization of unsaturated fat in comparison to satu- tion of FA is more limiting than is hydrolysis, because rated fat, while the higher contribution of the jejunum the main differences were observed in the jejunum and could explain the better utilization of the unsaturated ileum, where the absorption of FA takes place. The re- fat at 35 d. This suggests that the role of the ileum in sults observed in the present study also showed that the FA absorption is important and determines the global hydrolysis of fat is not the most limiting factor for fat fat utilization. utilization in young chickens. The absorption of fat has been reported to be neg- Thus, the dynamics of both FA digestibility and lipid ligible in the hindgut of poultry (Renner, 1965). Thus, class composition along the GIT was influenced by the after the ileum, no FA absorption is expected, and the dietary fat source and by the age of the chickens. The increase in FA digestibility coefficients (less than 10%) present results corroborate the better utilization of un- observed in the excreta, in comparison to the coeffi- saturated fat, and suggest that, regardless of the fat cients observed in the ileum, could be related to bac- source of the diet, the most limiting step in fat utiliza- terial activity (Lan et al., 2005). On the other hand, tion is the absorption process. the lower FFA percentage observed in the excreta for S both at 14 and 35 d also supports the higher utilization of the unsaturated fat. REFERENCES In conclusion, the determination of lipid classes AOAC International. 2005. Official Methods of Analysis of AOAC offers a wider outlook of fat utilization along International. 18th ed. AOAC International, Gaithersburg, MD. FAT DIGESTION ALONG THE DIGESTIVE TRACT 1353 Barros, M., L. F. Fleuri, and G. A. Macedo. 2010. Seed lipases: Ravindran, V., P. Tancharoenrat, F. Zaefarian, and G. Ravindran. sources, applications and properties - A review. Braz. J. Chem. 2016. Fats in poultry nutrition: digestive physiology and factors Eng. 27:15–29. influencing their utilisation. Anim. Feed Sci. Technol. 213:1–21. Clément, J. 1980. Intestinal absorption of triglycerols. Reprod. Nutr. Renner, R. 1965. Site of fat absorption in the chick. Poult. Sci. Dévelop. 20:1285–1307. 44:861–864. Fundación Española para el Desarrollo de la Nutrición Animal. 2008. Renner, R., and F. W. Hill. 1961. Factors affecting the absorbability Necesidades Nutricionales Para Avicultura: Pollos de Carne y of saturated fatty acids in the chick. J. Nutr. 74:254–258. Aves de Puesta. FEDNA, Madrid, Spain. Sacranie, A., B. Svihus, V. Denstadli, B. Moen, P. A. Iji, and M. Gong, D. H., B. Turner, K. R. Bhaskar, and J. T. Lamont. 1990. Choct. 2012. The effect of insoluble fiber and intermittent feeding Lipid binding to gastric mucin: protective effect against oxygen on gizzard development, gut motility, and performance of broiler radicals. Am. J. Physiol. Gastrointest. Liver Physiol. 259:G681– chickens. Poult. Sci. 91:693–700. G686. Sacranie, A., P. A. Iji, and M. Choct. 2005. Reflux of digesta and its Guardiola, F., R. Codony, M. Rafecas, J. Boatella, and A. López. implications for nutrient digestion and bird health. Proc. Aust. 1994. Fatty acid composition and nutritional value of fresh eggs, Poult. Sci. Symp. 17:171–174 (abstr.). from large- and small-scale farms. J. Food Compos. Anal. 7:171– Short, F. J., P. Gorton, J. Wiseman, and K. N. Boorman. 1996. 188. Determination of titanium dioxide added as an inert marker in Hurwitz, S., A. Bar, M. Katz, D. Sklan, and P. Budowski. 1973. Ab- chicken digestibility studies. Anim. Feed Sci. Technol. 59:215–221. sorption and secretion of fatty acids and bile acids in the intestine Sklan, D. 2001. Development of the digestive tract of poultry. Worlds of the laying fowl. J. Nutr. 103:543–547. Poult. Sci. J. 57:415–428. Kapranchikov, V. S., N. A. Zherebtsov, and T. N. Popova. 2004. Sklan, D., B. Shachaf, J. Baron, and S. Hurwith. 1978. Retrograde Purification and characterization of lipase from wheat (Triticum movement of digesta in the duodenum of the chick: extent, fre- aestivum L.) germ. Appl. Biochem. Microbiol. 40:84–88. quency, and nutritional implications. J. Nutr. 108:1485–1490. Katongole, J. B., and B. E. March. 1980. Fat utilization in relation Worlds Poult. Sci. J. Vol. 57:415-428. to intestinal fatty acid binding protein and bile salts in chicks of Sklan, D., P. Budowski, I. Ascarelli, and S. Hurwitz. 1973. Lipid different ages and different genetic sources. Poult. Sci. 59:819– absorption and secretion in the chick: effect of raw soybean meal. 827. J. Nutr. 103:1299–1305. Krogdahl, A. 1985. Digestion and absorption of lipids in poultry. J. Smulikowska, S., and A. Mieczkowska. 1996. Effect of rye level, fat Nutr. 115:675–685. source and enzyme supplementation on fat utilization, diet me- Lan, Y., M. W. A. Verstegen, S. Tamminga, and B. A. Williams. tabolizable energy, intestinal viscosity and performance of broiler 2005. The role of the commensal gut microbial community in chickens. J. Anim. Feed Sci. 5: 379–393. broiler chickens. Worlds Poult. Sci. J. Vol. 61:95–104. Tancharoenrat, P., V. Ravindran, F. Zaefarian, and G. Ravindran. Lindsay, O. B., J. Biely, and B. E. March. 1969. Excretion of 2014. Digestion of fat and fatty acids along the gastrointestinal bile acids by cockerels fed different lipids. Poult. Sci. 48:1216– tract of broiler chickens. Poult. Sci. 93:371–379. 1222. Tuchweber, B., I. M Yousef, G. Ferland, and A. Perea. 1996. Nutri- Meng, X., B. A. Slominsky, and W. Guenter. 2004. The effect of fat tion and bile formation. Nutr. Res. 16, 1041–1080. type, carbohydrase, and lipase addition on growth performance Uni, Z., Y. Noy, and D. Sklan. 1995. Posthatch Changes in Morphol- and nutrient utilization of young broilers fed wheat-based diets. ogy and Function of the Small Intestines in Heavy- and Light- Poult. Sci. 83:1718–1727. Strain Chicks. Poult. Sci. 74:1622–1629. Nitsan, Z., G. Ben-Avraham, Z. Zoref, and I. Nir. 1991. Growth and Van Kuiken, B. A., and W. D. Behnke. 1994. The activation of development of the digestive organs and some enzymes in broiler porcine pancreatic lipase by cis-unsaturated fatty acids. Biochim. chicks after hatching. Br. Poult. Sci. 32:515–523. Biophys. Acta 1214:148–160. Noy, Y., and D. Sklan. 1995. Digestion and absorption in the young Whitehead, G. C., and C. Fisher. 1975. The utilisation of various chick. Poult. Sci. 74:366–373. fats by turkeys of different ages. Br. Poult. Sci. 16:481–485.