Influence of free fatty acid content and degree of fat saturation in laying hen diets on egg quality, yolk fatty acid profile, and cholesterol content M. Palomar,* M. D. Soler ,* A. Tres ,y A. C. Barroeta ,z M. Mu~ noz-N un~ez,y and C. Garces-Narro *,1 * AviFeed Science, Department of Animal Production and Health, Faculty of Veterinary Medicine, Universidad CEU Cardenal Herrera − CEU Universities, E-46115 Alfara de Patriarca, Valencia, Spain; yNutrition, Food Science and Gastronomy Department, Faculty of Pharmacy and Food Science, Universitat de Barcelona, E-08921 Santa Coloma de Gramenet, Barcelona, Spain; and zAnimal Nutrition and Welfare Service, Department of Animal and Food Science, Faculty of Veterinary Medicine, Universitat Aut onoma de Barcelona, E-08193 Bellaterra, Barcelona, Spain ABSTRACT The aim of the present study was to diets presented higher yolk:albumen ratio than soybean evaluate the effect of dietary free fatty acid (FFA) diets (P < 0.001), but the effect of FFA did not follow a content and the degree of saturation on egg quality, linear trend. Hens fed soybean diets laid eggs with yolk fatty acid (FA) profile, and yolk cholesterol con- higher Haugh units (HU) than palm diets (P < 0.001), tent. For a 15-wk period, a total of 144 laying hens (19- although increasing the dietary FFA% reduced the HU wk-old) were randomly assigned to 8 treatments values in both (linear, P < 0.001). Palm diets enhanced arranged in a 2 £ 4 factorial design, with 2 sources of shell quality with greater resistance to breakage, and crude oil (soybean oil and palm oil) and 4 levels of FFA higher dry matter and ash content than soybean diets (10, 20, 30, and 45%). The dietary treatments were (P < 0.05). No differences in egg chemical composition achieved by progressively substituting the original oils and yolk cholesterol content were found (P > 0.05). with equivalent amounts of their corresponding acid The saturation degree had a significant effect on all the oils (soybean acid oil and palm fatty acid distillate, analyzed yolk FA (P < 0.001) except for arachidonic respectively). No differences in ADFI or egg mass were acid (C20:4 n-6), whereas increasing the FFA content found. However, dietary FFA reduced egg production did not affect to a great extent. These results show (linear, P < 0.05) and increased the feed conversion that varying dietary FFA level did not affect egg qual- ratio (linear, P < 0.05). Higher levels of FFA in soy- ity and yolk composition as much as the dietary fat bean diets resulted in higher egg weight with higher source did, supporting the use of acid oils and fatty albumen and yolk weights (linear, P < 0.01). Palm acid distillates as fat ingredients for feed. Key words: acid oil, egg weight, fat by-product, fatty acid distillate, yolk fatty acid profile 2023 Poultry Science 102:102236 https://doi.org/10.1016/j.psj.2022.102236 INTRODUCTION A diverse variety of fat sources are available to pro- vide lipids in laying hen feed. Certain types of fat by- Fats and oils have the highest caloric values among all products, such as acid oils (AO) and FA distillates the ingredients used in poultry diets. Adding supplemen- (FAD), are an example of unconventional feed resour- tal dietary fat also confers other benefits, including pro- ces that are of growing interest in animal feeding due to viding essential fatty acids (FA), such as linoleic acid their market availability, competitive price, and poten- (LA), and fat-soluble vitamins, while also improving tial environmental sustainability (Varona et al., 2021a, palatability, and allowing better nutrient digestion and c). These fat by-products come from the chemical (AO) absorption (Mateos and Sell, 1981; Grobas et al., 1999b; or the physical (FAD) refining processes of edible oils Bouvarel et al., 2010; Ravindran et al., 2016; and fats (European Commission, 2013). Both by-prod- Palomar et al., 2020). ucts are characterized by having a high proportion of free fatty acids (FFA; 31.7−93.6%) and a FA profile Ó 2022 The Authors. Published by Elsevier Inc. on behalf of Poultry consistent with that of the corresponding crude oil Science Association Inc. This is an open access article under the CC (Varona et al., 2021a). However, much variability in BY-NC-ND license (http://creativecommons.org/licenses/by-nc-nd/ their composition has been reported, including energy- 4.0/). diluting compounds such as moisture, impurities, and Received February 18, 2022. Accepted October 2, 2022. unsaponifiable material (collectively referred to as 1 Corresponding author: cgarces@uchceu.es MIU), which can affect their energy value (Nuchi et al., 1 2 PALOMAR ET AL. 2009; Roll et al., 2018b; Varona et al., 2021b). Limited Herrera-CEU Teaching and Research Farm (N aquera, knowledge of the composition and use of these fat by- Valencia, Spain). products in laying hen feed restricts their routine use by A total of 144 laying hens (Lohmann Brown-Classic; many commercial producers. 19 wk old; average body weight of the flock: 1,745 § It is well known that the FA composition of the yolk is 117 g, mean § standard deviation) obtained from a com- strongly dependent on dietary lipid sources (Cruick- mercial farm (Huevos Guillen, Valencia, Spain) were shank, 1934; Baucells et al., 2000; Meluzzi et al., 2000; randomly divided into 8 groups, each including 6 repli- Bou et al., 2009; Oliveira et al., 2010). The amount and cates with 3 birds per replicate. The birds were kept in a type of supplemental fat, as well as the LA content of 3-tier battery cage, with each cage (76.2 £ 63.0 cm2 the diet, have been shown to affect egg weight with a minimum height of 45.0 cm) containing one repli- (Shutze and Jensen, 1963; Scragg et al., 1987; White- cate. Throughout the study, feed and water were sup- head, 1995; Grobas et al., 1999b; Safaa et al., 2008). In plied for ad libitum consumption, and the animals addition, dietary lipids may influence other physico- were raised under the conditions recommended by the chemical characteristics of eggs, such as the yolk:albu- breeder (Lohmann, 2019). The feeding period lasted men ratio, (Whitehead et al., 1991; Grobas et al., 1999a, 15 wk. 2001; D€ anicke et al., 2000), the cholesterol content of The birds received a barley-soybean meal-based feed the yolk (Sim and Bragg, 1997; Wen et al., 2019), yolk in mash form formulated to meet or exceed FEDNA’s color (Perez-Bonilla et al., 2011; Zhou et al., 2019), and (Fundacion Espa~ nola para el Desarrollo de la Nutrici on eggshell quality (Millet et al., 2006; Cherian et al., Animal, 2018) recommendations and to minimize basal 2007). fat levels, as shown in Table 1. All experimental diets The chemical characteristics of oils and fats − particu- consisted of a basal diet (94%) and an experimental fat larly FA composition, degree of saturation (expressed as or fat blend (6%). Four different fats were used: 2 soy- the ratio of unsaturated to saturated fatty acids [UFA: bean sources (crude SO and SAO, both provided by SFA]), and FFA content − determine their nutritional Riosa S.A., Jaen, Spain) and 2 palm sources (crude PO value (Ravindran et al., 2016). Nevertheless, there is a and PFAD, both provided by Lípidos Santiga S.A., Bar- dearth of information on the possible influence of dietary celona, Spain). As Table 2 shows, a 2 £ 4 factorial design FFA content on egg quality and yolk composition. It has was used, with 8 dietary treatments being created been reported that the UFA:SFA ratio of a diet has a through the use of 2 fat sources (soybean or palm) and 4 greater effect than FFA content on the fat absorption dietary levels of FFA (10, 20, 30, or 45%). The different process (Vilarrasa et al., 2015; Rodriguez-Sanchez et al., levels of FFA for each fat source were achieved by using 2019a,b, 2021; Jimenez-Moya et al., 2021a,b). However, crude oil for 10% FFA and then progressively replacing most of the data available on the use of AO and FAD the crude oil with equivalent amounts of the correspond- concerns broiler chickens. Although some studies have ing AO or FAD. considered the replacement of soybean oil (SO) with soybean AO (SAO) in the feed of laying hens (Pardío et al., 2005; Perez-Bonilla et al., 2011; Analytical Determination of Oils and Irandoust et al., 2012; Irandoust and Ahn, 2015), the Experimental Diets effect of increasing the dietary FFA content has yet to be addressed. A better understanding of the effects of Oil samples were analyzed in triplicate following the FFA on hen nutrition could enable the use of these fat method used by Varona et al. (2021b) for the analysis of by-products to be increased and optimized. AO and FAD in animal nutrition (Table 3). Analytical Therefore, the study described here was conducted in determination of the experimental diets was performed order to assess the effect of dietary FFA content and fat at least in duplicate according to the AOAC Interna- saturation degree on egg quality and lipid composition tional (2005) method. Gross energy was determined by of the yolk. The experimental diets were obtained by an adiabatic bomb calorimeter (Parr 6300 Calorimeter, gradually replacing SO with SAO, or palm crude oil Parr Instrument Company, Moline, IL). Crude, neutral, (PO) with equivalent graded levels of palm FAD and acid detergent fiber levels were determined using a (PFAD). fiber analyzer (A2000, ANKOM Technology, Macedon, NY). Calcium and total phosphorus were determined via ICP-OES (Optima 3200 RL, Perkin Elmer, Wal- MATERIALS AND METHODS tham, MA). The amino acid content was analyzed by Animals and Diets chromatography (Hewlett-Packard 1100, Waldbronn, Germany). The FA profile of the feed was determined All the experimental procedures applied in this study via GC-FID as is described below concerning the yolk followed the Directive 2010/63/EU on the protection of FA profile. The lipid-class composition was determined animals used for scientific purposes (European Parlia- after lipid extraction by size-exclusion chromatography ment and of the Council, 2010) and were approved by (Agilent 1100 HPLC, Agilent Technologies, Santa the Animal Research Ethics Committee of the Universi- Clara, CA), as described by Rodriguez-Sanchez et al. dad Cardenal Herrera-CEU (CEEA 17/018). The ani- (2019b). The lipid analysis of the experimental diets is mal study was conducted at the Universidad Cardenal shown in Table 4. FAT BY-PRODUCTS IN LAYING HEN DIETS 3 Table 1. Ingredient composition and determined analysis of the experimental diets (g/100 g on a DM basis, unless otherwise indicated). Soybean Palm Diets S10 S20 S30 S45 P10 P20 P30 P45 Ingredient composition Barley 49.9 Soybean meal, 47.5% crude protein 24.3 Corn 6.9 Calcium carbonate, fine-grained 6.8 Experimental fats1 6.0 Calcium carbonate, coarse-grained 2.1 Sunflower meal, 36% crude protein 1.9 Monocalcium phosphate 1.1 Vitamin and mineral premix2 0.4 Sodium chloride 0.3 Methionine hydroxy analogue 0.2 Sodium bicarbonate 0.1 Determined analysis Gross energy, MJ/kg 17.68 17.52 17.41 17.36 16.98 17.23 17.15 16.70 Dry matter 90.9 90.9 91.1 91.0 91.2 91.0 90.8 90.9 Crude protein 17.4 17.4 17.3 17.3 17.3 17.3 17.3 17.3 Lys 1.00 1.01 1.00 0.98 0.99 1.01 1.00 0.98 Met 0.48 0.48 0.46 0.46 0.47 0.47 0.48 0.46 Met + Cys 0.80 0.80 0.79 0.80 0.77 0.79 0.80 0.77 Thr 0.71 0.70 0.71 0.72 0.69 0.71 0.70 0.70 Ash 13.9 14.3 14.2 14.0 14.0 14.2 14.1 13.9 Ether extract 7.9 7.8 7.7 7.8 7.8 7.8 7.9 7.6 Linoleic acid 2.9 3.0 2.5 2.7 1.2 1.2 1.1 1.1 Crude fiber 3.5 3.5 3.7 3.6 3.8 3.4 3.5 3.4 Neutral detergent fiber 12.2 11.5 12.2 11.6 11.3 11.8 11.1 11.3 Acid detergent fiber 3.8 3.6 3.9 3.7 3.7 3.7 3.8 3.7 Calcium 3.98 4.39 4.24 4.11 4.06 4.27 4.20 4.14 Total phosphorus 0.66 0.67 0.63 0.67 0.68 0.62 0.69 0.66 1 Soybean oil, soybean acid oil, palm oil or palm fatty acid distillate in different proportions (see Table 2). 2 Premix provides per kg of feed: enzymatic complex (Setnazyme: Endo 1-4 Beta-Xylanase, 12,000 BXU/g; 6-phytase 100, 300 PPU/g), 1,000 mg; cho- line chloride 75%, 500 mg; red synthetic pigment (Roxafil 30/10), 300 mg; butylated hydroxytoluene, 100 mg; vitamin A, 9,000 IU; vitamin D3, 3,000 IU; vitamin E, 13 IU; vitamin B1, 1 mg; vitamin B2, 4 mg; vitamin B6, 1.8 mg; vitamin B12, 10 mg; vitamin K3, 1.7 mg; folic acid, 0.3 mg; niacin, 20 mg; pan- tothenic acid, 8 mg; biotin, 52 mg; Fe (from FeSO4¢7H2O), 32 mg; Cu (from CuSO4¢5H2O), 7 mg; Zn (from ZnO), 65 mg; Mn (from MnO), 85 mg; Se (from Na2SeO3), 0.35 mg; I (from Ca(I2O3)2), 0.7 mg. Production Performance per treatment (15 fresh eggs per replicate) were col- lected to determine egg quality. The weight of each Egg production (including the number of eggs pro- egg was recorded. Shell with membranes and yolk duced and individual egg weights) and feed intake were were separated carefully and weighed. The albumen recorded weekly for each replicate cage. From these weight was obtained subtracting the yolk and eggshell data, ADFI, egg production, egg mass, and cumulative weight from the total egg weight. The yolk:albumen feed conversion ratio (FCR) per kilogram of eggs were ratio was calculated according to the procedure calculated for the last weeks of the experiment (33 described by Hussein et al. (1992). Based on the egg −34 wk of age). weight and thick albumen height, the Haugh unit val- ues (HU) were calculated according to Haugh’s for- mula (1937). Yolk color, as measured by the DSM Egg Quality yolk color score (Vuilleumier, 1969), was determined During the last 2 wk (33−34 wk of age; 14−15 wk of in these eggs using CIELAB values. Eggshell thickness the experiment) a total of 90 randomly selected eggs was measured using a micrometer (Mitutoyo 293-130, Kawasaki, Japan) and eggshell breaking strength was Table 2. Oil blends used in the experimental diets. also recorded. The FUTURA Egg-Quality-Measuring- System 3/A (Br€oring, Lohne, Deutschland) was used Soybean Palm for the tests. Diets S10 S20 S30 S45 P10 P20 P30 P45 Theoretical 10 20 30 45 10 20 30 45 FFA1% Proportion in oil blend2, % Chemical Composition of the Eggs Crude soybean oil 100 70 30 - - - - - Soybean acid oil - 30 70 100 - - - - At the end of the experiment, 6 eggs from each repli- Crude palm oil - - - - 100 80 53 33 Palm fatty acid - - - - - 20 47 66 cate were randomly collected. Egg components, including distillate albumen, yolk, and shell, were separately homogenized in 1 Free fatty acids. a pool for later testing. Yolk samples were stored at 2 All oil blends were added at 6% to the basal diet. −20°C in glass vials until lipid analysis was performed. 4 PALOMAR ET AL. Table 3. Quality traits, fatty acid, and lipid-class composition of the experimental fats. Palm fatty acid Soybean oil Soybean acid oil Palm oil distillate MIU, g/100 g 0.59 4.95 0.52 2.13 Moisture, g/100 g 0.05 0.56 ND1 0.12 Insoluble impurities, g/100 g 0.17 1.97 0.21 0.27 Unsaponifiable matter, g/100 g 0.37 2.42 0.31 1.74 Peroxide value, meq O2/kg 4.5 0.7 3.9 1.6 p-Anisidine value 2.3 15.2 5.1 64.3 Fatty acid composition, % Myristic acid (C14:0) 0.1 0.1 1.2 1.5 Palmitic acid (C16:0) 11.9 11.7 44.3 46.2 Stearic acid (C18:0) 4.8 4.2 4.2 4.6 Oleic acid (C18:1 n-9) 23.1 31.9 37.8 36.5 Vaccenic acid (C18:1 n-7) 1.5 1.3 0.9 0.9 Linoleic acid (C18:2 n-6) 52.8 46.1 10.2 8.8 Linolenic acid (C18:3 n-3) 4.2 1.8 0.3 0.3 Minor fatty acids2 1.5 2.5 1.1 1.1 Trans fatty acids (C18:1) ND 0.4 ND 0.2 Saturated fatty acids 17.9 17.8 50.4 52.9 Monounsaturated fatty acids 25.0 33.7 39.0 37.7 Polyunsaturated fatty acids 57.1 48.1 10.6 9.2 UFA:SFA3 4.6 4.6 1.0 0.9 Lipid-class composition, % Triacylglycerols 94.3 29.1 83.6 6.2 Diacylglycerols 3.8 15.8 9.7 5.0 Monoacylglycerols 0.2 1.3 0.4 1.6 Free fatty acids 1.7 53.8 6.3 87.2 Gross energy, MJ/kg 39.69 39.61 38.76 39.43 1 Non-detectable value. 2 Minor fatty acids identified and quantified: C15:0, C16:1 n-7, C16:1 n-9, C17:0, C18:3 n-6, C20:0, C20:1 n-9, C20:2 n-6, C20:3 n-3, C20:3 n-6, C20:4 n- 6, C20:5 n-3, C22:0, C22:4 n-6, C22:5 n-3, C22:6 n-3, C24:0, C24:1. 3 Ratio of unsaturated to saturated fatty acids. Eggs were chemically characterized for DM, ash, CP, AOAC International (2005) (Methods 925.31 and and ether extract (EE). The DM content was deter- 925.32, respectively). Chemical analyses were carried mined according to Sun et al. (2019). Eggshell calcium out at least in duplicate. was tested using ICP-OES (Optima 3200 RL, Perkin Elmer). Albumen and yolk samples were freeze-dried prior to determining CP, using the Kjeldahl method, Fatty Acid Profile and Cholesterol Content of and EE, using Soxhlet analysis in accordance with Egg Yolk Table 4. Fatty acid profile and lipid class composition of the Regarding the lipid analysis, the yolk FA composition experimental diets. and cholesterol content were determined using a gas chromatograph (4890D, Agilent Technologies), Soybean Palm equipped with a flame ionization detector and a capillary Diets S10 S20 S30 S45 P10 P20 P30 P45 column (GC-FID). For FA composition, total yolk lip- Fatty acid composition, % ids were extracted in chloroform-methanol (2:1) (vol/ Palmitic acid (C16:0) 12.2 12.6 12.5 12.8 37.7 39.3 38.6 40.6 vol) according to Folch’s method (Folch et al., 1957). Stearic acid (C18:0) 4.3 4.2 4.0 4.0 4.0 4.1 4.1 4.2 Oleic acid (C18:1 n-9) 22.2 23.8 26.1 29.1 34.3 34.1 33.4 33.0 The extracted fat was transferred by dissolving it in Vaccenic acid (C18:1 n-7) 1.5 1.4 1.4 1.4 1.1 1.1 1.1 1.2 diethyl ether in a glass test tube with an internal stan- Linoleic acid (C18:2 n-6) 50.8 49.0 49.0 46.3 19.2 17.8 18.9 17.1 dard (heneicosanoic acid methyl ester, C21:0) (Sigma- Linolenic acid (C18:3 n-3) 6.8 5.1 3.9 2.2 1.0 1.0 1.1 1.0 Minor fatty acids1 2.0 3.4 2.5 3.2 2.6 2.4 2.6 2.7 Aldrich Co., St. Louis, MI), the solvent was evaporated Trans fatty acids (C18:1) 0.2 0.5 0.6 0.9 0.1 0.1 0.1 0.1 using N2 at 30°C and the FA methyl esters were Saturated fatty acids 17.9 18.5 18.3 19.0 43.6 45.4 44.8 46.8 obtained as described by Guardiola et al. (1994). There- Monounsaturated fatty acids 24.1 25.7 28.0 31.1 35.8 35.6 34.9 34.6 Polyunsaturated fatty acids 57.8 55.3 53.1 49.0 20.5 18.9 20.2 18.5 after, FA methyl esters were analyzed using GC-FID as UFA:SFA2 4.6 4.4 4.4 4.2 1.3 1.2 1.2 1.1 described by Varona et al. (2021b) and identified by Lipid-class composition % matching their retention times with those of standards Triacylglycerols 85.4 71.4 59.2 41.4 79.0 68.6 57.4 44.1 Diacylglycerols 5.2 8.2 10.2 13.4 9.7 8.8 8.0 7.5 (Sigma-Aldrich Co.). The results were expressed using Monoacylglycerols 0.4 1.3 0.8 1.7 0.7 0.4 0.5 0.5 internal normalization (area %). Cholesterol content Free fatty acids 9.0 19.1 29.8 43.5 10.6 22.2 34.1 47.9 was determined after silylation of the unsaponifiable 1 Minor fatty acids identified and quantified: C14:0, C15:0, C16:1 n-7, matter following the methodology described by C16:1 n-9, C17:0, C18:3 n-6, C20:0, C20:1 n-9, C20:2 n-6, C20:3 n-3, Tres et al. (2020), with slight modifications. The quanti- C20:3 n-6, C20:4 n-6,, C20:5 n-3, C22:0, C22:4 n-6, C22:5 n-3, C22:6 n-3, C24:0, C24:1. fication was performed using 5a-cholestane as an inter- 2 Ratio of unsaturated to saturated fatty acids. nal standard and the corresponding calibration curve FAT BY-PRODUCTS IN LAYING HEN DIETS 5 with standards (Sigma-Aldrich Co.). Chemical analyses Quadratic response was also calculated in the case of an were carried out at least in duplicate. inadequate fit for the linear effect. Results in tables are reported as means, and differen- ces were considered significant when P < 0.05. All data analysis was performed using SPSS statistics (27.0.1.0, Statistical Analysis IBM, Armonk, NY, 2020). The experiment was conducted using a completely randomized design with 8 treatments and 6 replicates, RESULTS each containing 3 laying hens. The experimental unit was the replicate for all measurements. Before analysis, Production Performance the normality of the data, and homogeneity of the vari- The mean values for the laying hens’ production per- ance were verified. Egg quality traits, yolk FA composi- formance are shown in Table 5. ADFI and egg mass tion, and cholesterol content were subjected to a two- were not significantly different among the grouped treat- way ANOVA using the GLM procedure. The model ments. However, egg production (linear and quadratic, included the dietary fat source (soybean or palm), and P < 0.05) and FCR (linear, P < 0.01) were negatively the FFA level (10, 20, 30, or 45%) as the main factors, as affected by increasing levels of FFA; hens fed dietary well as their interaction. The effect of the experimental treatments with 45% FFA presented the lowest egg pro- diet was also evaluated by a one-way ANOVA when the duction and the highest FCR at the end of the trial. interaction fat source £ FFA level was significant. Dif- ferences among treatment means were tested using Tukey’s test for multiple comparisons. Egg Quality In addition, orthogonal polynomial contrasts were used to determine the linear effect of increasing levels of The effects of the added fat source and the dietary FFA in hen diets when the effect of FFA content was FFA content on egg weight and the relative weights of significant. If there was no interaction (fat the egg components at the end of the trial are shown in source £ FFA content), the linear response to dietary Table 6. A significant interaction (P < 0.01) between FFA content was evaluated for both fat sources the fat source and the FFA content was observed in together; on the other hand, when a significant interac- total egg weight and the relative albumen and yolk tion was found, the linear contrast analysis was per- weights. Thus, an increase in egg size accompanied the formed separately for soybean and palm diets. increase in FFA content in the case of soybean diets Table 5. Effects of fat source and dietary free fatty acid content on hen productive performance at the end of the trial (33−34 wk of age). Item ADFI, g/hen Egg production, egg/hen/d Egg mass, g/d FCR1 Experimental diet S10 93.97 0.94 55.86 1.69 S20 101.24 0.95 58.01 1.75 S30 102.15 0.94 56.19 1.83 S45 98.15 0.84 53.29 1.92 P10 100.59 0.92 56.04 1.80 P20 96.98 0.94 57.27 1.72 P30 105.89 0.96 58.16 1.83 P45 102.63 0.89 53.79 1.93 Fat source Soybean 98.88 0.92 55.84 1.80 Palm 101.52 0.93 56.32 1.82 FFA2 content, % 10 97.28 0.93a 55.95 1.75b 20 99.11 0.94a 57.64 1.74b 30 104.02 0.95a 57.17 1.83ab 45 100.39 0.86b 53.54 1.92a S.E.M 1.01 0.02 0.98 0.04 Effects, P-values Fat source 0.183 0.525 0.741 0.612 FFA content 0.107 0.011 0.196 0.026 Fat source £ FFA 0.231 0.684 0.930 0.750 Linear contrast,3 P-values Overall - 0.032 - 0.005 Quadratic contrast,4 P-values Overall - 0.017 - - 1 Feed conversion ratio. 2 Free fatty acid. 3 Linear responses to dietary free fatty acid content. 4 Quadratic responses to dietary free fatty acid content. a-b Means within each variable with more than two levels (experimental diet or free fatty acid content) not sharing a common superscript differ accord- ing to Tukey’s test (P < 0.05). 6 PALOMAR ET AL. Table 6. Effects of fat source and dietary free fatty acid content on egg weight and their relative proportions at the end of the trial (n = 90 eggs per treatment). Item Egg weight, g Albumen, g Yolk, g Shell, g Yolk:albumen Experimental diet S10 60.01bc 39.58bcd 14.34b 6.06 0.364bc S20 60.95b 40.65b 14.34b 5.95 0.354cd S30 59.98bc 39.81bcd 14.33b 5.82 0.362bc S45 63.84a 42.88a 14.83a 6.04 0.347d P10 60.98b 40.29bc 14.63ab 6.04 0.366bc P20 59.00c 38.75d 14.28b 5.98 0.370ab P30 60.20bc 39.18cd 14.88a 6.13 0.382a P45 60.14bc 39.51bcd 14.62ab 6.01 0.372ab Fat source Soybean 60.53 40.61 14.44 5.96 0.357 Palm 60.02 39.37 14.60 6.05 0.373 FFA1 content, % 10 60.48b 39.93b 14.48b 6.05 0.365ab 20 59.99b 39.71b 14.31ab 5.97 0.362b 30 60.09b 39.49b 14.61a 5.98 0.372a 45 61.98a 41.18a 14.72a 6.02 0.359b S.E.M 0.33 0.26 0.10 0.04 0.003 Effects, P-values Fat source <0.001 <0.001 0.074 0.066 <0.001 FFA content <0.001 <0.001 0.002 0.216 <0.001 Fat source £ FFA <0.001 <0.001 0.002 0.101 0.006 Linear contrast,2 P-values Overall - - - - - Soybean <0.001 <0.001 0.010 - 0.003 Palm 0.451 0.198 0.257 - 0.066 Quadratic contrast,3 P-values Overall - - - - - Soybean <0.001 <0.001 0.038 - 0.474 Palm 0.010 0.003 0.653 - 0.041 1 Free fatty acid. 2 Linear responses to dietary free fatty acid content. 3Quadratic responses to dietary free fatty acid content. a-d Means within each variable with more than two levels (experimental diet or free fatty acid content) not sharing a common superscript differ accord- ing to Tukey’s test (P < 0.05). only (linear and quadratic, P < 0.001). The largest albu- The chemical composition of the eggs was little men and egg weights were recorded for hens on the S45 affected by the supplemental fat source, FFA content, diet (63.84 g total egg weight), with the other soybean or any interaction between them (Table 8). In fact, the diets showing values similar to those for palm treat- only differences detected were for shell dry matter (%) ments. Consequently, this was also reflected in the yolk: and ash (%), with these being found to be higher in the albumen ratio, with significantly lower values being eggs from palm diets compared with those fed soybean observed for soybean diets. In contrast, shell weight was diets (P < 0.01). not affected by differences in the added fat source or die- tary FFA content. With regard to the most important egg quality traits Fatty Acid Profile and Cholesterol Content of (Table 7), significant differences were found in the albu- Egg Yolk men HU, with the highest values being recorded in soy- bean diets, and the lowest in eggs from palm diets (P < The FA composition of egg yolks from all experimen- 0.001). Increasing the FFA content up to 30% reduced tal groups is summarized in Table 9, with additional HU values in both fat sources, showing a significant lin- detailed data presented in Supplementary Table 1. ear and quadratic effect (P < 0.01). No interaction Analysis of the FA profile in yolks showed that the sup- between fat source and FFA content was observed in plemental fat source had a significant effect on all FA this respect. Concerning yolk color, a significant interac- presented (P < 0.001), except for arachidonic acid tion was found for fat source £ FFA content, as well as (C20:4 n-6). for the effect of FFA content (P < 0.01). These results Regarding SFA and monounsaturated fatty acids presented a significant quadratic effect (P < 0.05) but (MUFA), the proportions of palmitic (C16:0) and oleic were not accompanied by a linear trend for any of the 2 (C18:1 n-9) acids, as well as the total SFA and total fat sources (P > 0.05). Yolk pigmentation was signifi- MUFA, were significantly greater for the eggs laid by cantly greater in eggs from palm diets in comparison to hens fed palm diets, whereas stearic acid (C18:0) was soybean diets (P < 0.001). Regarding eggshell quality, higher in soybean diets (P < 0.001). Furthermore, no influence by the dietary treatments was found for increasing dietary content of FFA led to linear increases shell thickness. However, shell breaking strength was in the proportions of palmitoleic (C16:1 n-7) acid in egg higher for palm diets (P < 0.05). yolks from palm diets (P < 0.05). FAT BY-PRODUCTS IN LAYING HEN DIETS 7 Table 7. Effects of fat source and dietary free fatty acid content of 1,403 mg/100 g in the yolks from soybean diets, and on egg quality traits (n = 90 eggs per treatment). 1,371 mg/100 g in those from palm diets. No effect for Eggshell fat source, FFA content or interaction was detected. Albumen Yolk Furthermore, no significant differences were found either Item HU1 Color2 Thickness, mm Strength, N in the cholesterol content of the edible part of the egg, Experimental diet S10 91.5 13.38b 0.375 45.2 obtained from the relative proportions of yolk and albu- S20 88.3 13.48ab 0.369 43.3 men in each treatment group. S30 86.7 13.16c 0.364 42.8 S45 89.1 13.46ab 0.365 46.0 P10 89.0 13.51ab 0.370 45.5 P20 86.7 13.44ab 0.365 47.2 DISCUSSION P30 84.8 13.44ab 0.373 44.6 P45 87.6 13.62a 0.367 45.3 Fat source There has been increasing interest in recent years in Soybean 88.9 13.37 0.368 44.3 the use of AO and FAD as fat sources for animal feed. Palm 87.0 13.50 0.369 45.6 However, most of the available data derives from their FFA3 content, % 10 90.3a 13.45a 0.372 45.2 use with broilers (Wiseman and Salvador, 1991; 20 87.5bc 13.46a 0.367 45.2 Blanch et al., 1996; Vilarrasa et al., 2015; Rodriguez- 30 85.7c 13.31b 0.368 43.7 Sanchez et al., 2019a,b, 2021; Jimenez-Moya et al., 45 88.1b 13.54a 0.366 45.6 S.E.M 0.66 0.04 0.003 0.69 2021a,b) rather than laying hen. Effects, P-values In this study, the replacement of SO with SAO Fat source < 0.001 < 0.001 0.760 0.026 induced a significant increase in egg weight, specifically FFA content < 0.001 < 0.001 0.197 0.076 Fat source £ FFA 0.890 0.009 0.057 0.061 in the S45 group. In contrast, as dietary FFA content Linear contrast,4 increased, egg production decreased and FCR increased. P-values Consequently, no differences were observed in terms of Overall 0.002 - - - Soybean - 0.647 - - the egg mass between the experimental groups. Palm - 0.099 - - Grobas et al. (2001) obtained significantly higher egg Quadratic contrast,5 weight when SO was used in laying hen feed compared P-values Overall <0.001 - - - to other sources of fat (tallow, linseed, and olive oil). Soybean - 0.041 - - However, differences in production performance or egg Palm - 0.007 - - weight laid by birds fed SO or SAO have not been 1 Haugh Units. observed in similar studies (Pardío et al., 2005; Perez- 2 Yolk color, as measured by the DSM yolk color score (Vuilleumier, Bonilla et al., 2011; Irandoust et al., 2012; Roll et al., 1969). 3 Free fatty acid. 2018a). In our study, however, differences in egg size are 4 Linear responses to dietary free fatty acid content. almost entirely attributable to the high weight recorded 5 Quadratic responses to dietary free fatty acid content. a-c for hens fed S45 (63.84 g), with the remaining groups Means within each variable with more than two levels (experimental diet or free fatty acid content) not sharing a common superscript differ presenting considerably lower average values (59.00 according to Tukey’s test (P < 0.05). −60.95 g) and egg mass remained unaffected. It has been reported that egg weight largely depends on the amount and type of fat used in feeding, and this effect is primarily attributed to the LA content of the diet (Shutze and Jensen, 1963; Scragg et al., 1987; On the other hand, yolks from soybean diets had sig- Grobas et al., 1999a,b, 2001; Safaa et al., 2008). In the nificantly higher content of n-6 polyunsaturated fatty current trial, the amount of supplemental fat cannot be acids (PUFA), n-3 PUFA, and total PUFA (P < a factor, since all the oil blends contributed 6% to the 0.001), being approximately twice as high as those from basal diet. In order to optimize egg production and palm diets. Moreover, a significant effect of FFA and ensure a rapid increase in egg size, many researchers sug- interaction between the dietary fat source and FFA con- gest LA levels around 1.2% (Grobas et al., 1999a; tent (P < 0.001) was found for n-3 PUFA; increasing Safaa et al., 2008; FEDNA, 2018). Discrepancies still dietary content of FFA caused a substantial decrease in exist today between researchers and most commercial linolenic (LNA, C18:3 n-3) and DHA (C22:6 n-3) acid management guidelines, which usually recommend die- content in yolks from soybean diets only (linear, P < tary LA levels above 1.8% at the beginning of the laying 0.001), whereas no relationship was found between vary- period (Lohmann, 2019). In the present trial, the mean ing levels of FFA and the percentage of n-3 PUFA for LA content of diets containing soybean (S10, S20, S30, yolks from palm diets. Eggs from hens fed soybean diets and S45) or palm oils (P10, P20, P30, and P45) was showed higher content of LA (C18:2 n-6) (P < 0.001) 2.8% and 1.2%, respectively, with the values of soybean than those that received palm diets (24.1 vs. 12.1, diets being above the hens’ requirements (Table 1). respectively), but in this case no association was found Therefore, the high egg size recorded for hens fed S45 with different FFA levels, and no interaction was cannot be attributed to the LA content either. observed. As was the case in D€anicke et al. (2000), in the current The cholesterol content in the egg yolks of all experi- trial the increase of egg weight in soybean diets was mental groups (Table 10) was similar, with mean values accompanied by a simultaneous increase in the albumen 8 PALOMAR ET AL. Table 8. Effects of fat source and dietary free fatty acid content on the eggs’ chemical composition (n = 6 pools per treatment). Albumen Yolk Eggshell Item DM, % Ash, % CP, % DM, % Ash, % CP, % EE1, % DM, % Ash, % Ca, mg/g Experimental diet S10 12.3 0.83 9.8 51.3 1.95 15.3 28.8 98.7 93.74 383.5 S20 12.4 0.84 9.9 51.3 1.96 15.2 29.1 98.5 93.57 388.1 S30 12.2 0.83 9.8 51.3 1.84 15.5 29.0 98.7 93.48 386.5 S45 12.2 0.84 9.7 51.5 2.04 15.6 29.1 98.7 93.44 387.5 P10 12.2 0.86 9.6 51.4 2.04 15.3 29.22 98.8 94.00 383.2 P20 12.3 0.86 9.7 51.6 1.99 15.1 29.0 98.8 93.96 383.2 P30 12.0 0.85 9.5 51.7 2.01 15.2 29.3 98.8 93.97 380.4 P45 12.2 0.84 9.7 51.8 2.04 15.3 29.0 98.8 93.82 381.8 Fat source Soybean 12.3 0.84 9.8 51.4 1.95 15.4 29.0 98.7 93.56 386.4 Palm 12.2 0.85 9.6 51.6 2.04 15.2 29.1 98.8 93.94 382.1 FFA2 content, % 10 12.2 0.84 9.7 51.4 2.00 15.3 29.0 98.8 93.87 383.4 20 12.4 0.85 9.8 51.5 1.98 15.1 29.1 98.7 93.77 385.7 30 12.1 0.84 9.7 51.5 1.92 15.4 29.2 98.8 93.73 383.4 45 12.2 0.84 9.7 51.7 2.04 15.5 29.0 98.7 93.63 384.6 S.E.M 0.16 0.01 0.14 0.17 0.06 0.15 0.28 0.06 0.16 2.79 Effects, P-values Fat source 0.428 0.116 0.127 0.069 0.127 0.095 0.572 0.002 0.005 0.068 FFA content 0.656 0.866 0.734 0.497 0.370 0.349 0.939 0.162 0.633 0.875 Fat source £ FFA 0.902 0.803 0.908 0.903 0.611 0.772 0.885 0.160 0.937 0.795 1 Ether extract. 2 Free fatty acid. weight. Whitehead (1991) and Grobas et al. (1999a,b, to determine whether the negative impact of diets 2001) also found that supplemental fat exerted a benefi- including SAO was due to their FFA level or their MIU cial effect on egg weight, which was primarily due to an composition. In fact, the MIU content of AO and FAD increase in albumen weight. It has been suggested that could be one of the reasons for the controversial results this could be attributed to the influence of dietary fat on of previous studies, since MIU values are often not the estrogen metabolism, which is mainly responsible for reported and, therefore, any negative effect found when albumen secretion (Whitehead, 1995). Nevertheless, the including these fat by-products has been directly attrib- mechanism by which the supplemental fat source influ- uted to their high level of FFA. ences egg size remains unclear. Most of the trials con- These results should be interpreted with caution, since ducted up to now have focused only on egg weight, with in this trial no increase in egg size was observed in the insufficient attention being paid to the concurrent S30 group and previous studies reported no differences changes in yolk and albumen weights. in egg weight. Therefore, further research is needed to In addition to being rich in FFA, SAO have higher clarify the mechanisms involved in egg size. MIU content than crude oils and PFAD. The unsaponifi- Regarding albumen quality, higher HU were recorded able matter and insoluble impurities dilute the energy for hens fed soybean diets (P < 0.001). Additionally, an content of oils, but also include variable amounts of increase in the FFA content resulted in a linear decrease some compounds that could affect egg weight. Toco- in HU values in both fat sources (P < 0.05). These pherols and phytosterols are unsaponifiable matter that results contrast with those obtained by Grobas et al. can be removed from crude oils in refining procedures, (1999a,b) and Safaa et al. (2008), who did not observe but which then accumulates in their by-products (Mes- any effect for the type of supplemental fat on the HU of sina, 2010; Varona et al., 2021a). However, no effect on eggs. We are unable to explain this discrepancy with our egg weight has been found for these compounds results. Nonetheless, even the group with the lowest HU (Shi et al., 2014). Another factor to consider is the (P30; 84.8 HU) presented a HU score >72, indicating amount of insoluble impurities in SAO, which is also high freshness (AA quality) according to US Depart- higher than that for PFAD and crude oils. The content ment of Agriculture standards (United States Depart- and composition of these insoluble impurities have been ment of Agriculture USDA, 2000). Differences in yolk reported to be highly variable and might include some color between soybean and palm diets (P < 0.001) were phospholipids such as lecithins (Varona et al., 2021a). expected, as crude PO possesses greater amounts of In research with laying hens, Mandalawi et al. (2015) beta-carotene (Khaskheli and Chou, 2020): the more found that replacing animal fat with lecithin (40 g/kg) carotenoids in the diet, the redder and darker the yolk increased egg weight. Thus, it is possible that the MIU (Seuss-Baum et al., 2011). Moreover, the perception of components may have played unidentified roles in affect- the intensity of the color of the yolks depends directly on ing egg weight and egg quality. Unfortunately, it has not the quantity of carotenoids consumed by the bird been possible to analyze any such effect in this study, (Bouvarel et al., 2011). Pardío et al., (2005) and nor has it been possible, due to the experimental design, Irandoust et al. (2012) compared diets that included SO Table 9. Mean composition of fatty acids in egg yolks according to the fat source and the dietary free fatty acid content of the diet (% of total fatty acids) (n = 6 pools per treatment). Item C16:0 C18:0 C16-1 n-7 C18:1 n-9 C18:1 n-7 C18:2 n-6 C18:3 n-3 C20:4 n-6 C22:6 n-3 SFA1 MUFA2 n-3 n-6 PUFA3 Experimental diet S10 22.9 8.6 1.7c 34.4 1.4cd 23.9 1.6a 1.8 1.4a 31.8 38.5 3.2a 26.5 29.6 S20 22.6 8.5 1.5c 34.0 1.3d 25.0 1.2ab 1.9 1.3a 31.4 37.9 2.7ab 27.8 30.5 S30 22.7 8.4 1.6c 35.9 1.4cd 23.4 1.0bc 1.8 1.1b 31.4 39.9 2.2b 26.3 28.5 S45 22.6 8.3 1.5c 36.5 1.4d 24.0 0.6cd 1.9 0.7c 31.1 40.3 1.5c 26.9 28.4 P10 25.3 7.6 2.2b 45.6 1.5bc 13.2 0.4d 1.9 0.8c 33.3 49.4 1.3c 16.0 17.3 P20 26.0 7.3 2.6a 45.2 1.7a 11.8 0.3d 1.8 0.7c 33.7 50.5 1.1c 14.5 15.6 P30 26.0 7.3 2.6a 45.3 1.5bc 11.9 0.3d 1.8 0.7c 33.6 50.4 1.1c 14.8 15.9 P45 26.0 7.4 2.7a 45.1 1.7ab 11.6 0.3d 1.8 0.7c 33.7 50.5 1.1c 14.5 15.6 Fat source Soybean 22.7 8.4 1.6 35.2 1.4 24.1 1.1 1.8 1.1 31.5 39.1 2.4 26.9 29.3 Palm 25.8 7.4 2.5 45.0 1.6 12.1 0.3 1.8 0.7 33.6 50.2 1.2 14.9 16.1 FFA4 content, % 10 24.1 8.1 1.9 39.5 1.5 18.5 1.0a 1.8 1.1a 32.5 43.9 2.2a 21.2 23.4 FAT BY-PRODUCTS IN LAYING HEN DIETS 20 24.3 7.9 2.1 39.6 1.5 18.4 0.7ab 1.8 1.0a 32.6 44.2 1.9ab 21.2 23.1 30 24.4 7.8 2.1 40.6 1.5 17.6 0.6ab 1.8 0.9ab 32.5 45.1 1.7ab 20.5 22.2 45 24.3 7.8 2.1 40.8 1.5 17.8 0.5b 1.9 0.7b 32.4 45.4 1.3b 20.7 22.0 S.E.M 0.32 0.14 0.11 0.93 0.04 1.09 0.12 0.03 0.05 0.24 1.04 0.16 1.09 1.22 Effects, P-values Fat source <0.001 <0.001 <0.001 <0.001 <0.001 <0.001 <0.001 0.599 <0.001 <0.001 <0.001 <0.001 <0.001 <0.001 FFA content 0.736 0.121 0.303 0.060 0.520 0.348 <0.001 0.920 <0.001 0.923 0.065 <0.001 0.545 0.131 Fat source £ FFA 0.156 0.563 <0.001 0.141 0.002 0.202 0.002 0.062 <0.001 0.061 0.212 <0.001 0.116 0.253 Linear contrast,5 P-values Overall - - - - - - - - - - - - - - Soybean - - 0.160 - 0.270 - <0.001 - <0.001 - - <0.001 - - Palm - - 0.001 - 0.066 - 0.298 - 0.207 - - 0.212 - - 1 Saturated fatty acids. 2 Monounsaturated fatty acids. 3 Polyunsaturated fatty acids. 4 Free fatty acid. 5 Linear responses to dietary free fatty acid content. a-d Means within each variable with more than two levels (experimental diet or free fatty acid content) not sharing a common superscript differ according to Tukey’s test (P < 0.05). 9 10 PALOMAR ET AL. Table 10. Effects of fat source and free fatty acid content on yolk greater contribution of essential FA (LA and LNA) in cholesterol content (n = 6 pools per treatment). soybean diets was observed, which led not only to a Cholesterol, Cholesterol, higher content of these FA in the soybean egg yolks (P Item mg/100 g of yolk mg/100 g of egg1 < 0.001), but also influenced the content of the long- Experimental diet chain FAs synthesized from these precursors, such as S10 1,408 368 DHA. These results are consistent with those of S20 1,416 369 Cherian and Sim (1991) and Baucells et al. (2000), who S30 1,408 373 S45 1,380 346 examined the relationships among different n-3 and n-6 P10 1,345 354 PUFA in laying hen diets. P20 1,394 369 In the present trial, the inclusion of SAO in the diet P30 1,352 368 P45 1,393 369 decreased the n-3 PUFA % content of egg yolks (linear, Fat source P < 0.001), with the most pronounced reduction occur- Soybean 1403 364 ring in the S45 group. This can be attributed to the Palm 1,371 365 FFA2 content, % decreasing LNA content in the feed as SO was replaced 10 1,377 361 by SAO, because the SAO used in this study presented 20 1,405 369 lower LNA than the crude SO. Yolk n-3 PUFA reduc- 30 1,380 371 45 1,387 358 tion occurred not only for the LNA content, but also for S.E.M 25.53 8.12 that of the long-chain n-3 PUFA derived from it, such as Effects, P-values DHA. Pardío et al. (2005) also found lower n-3 PUFA Fat source 0.134 0.860 FFA content 0.776 0.439 content in yolks when comparing different combinations Fat source £ FFA 0.561 0.237 of soybean soapstock and SO after 15 wk of feeding. In 1 Edible portion of the egg. contrast, Irandoust and Ahn (2015) reported that total 2 Free fatty acid. PUFA and n-3 PUFA content were not affected by vary- ing the added oil source (35 g/kg of SO or SAO). This could be due to the fact that almost twice as much fat and SAO, but did not find significant differences in any was added to the feed in our study (60 g/kg) and to dif- of the egg quality traits studied. In contrast, in this ferences in the FA composition of the oils used, espe- study, eggshell quality was also found to be affected by cially LNA. In fact, Grobas et al. (2001) reported that the fat source added. Eggs obtained from palm dietary both the particular fat source added to the feed (tallow, groups of laying hens had the highest resistance to linseed, olive, and SO), and the amount that was breakage (P < 0.05), which is probably due to the lower included (50 or 100 g/kg) influenced the yolk FA profile. weight recorded for these eggs (P < 0.001). Even when Regarding palm diets, the increasing levels of PFAD they derive from the same source, AO and FAD vary had little effect on the FA composition of the yolk; there widely in terms of chemical composition, quality, and was only a linear increase in the proportions of palmito- oxidative status (Varona et al., 2021a). This variability leic (C16:1 n-7) acid as the FFA content increased. The in composition further complicates the study of the use degree of dietary fat saturation and the FA composition of these fat by-products, as it could be responsible for of the feed notably modified the composition of the yolk. the differences in results reported. However, increasing the FFA content did not have a The internal chemical composition of the egg did not notable impact on the yolk FA profile. Differences differ between the experimental treatments as expected, observed among eggs from soybean groups with different since the biological purpose of the egg is to provide an FFA% were related to the lower LNA content in SAO optimal nutritional environment for the developing compared with crude SO (1.8 and 4.2%, respectively). embryo. On the opposite side, yolk FA composition can Although cholesterol deposition in the yolk can be be markedly influenced by hen dietary manipulation affected by nutrition (Sim and Bragg, 1997; Wen et al., (Cruickshank, 1934; Meluzzi et al., 2000; Grobas et al., 2019), it is especially resistant to dietary changes since 2001; Bou et al., 2009; Oliveira et al., 2010). In general, hens are capable of meeting the minimum requirements the supplemental fat source added to the feeds notably for embryo development (Faitarone et al., 2013). In the modified the FA profile of the yolk, whereas the FFA present trial, no relationship was found between the content had limited effect on the FA composition. In amount of cholesterol found in the yolks and the differ- this sense, the yolks from soybean groups presented a ent experimental diets. The UFA:SFA ratio of the fat lower proportion of SFA and MUFA (P < 0.001), consis- sources used, as well as the replacement by their respec- tent with the lower levels of these FA in soybean diets in tive AO or FAD, did not have a negative impact on yolk comparison with palm diets. However, it should be noted cholesterol content, even when hens were fed with a low that the proportional difference in SFA between soybean UFA:SFA ratio and a high FFA% diet. Similarly, and palm was much less prominent in the yolks than in Irandoust and Ahn (2015) did not find any differences the feed itself. This finding is consistent with other stud- when comparing diets that included SO or SAO. ies (Cruickshank, 1934; Meluzzi et al., 2000; Petrovic In conclusion, the dietary fat type (UFA:SFA ratio) et al., 2012; Irandoust and Ahn, 2015), in which yolk affected egg weight, egg quality, and yolk FA composi- SFA were less affected by dietary manipulation than tion to a greater extent than the FFA content. Laying PUFA. With regard to PUFA, the influence of the hens consuming diets containing crude PO (low UFA: FAT BY-PRODUCTS IN LAYING HEN DIETS 11 SFA ratio) were hardly affected by increasing the levels Bouvarel, I., Y. Nys, and P. Lescoat. 2011. Hen nutrition for sustained egg of PFAD. However, it was observed that hens fed soy- quality. Pages 261−299 in Improving the Safety and Quality of Eggs and Egg Products Volume 1. Y. Nys, M. Bain and V. Van Immerseel, bean diets (high UFA:SFA ratio) showed a linear eds. 1st ed.. Woodhead Publishing Limited, Cambridge, UK. response to increasing levels of FFA, especially when Bouvarel, I., Y. Nys, M. Panheleux, and P. Lescoat. 2010. Comment SO was completely replaced by its by-product l’alimentation des poules influence la qualite des úufs. INRA Prod. (FFA = 45%). Therefore, the influence of SAO on egg Anim. 23:167–182. Cherian, G., D. Gonzalez, K. S. Ryu, and M. P. Goeger. 2007. Long- weight and egg quality requires more detailed study, as term feeding of conjugated linoleic acid and fish oil to laying hens: the higher MIU content found in SAO in comparison effects on hepatic histopathology, egg quality and lipid compo- with PFAD may have contributed to these results. Par- nents. J. Appl. Poult. Res. 16:420–428. ticular attention should be paid to examining the effect Cherian, G., and J. S. Sim. 1991. Effect of feeding full fat flax and canola seeds to laying hens on the fatty-acid composition of eggs, of SAO on egg size and the relationship of dietary sup- embryos, and newly hatched chicks. Poult. Sci. 70:917–922. plemental fat with albumen weight. The results pre- Cruickshank, E. M. 1934. Studies in fat metabolism in the fowl. Bio- sented here serve to increase the available knowledge chem. J. 28:965–977. about the use of AO and FAD in commercial layers, D€anicke, S., I. Halle, H. Jeroch, W. B€ ottcher, P. Ahrens, R. Zachmann, and S. G€ otze. 2000. Effect of soy oil supplementa- highlighting the value of these by-products as alterna- tion and protein level in laying hen diets on praecaecal nutrient tive lipid sources and thus contributing to the transition digestibility, performance, reproductive performance, fatty acid toward a more sustainable egg industry. composition of yolk fat, and on other egg quality parameters. Eur. J. Lipid Sci. Technol. 102:218–232. European Commission. 2013. Commission Regulation (EU) 68/2013 of 16 January 2013 and its amendments on the Catalogue of feed ACKNOWLEDGMENTS materials. Off. J. Eur. Union 29:1–64. European Parliament and of the Council. 2010. Directive 2010/63/ This research was carried out with the financial sup- EU of the of 22 September 2010 on the protection of animals used port of the Generalitat Valenciana and the European for scientific purposes. Off. J. Eur. Union 276,33−79. Social Fund (GV/188/2018), a pre-doctoral research Faitarone, A. B. G., E. A. Garcia, O. R. De Roça, A. H. Ricardo, E. N. De Andrade, K. Pelícia, and F. Vercese. 2013. Cholesterol grant from the Generalitat Valenciana and the Euro- levels and nutritional composition of commercial layers eggs fed pean Social Fund (ACIF/2019/201), and a post-doc- diets with different vegetable oils. Braz. J. Poult. Sci. 15:31–38. toral contract as part of the Ramon y Cajal program Folch, J., M. Lees, and G. H Sloane Stanley. 1957. A simple method from the Spanish Government’s Ministerio de Ciencia e for the isolation and purification of total lipides from animal tis- sues. J. Biol. Chem. 226:497–509. Innovacion (RYC-2017-23601). The authors wish to Fundaci on Espa~ nola para el Desarrollo de la Nutrici on Animal. 2018. thank Jose A. García-Bautista, Patricia Pedrola and Necesidades Nutricionales Para Avicultura: Normas FEDNA. 2nd Cristian Colonna for their technical assistance, and ed. FEDNA, Madrid, Spain. Greg Hunt for his help in proofreading the manuscript Grobas, S., G. G. Mateos, and J. Mendez. 1999a. Influence of dietary linoleic acid on production and weight of eggs and egg components and resolving language-related issues. in young brown hens. J. Appl. Poult. Res. 8:177–184. Grobas, S., J. Mendez, C. De Blas, and G. G. Mateos. 1999b. Laying hen productivity as affected by energy, supplemental fat, and lino- DISCLOSURES leic acid concentration of the diet. Poult. Sci. 78:1542–1551. Grobas, S., J. Mendez, R. Lazaro, C. De Blas, and The authors declare no conflict of interest. None of the G. G. Mateos. 2001. Influence of source and percentage of fat authors of this study has a conflict of interest related to added to diet on performance and fatty acid composition of egg yolks of two strains of laying hens. Poult. Sci. 80:1171–1179. employment, consultancies, grants, or other funding. Guardiola, F., R. Codony, M. Rafecas, J. Boatella, and A. L opez. 1994. Fatty acid composition and nutritional value of fresh eggs, from large- and small-scale farms. J. Food Compos. SUPPLEMENTARY MATERIALS Anal. 7:171–188. Haugh, H. 1937. The Haugh Unit for Measuring Egg Quality. U.S. Supplementary material associated with this article Egg Poult. Mag. 43:552–555. can be found, in the online version, at doi:10.1016/j. Hussein, S. M., R. H. Harms, and D. M. Janky. 1992. Comparison of techniques for egg component yields. Poult. Sci. 71:373–377. psj.2022.102236. IBM Corp. 2020. IBM SPSS Statistics for Macintosh, Version 27.0. IBM Corp., Armonk, NY. Irandoust, H., and D. U. Ahn. 2015. Influence of soy oil source and REFERENCES dietary supplementation of vitamins E and C on the oxidation sta- tus of serum and egg yolk, and the lipid profile of egg yolk. Poult. American Oil Chemists’ Society. 2005. Official Methods of Analysis of Sci. 94:2763–2771. AOAC International. 18th ed. AOAC International, Gaithersburg, Irandoust, H., A. H. Samie, H. R. Rahmania, M. A. Edrissa, and MD. G. G. Mateos. 2012. Influence of source of fat and supplementation Baucells, M. D., N. Crespo, A. C. Barroeta, S. Lopez-Ferrer, and of the diet with vitamins E and C on performance and egg quality M. A. Grashorn. 2000. Incorporation of different polyunsaturated of laying hens from forty four to fifty six weeks of age. Anim. Feed fatty acids into eggs. Poult. Sci. 79:51–59. Sci. Technol. 177:75–85. Blanch, A., A. C. Barroeta, M. D. Baucells, X. Serrano, and Jimenez-Moya, B., A. C. Barroeta, F. Guardiola, M. D. Soler, F. Puchal. 1996. Utilization of different fats and oils by adult R. Rodriguez-Sanchez, and R. Sala. 2021a. Replacement of palm chickens as a source of energy, lipid and fatty acids. Anim. Feed oil with soybean acid oil in broiler chicken diet: fat digestibility Sci. Technol. 61:335–342. and lipid class content along the intestinal tract. Animals 11:2586. Bou, R., R. Codony, A. Tres, E. A. Decker, and F. Guardiola. 2009. Jimenez-Moya, B., A. C. Barroeta, A. Tres, M. D. Soler, and Dietary strategies to improve nutritional value, oxidative stability, R. Sala. 2021b. Soybean oil replacement by palm fatty acid distil- and sensory properties of poultry products. Crit. Rev. Food Sci. late in broiler chicken diets: fat digestibility and lipid-class content Nutr. 49:800–822. along the intestinal tract. Animals 11:1035. 12 PALOMAR ET AL. Khaskheli, A. A., and L. Chou. 2020. Evaluation of fatty acid compo- oils affect their nutritive value in broiler chicken diets. Animal sition, metabolizable energy, nutrients digestibility of crude palm 12:2040–2048. oil fat crystals in turkeys. Anim. Feed Sci. Technol. 270:114734. Safaa, H. M., M. P. Serrano, D. G. Valencia, X. Arbe, Lohmann. 2019. Lohmann Brown-Classic Layers Management Guide E. Jimenez-Moreno, R. Lazaro, and G. G. Mateos. 2008. Effects of Cage Housing. Lohmann Breeders, Cuxhaven, Germany. the levels of methionine, linoleic acid, and added fat in the diet on Mandalawi, H. A., R. Lazaro, M. Red on, J. Herrera, D. Menoyo, and productive performance and egg quality of brown laying hens in G. G. Mateos. 2015. Glycerin and lecithin inclusion in diets for the late phase of production. Poult. Sci. 87:1595–1602. brown egg-laying hens: effects on egg production and nutrient Scragg, R. H., N. B. Logan, and N. Geddes. 1987. Response of egg digestibility. Anim. Feed Sci. Technol. 209:145–156. weight to the inclusion of various fats in layer diets. Br. Poult. Sci. Mateos, G. G., and J. L. Sell. 1981. Nature of the extrametabolic 28:15–21. effect of supplemental fat used in semipurified diets for laying Seuss-Baum, I., F. Nau, and C. Guerin-Dubiard. 2011. The nutri- hens. Poult. Sci. 60:1925–1930. tional quality of eggs. Pages 201−236 in Improving the Safety and Meluzzi, A., F. Sirri, G. Manfreda, N. Tallarico, and A. Franchini. 2000. Quality of Eggs and Eggs Products Volume 2. F. Van Immerseel, Effects of dietary vitamin E on the quality of table eggs en- riched Y. Nys and M. Bain, eds. Woodhead Publishing Ltd, Cambridge, with n-3 long chain fatty acids. Poult. Sci. 79:539–545. UK. Messina, M. 2010. A brief historical overview of the past two decades Shi, S. R., Y. R. Shen, L. L. Chang, C. J. Zhou, Z. Bo, Z. Y. Wang, of soy and isoflavone research. J. Nutr. 140:1350–1354. H. B. Tong, and J. M. Zou. 2014. Safety evaluation of phytosterols Millet, S., K. De Ceulaer, M. Van Paemel, K. Raes, S. De Smet, and in laying hens: effects on laying performance, clinical blood param- G. P. J. Janssens. 2006. Lipid profile in eggs of Araucana hens com- eters, and organ development. Poult. Sci. 93:545–549. pared with Lohmann selected Leghorn and ISA Brown hens given Shutze, J. V., and L. S. Jensen. 1963. Influence of linoleic acid on egg diets with different fat sources. Br. Poult. Sci. 47:294–300. weight. Poult. Sci. 42:921–924. Nuchi, C. D., F. Guardiola, R. Bou, P. Bondioli, L. Della Bella, and Sim, J. S., and D. B. Bragg. 1997. Effect of dietary factors on serum R. Codony. 2009. Assessment of the levels of degradation in fat co- and egg yolk cholesterol levels of laying hens. Poult. Sci. 56:1616– and byproducts for feed uses and their relationships with some 1621. lipid composition parameters. J. Agric. Food Chem. 57:1952–1959. Sun, C., J. Liu, N. Yang, and G. Xu. 2019. Egg quality and egg albu- Oliveira, D. D., N. C. Baiao, S. V. Cançado, R. Grimaldi, men property of domestic chicken, duck, goose, turkey, quail, and M. R. Souza, L. J. C. Lara LJC, and A. M. Q. Lana. 2010. Effects pigeon. Poult. Sci. 98:4516–4521. of lipid sources in the diet of laying hens on the fatty acid profiles Tres, A., N. Tarnovska, E. Varona, B. Quintanilla-Casas, S. Vichi, of egg yolks. Poult. Sci. 89:2484–2490. A. Gibert, E. Vilchez, and F. Guardiola. 2020. Determination and Palomar, M., M. D. Soler, E. Roura, R. Sala, O. Piquer, and comparison of the lipid profile and sodium content of gluten-free C. Garces-Narro. 2020. Degree of saturation and free fatty acid and gluten-containing breads from the Spanish market. Plant content of fats determine dietary preferences in laying hens. Ani- Foods Hum. Nutr. 75:344–354. mals 10:2437. United States Department of Agriculture (USDA). 2000. United Pardío, V. T., L. A. Landín, K. N. Waliszewski, F. Perez-Gil, L. Díaz, States Standards, Grades, and Weight Classes for Shell Eggs AMS and B. Hernandez. 2005. The effect of soybean soapstock on the 56. Foreign Agricultural Service, Washington DC, USA. quality parameters and fatty acid composition of the hen egg yolk. Varona, E., A. Tres, M. Rafecas, S. Vichi, A. C. Barroeta, and Poult. Sci. 84:148–157. F. Guardiola. 2021a. Composition and nutritional value of acid Perez-Bonilla, A., M. Frikha, S. Mirzaie, J. Garcia, and oils and fatty acid distillates used in animal feeding. Animals G. G. Mateos. 2011. Effects of the main cereal and type of fat of 11:196. the diet on productive performance and egg quality of brown-egg Varona, E., A. Tres, M. Rafecas, S. Vichi, A. C. Barroeta, and laying hens from 22 to 54 weeks of age. Poult. Sci. 90:2801–2810. F. Guardiola. 2021b. Methods to determine the quality of acid oils Petrovic, M., M. Gacic, V. Karacic, Z. Gottstein, H. Mazija, and and fatty acid distillates used in animal feeding. MethodsX H. Medic. 2012. Enrichment of eggs in n-3 polyunsaturated fatty 8:101334. acids by feeding hens with different amount of linseed oil in diet. Varona, E., A. Tres, M. Rafecas, S. Vichi, R. Sala, and Food Chem 135:1563–1568. F. Guardiola. 2021c. Oxidative quality of acid oils and fatty acid Ravindran, V., P. Tancharoenrat, F. Zaefarian, and G. Ravindran. 2016. distillates used in animal feeding. Animals 11:2559. Fats in poultry nutrition: digestive physiology and factors influencing Vilarrasa, E., R. Codony, E. Esteve-Garcia, and A. C. Barroeta. 2015. their utilisation. Anim. Feed Sci. Technol. 213:1–21. Use of re-esterified oils, differing in their degree of saturation and Rodriguez-Sanchez, R., A. Tres, R. Sala, C. Garces-Narro, molecular structure, in broiler chicken diets. Poult. Sci. 94:1527– F. Guardiola, J. Gasa, and A. C. Barroeta. 2019a. Effects of die- 1538. tary free fatty-acid content and saturation degree on lipid-class Vuilleumier, J. P. 1969. The ‘Roche Yolk Color Fan’ an instrument composition and fatty-acid digestibility along the gastrointestinal for measuring yolk color. Poult. Sci. 48:767–779. tract in broiler starter chickens. Poult. Sci. 98:4929–4941. Wen, Z., Y. Wu, Z. Qi, X. Li, F. Li, X. Wu, and P. Yang. 2019. Rub- Rodriguez-Sanchez, R., A. Tres, R. Sala, F. Guardiola, and ber seed oil supplementation enriches n-3 polyunsaturated fatty A. C. Barroeta. 2019b. Evolution of lipid classes and fatty acid acids and reduces cholesterol contents of egg yolks in laying hens. digestibility along the gastrointestinal tract of broiler chickens fed Food Chem 301:125198. different fat sources at different ages. Poult. Sci. 98:1341–1353. Whitehead, C. C. 1995. Plasma oestrogen and the regulation of egg Rodriguez-Sanchez, R., A. Tres, R. Sala, M. D. Soler, F. Guardiola, weight in laying hens by dietary fats. Anim. Feed Sci. Technol. and A. C. Barroeta. 2021. Effects of free-fatty-acid content and 53:91–98. saturation degree of the dietary oil sources on lipid-class content Whitehead, C. C., A. S. Bowman, and H. D. Griffin. 1991. The effects and fatty-acid digestibility along the gastrointestinal tract in of dietary fat and bird age on the weights of eggs and egg compo- broilers from 22 to 37 days of age. Poult. Sci. 9:101261. nents in the laying hen. Br. Poult. Sci. 32:565–574. Roll, A. P., J. Forgiarini, E. G. Xavier, D. C. N. Lopes, V. F. B. Roll, Wiseman, J., and F. Salvador. 1991. The influence of free fatty acid and F. Rutz. 2018a. Replacing soybean oil with increasing levels of content and degree of saturation on the apparent metabolizable soybean acid oil in diets of coturnix quails (Coturnix coturnix energy value of fats fed to broilers. Poult. Sci. 70:573–582. coturnix) and the effects on egg quality. Acad. Bras. Cienc. Zhou, L., X. M. Ding, J. P. Wang, S. P. Bai, Q. F. Zeng, Z. W. Su, 90:529–539. Y. Xuan, and K. Y. Zhang. 2019. Ageing corn results in the pale- Roll, A. P., E. Vilarrasa, A. Tres, and A. C. Barroeta. 2018b. The dif- coloured egg yolks with decreased polyunsaturated fatty acids. ferent molecular structure and glycerol-to-fatty acid ratio of palm Anim. Feed Sci. Technol. 256:114242.