
==== Front
Poult Sci
Poult Sci
Poultry Science
0032-5791
1525-3171
Elsevier

S0032-5791(24)00756-9
10.1016/j.psj.2024.104177
104177
METABOLISM AND NUTRITION
Effects of dietary free fatty acid content and degree of fat saturation on tibia bone properties of laying hens
Palomar M. *
Soler M.D. *
Benavides-Reyes C. †
Rodríguez-Navarro A.B. †
García-Bautista J.A. *
Orozco A. *
Garcés-Narro C. cgarces@uchceu.es
*1
⁎ AviFeed Science, Department of Animal Production and Health, Facultad de Veterinaria, Universidad CEU Cardenal Herrera – CEU Universities, Alfara de Patriarca, Valencia E-46115, Spain
† Department of Mineralogy and Petrology, University of Granada, Granada E-18071, Spain
1 Corresponding author. cgarces@uchceu.es
06 8 2024
11 2024
06 8 2024
103 11 1041778 5 2024
1 8 2024
© 2024 The Authors
2024
https://creativecommons.org/licenses/by-nc-nd/4.0/ This is an open access article under the CC BY-NC-ND license (http://creativecommons.org/licenses/by-nc-nd/4.0/).
Acid oils and fatty acid distillates are fat by-products of the refining process of edible oils and are characterized by their high proportion of free fatty acids (FFA). While lipids are essential in poultry diets, their chemical structure may interfere with calcium absorption. Therefore, this study investigated the effects of dietary FFA content and the degree of fat saturation on bone metabolism in commercial layers. For 15-wk, a total of 144 laying hens (19-wk-old) were randomly assigned to 8 treatments (6 replicates with 3 birds each), which were obtained by gradually replacing crude soybean oil (rich in unsaturated fatty acids [UFA]) with soybean acid oil (rich in UFA and FFA), or crude palm oil (rich in saturated fatty acids [SFA]) with palm fatty acid distillate (rich in SFA and FFA). Following a 2 × 4 factorial design, 4 UFA-rich and 4 SFA-rich diets were created with varying FFA content: 10, 20, 30, and 45%. Tibiae (6 birds/treatment) were collected at the end of the trial for the assessment of mineral composition, morphological properties, and mechanical characteristics. The data were analyzed using a 2-way ANOVA with the GLM procedure. Orthogonal polynomial contrasts were employed to determine the linear effect of increasing %FFA, with statistical significance set at P < 0.05. The degree of saturation was found to negatively impact on calcium and phosphorus bone content, with higher levels found in soybean-based diets (P < 0.001). A significant interaction was observed for medullary bone mineral content, showing a linear decrease as the dietary %FFA increased (P < 0.05) in palm diets. In contrast, morphological and mechanical bone traits, total ash content, and cortical bone mineral composition remained unaffected (P > 0.05). These results suggest that the degree of fat saturation exerts a greater impact than FFA content on bone mineral metabolism, supporting the commercial use of fat by-products rich in FFA in laying hen diets, at least during the early stages of the laying cycle.

Key words

acid oil
bone quality
calcium
fat by-product
fatty acid distillate
==== Body
pmcINTRODUCTION

The use of circular solutions in livestock farming is emerging as an environmentally sustainable approach to reducing production costs and ensuring the responsible utilization of natural resources. Consequently, and alongside the scarcity and price fluctuations of conventional raw materials, there is growing interest in incorporating unconventional ingredients into livestock nutrition where they are both environmentally friendly and affordable for producers (Abín et al., 2018; Leinonen, 2019). Attention has thus turned to re-purposing by-products of diverse agricultural, food processing, and industrial processes as ingredients in animal feed. For example, some fat by-products obtained from the refining of edible oils, such as acid oils (AO) and fatty acid distillates (FAD), may be of interest to the egg industry as alternative lipid sources (Wiseman et al., 1991; Blanch et al., 1996; Jimenez-Moya et al., 2021; Palomar et al., 2023a,b).

Throughout the various stages of the refining process, crude oils undergo the removal of free fatty acids (FFA) and other undesired compounds via both chemical and physical refining techniques. This leads to the formation of fat by-products with a characteristically elevated FFA content, ranging from 32 to 94% (European Commission, 2013; Varona et al., 2021). The choice between these 2 major refining methods depends on the composition of the crude oil and differs in how FFA are removed: either through neutralization with an alkali (chemical refining) or through steam distillation (physical refining) (Akoh and Min, 2008). Chemical refining, which produces AO as a fat by-product, is the most widely used technique for purifying vegetable oils rich in unsaturated fatty acids (UFA) and phospholipids, such as soybean oil (SO). On the other hand, physical refining, which yields FAD, is the primary processing method for oils rich in saturated fatty acids (SFA), such as palm oil (PO). Both SO and PO are extensively used in animal nutrition and are the 2 most produced oils worldwide (USDA, 2023).

Although FFA are naturally generated during lipid digestion, dietary FFA could potentially decrease the energy value of supplemental fat (Wiseman and Salvador, 1991; Vilà and Esteve-Garcia, 1996; Ravindran et al., 2016). One of the key factors contributing to this effect is the tendency of FFA to react with ionized metals, such as calcium (Ca), forming insoluble and indigestible Ca soaps (Leeson and Summers, 2005). These soaps not only affect fat digestibility but also impair Ca absorption, especially when high dietary levels of Ca and fats rich in SFA are included in feeds (Atteh and Leeson, 1985a,b; Tancharoenrat and Ravindran 2014).

Calcium plays an important role in eggshell formation, growth, skeletal development, and various biological functions of laying hens. Approximately two-thirds of the Ca deposited for eggshell formation in the uterus comes directly from diet (Bar, 2009; Hincke et al., 2012; Molnár et al., 2018). However, during the night, when dietary Ca sources are depleted due to the absence of feed intake, around 30 to 40% of the Ca allocated for eggshell mineralization is mobilized from the skeleton (Nys and Guyot, 2011; Nys and Le Roy, 2018). This daily cycle of bone resorption is facilitated by the development of medullary bone (MB) in female birds, which is driven by estrogens and acts as a labile source of Ca (Whitehead, 2004; Nys and Le Roy, 2018). Two weeks before the onset of egg production and during the laying period, MB is formed at the expense of cortical bone (CB), resulting in a progressive loss of structural bone (Whitehead, 2004; Rodríguez-Navarro et al., 2018; Alfonso-Carrillo et al., 2021; Gautron et al., 2021).

It is widely recognized that incorporating coarse calcium carbonate particles into laying hen diets enhances eggshell quality and bone mineralization by increasing the concentration of soluble Ca in the intestine during the night (Nys and Guyot, 2011; Molnár et al., 2018). Despite this, any disruption in Ca homeostasis can adversely impact bone mineralization. In fact, this a common challenge in laying flocks, leading to both economic losses and welfare issues (Whitehead, 2004; Gloux et al., 2019; Toscano et al., 2020).

Many dietary factors interacting with Ca digestibility have already been studied, including phytic acid content, dietary vitamin D3 levels, and calcium to phosphorus (P) ratio (Jalal and Scheideler, 2001; Lim et al., 2003; Adhikari et al., 2020). However, only a limited number of trials have been undertaken assessing the formation of indigestible Ca soaps and the effect of sources of added fat. In our previous research conducted on laying hens, it was observed that both the degree of fat saturation and the acidity level decreased Ca digestibility (Palomar et al., 2023a). However, there were no effects on eggshell commercial quality (Palomar et al., 2023b).

Thus, the overall aim of this study was to assess the effect of dietary FFA level and the degree of fat saturation on bone quality of laying hens. Our hypothesis was that AO and FAD represent valuable ingredients that could contribute to more sustainable egg production without compromising bone quality.

MATERIALS AND METHODS

Animal Ethics

All animal handling protocols were carried out in accordance with Directive 2010/63/EU concerning the protection of animals used for scientific purposes (European Parliament and of the Council, 2010) under the supervision of the Animal Research Ethics Committee of the Universidad Cardenal Herrera-CEU (CEEA 17/018). The animal study took place at the Teaching and Research Farm of the Universidad Cardenal Herrera-CEU in Náquera, Valencia, Spain.

Birds and Diets

The experimental design was detailed in a previous publication by Palomar et al. (2023a). In summary, a total of 144 Lohmann Brown-Classic laying hens obtained from a commercial layer farm were randomly assigned to 8 groups at 19 wk of age. Each group comprised 6 replicates, and each replicate consisted of a cage housing 3 birds (considered to be the experimental unit). The cages, measuring 76.2 × 63.0 cm with a minimum height of 45.0 cm, were equipped with a nest, perch, and 3 freshwater nipple drinkers. Throughout the 15-wk duration of the study, the hens had ad libitum access to feed and water, and they were raised under the conditions recommended by the breeder (Lohmann, 2019).

As presented in Table 1, the experimental diets were formulated to meet or exceed FEDNA's (Fundación Española para el Desarrollo de la Nutrición Animal) recommendations (2018) and consisted of a basal diet (94%) and an experimental fat or fat blend (6%). Four fats were used in this experiment: 2 soybean sources (crude soybean oil [SO] and soybean acid oil [SAO] from chemical refining, both provided by Riosa S.A., Jaén, Spain) and 2 palm sources (crude palm oil [PO] and palm fatty acid distillate [PFAD] from physical refining, both provided by Lípidos Santiga S.A., Barcelona, Spain). The 8 dietary treatments were obtained using a 2 × 4 factorial design with 2 fat sources (soybean – high UFA:SFA ratio; or palm – low UFA:SFA ratio), and 4 different levels of free fatty acids (FFA) (10, 20, 30, or 45%). The S10 treatment was considered a reference diet since it contained SO in a proportion commonly used in the egg industry (60 g/kg). All diets included fine-ground Ca carbonate (CaCO3) from limestone at 68 g/kg (< 0.7 mm) and coarse-ground at 21 g/kg (≥ 1 mm). They were stored in a cool, dry place until the trial began.Table 1 Ingredient composition and calculated analysis of the experimental diets (g/100g on a DM basis, unless otherwise indicated).

Table 1	Soybean	Palm	
Diets	S10	S20	S30	S45	P10	P20	P30	P45	
Ingredient composition									
 Barley	49.9	49.9	49.9	49.9	49.9	49.9	49.9	49.9	
 Soybean meal, 47.5% crude protein	24.3	24.3	24.3	24.3	24.3	24.3	24.3	24.3	
 Corn	6.9	6.9	6.9	6.9	6.9	6.9	6.9	6.9	
 Calcium carbonate, fine-grained	6.8	6.8	6.8	6.8	6.8	6.8	6.8	6.8	
 Crude soybean oil	6.0	4.2	1.8	-	-	-	-	-	
 Soybean acid oil	-	1.8	4.2	6.0	-	-	-	-	
 Crude palm oil	-	-	-	-	6.0	4.8	3.2	2.0	
 Palm fatty acid distillate	-	-	-	-	-	1.2	2.8	4.0	
 Calcium carbonate, coarse-grained	2.1	2.1	2.1	2.1	2.1	2.1	2.1	2.1	
 Sunflower meal, 36% crude protein	1.9	1.9	1.9	1.9	1.9	1.9	1.9	1.9	
 Monocalcium phosphate	1.1	1.1	1.1	1.1	1.1	1.1	1.1	1.1	
 Vitamin and mineral premix1	0.40	0.40	0.40	0.40	0.40	0.40	0.40	0.40	
 Sodium chloride	0.30	0.30	0.30	0.30	0.30	0.30	0.30	0.30	
 Methionine hydroxy analogue	0.20	0.20	0.20	0.20	0.20	0.20	0.20	0.20	
 Sodium bicarbonate	0.10	0.10	0.10	0.10	0.10	0.10	0.10	0.10	
Calculated analysis2									
 AME, MJ/kg	12.0	12.0	12.0	12.0	11.8	11.8	11.8	11.8	
 Dry matter	90.8	90.8	90.8	90.8	90.8	90.8	90.8	90.8	
 Crude protein	17.5	17.5	17.5	17.5	17.5	17.5	17.5	17.5	
 Digestible protein	15.0	15.0	15.0	15.0	15.0	15.0	15.0	15.0	
  Lys	0.91	0.91	0.91	0.91	0.91	0.91	0.91	0.91	
  Met	0.43	0.43	0.43	0.43	0.43	0.43	0.43	0.43	
  Met + Cys	0.75	0.75	0.75	0.75	0.75	0.75	0.75	0.75	
  Thr	0.66	0.66	0.66	0.66	0.66	0.66	0.66	0.66	
  Trp	0.22	0.22	0.22	0.22	0.22	0.22	0.22	0.22	
 Ash	12.5	12.5	12.5	12.5	12.5	12.5	12.5	12.5	
 Ether extract	7.5	7.5	7.5	7.5	7.5	7.5	7.5	7.5	
 Crude fiber	4.2	4.2	4.2	4.2	4.2	4.2	4.2	4.2	
 Calcium	3.8	3.8	3.8	3.8	3.8	3.8	3.8	3.8	
 Total phosphorus	0.73	0.73	0.73	0.73	0.73	0.73	0.73	0.73	
 Digestible phosphorus	0.45	0.45	0.45	0.45	0.45	0.45	0.45	0.45	
1 Premix provides per kg of feed: enzymatic complex (Setnazyme: Endo 1-4 Beta-Xylanase, 12000 BXU/g; 6-phytase 100, 300 PPU/g), 1000 mg; choline chloride 75%, 500 mg; red synthetic pigment (Roxafil 30/10), 300 mg; butylated hydroxytoluene, 100 mg; vitamin A, 9000 IU; vitamin D3, 3000 IU; vitamin E, 13 IU; vitamin B1, 1 mg; vitamin B2, 4 mg; vitamin B6, 1.8 mg; vitamin B12, 10 μg; vitamin K3, 1.7 mg; folic acid, 0.3 mg; niacin, 20 mg; pantothenic 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.

2 Based on data composition of ingredients provided by FEDNA (2021).

During the feed manufacturing process, representative samples were taken from all experimental treatments, ground (< 1 mm), and homogenized before performing the analytical determinations. All diets were evaluated at least in duplicate according to the established procedures of the AOAC International (2005). Dry matter (Method 934.01), ash (Method 942.05), crude protein (Method 954.01), and ether extract by Soxhlet extraction after 3N HCl acid hydrolysis (Method 954.02) were determined. Crude, neutral, and acid detergent fiber levels were measured with a fiber analyzer (A2000, ANKOM Technology, Macedon, NY), following the procedures of Van Soest et al. (1991). Calcium and total phosphorus were quantified via ICP-OES (Optima 3,200 RL, Perkin Elmer, Waltham, MA), as described by Short et al. (1996). The amino acid content was analyzed via chromatography (Hewlett-Packard 1100, Waldbronn, Germany). Gross energy was determined by an adiabatic bomb calorimeter (Parr 6300 Calorimeter, Parr Instrument Company, Moline, IL).

Regarding the lipid analysis, as detailed in Palomar et al. (2023b), the fatty acid (FA) profile (UFA:SFA ratio) was established by means of the direct transesterification method, using heneicosanoic acid (C21:0, Sigma-Aldrich Chemical Co., St. Louis, MO) as an internal standard. FA methyl esters were analyzed on a gas chromatograph (4890D, Agilent Technologies, Santa Clara, CA), equipped with a flame ionization detector and a capillary column (GC-FID). The lipid-class composition (FFA content) was determined after lipid extraction by size-exclusion chromatography (Agilent 1100 HPLC, Agilent Technologies). The results were expressed using internal normalization (area %).

Bone Quality Measurements

At the end of the trial, one hen per replicate was selected based on the average body weight of the flock and euthanized using 1 ml/hen of T-61 (Merck Sharp & Dohme Corp., Kenilworth, New Jersey, USA). Both tibiae of each bird (6 hens/treatment) were removed, cleaned of non-bone tissues (muscle, fat, tendon, etc.), frozen, and stored in individual plastic bags at –20°C before proceeding with further analyses and measurements.

The tibia bones from the right legs were boiled and thoroughly cleaned of any attached tissue. They were then weighed, dried at 109°C for 12 h, subjected to chemical cleaning in acetone for 48 h, dried again at 109°C for 12 h, and finally ashed in a muffle furnace overnight (550°C) to determine the percentage of ash content, following the method described by Asensio et al. (2020). The total bone Ca and P content were then tested using ICP-OES (Optima 3200 RL, Perkin Elmer, Waltham, MA).

Tibia bones from the left legs were utilized to evaluate the main morphological properties, including bone length, diameter, cortical thickness, and relative bone weight (expressed as a percentage of live body weight). Bone biomechanical properties, specifically tibia breaking strength and elasticity, were determined through a 3-point bending test using a material testing machine (TA.XTplus100, Stable Micro Systems, Godalming, United Kingdom), as described by Alfonso-Carrillo et al. (2021).

The percentage of mineral content (phosphate + carbonate) in cortical bone (CB) and medullary bone (MB) samples was estimated using thermogravimetric analysis (TGA), following the procedure described by Alfonso-Carrillo et al. (2021). In brief, carbonate content was assessed by measuring weight loss between 600 and 800°C in a furnace, while phosphate content was determined by the remaining weight at 800°C. Additionally, the mineral/organic matrix ratio (PO4/Amide I), indicating the degree of bone mineralization, was calculated using major absorption peaks identified by infrared spectroscopy. This was conducted with a FTIR spectrometer (FP-6200, JASCO International Co., Ltd., Tokyo, Japan) equipped with a diamond-attenuated total reflection accessory (ATR Pro ONE, JASCO International Co., Ltd., Tokyo, Japan) (Rodríguez-Navarro et al., 2018). For these analyses, CB and MB samples were collected from the mid-diaphysis of the tibiae, carefully separated using a scalpel, and homogenized by grinding in an agate ball mill.

Statistical Analysis

The experimental unit was the replicate for all measurements. Before analysis, the normality of the data (Shapiro-Wilk test) and homogeneity of the variance (Levene test) were verified. Bone quality traits and composition were subjected to a 2-way ANOVA using the GLM procedure. The model included the dietary fat source and the FFA level as the main factors, as well as their interaction. When the interaction fat source × FFA level was significant, differences among treatment means were tested using Tukey's test for multiple comparisons.

In addition, orthogonal linear contrasts of the GLM procedure were used for increasing levels of FFA when the effect of FFA content was significant. The contrast coefficients used were -0.671, -0.224, +0.224, and +0.671.

The results in the tables are reported as means and the pooled standard error of the mean. Differences were considered significant when P < 0.05. The statistical power was measured for the main effects and resulted strong enough (β < 0.2) using the Statistical Power Analysis. All the data analysis was performed using SPSS statistics (27.0.1.0, IBM, Armonk, NY, USA) (2020).

RESULTS

Characterization of the Experimental Diets

The detailed analysis of the experimental diets is shown in Table 2. All dietary treatments exhibited similar macronutrient contents, meeting the nutritional requirements of the birds. The main differences were related to their lipid composition. Two levels of UFA:SFA ratio (mean values = 4.4 and 1.2) were obtained. Concerning the lipid-class composition, both soybean and palm diets showed a progressive increase in the dietary free fatty acid (FFA) content. The average acidity levels achieved in the experimental diets were close to the theoretical ones previously formulated.Table 2 Determined analysis of the experimental diets (g/100g on a DM basis, unless otherwise indicated).

Table 2	Soybean	Palm	
Diets	S10	S20	S30	S45	P10	P20	P30	P45	
Determined analysis1									
 Gross energy, MJ/kg	17.7	17.5	17.4	17.4	17.0	17.2	17.2	16.7	
 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	
 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	4.0	4.4	4.2	4.1	4.1	4.3	4.2	4.1	
 Total phosphorus	0.66	0.67	0.63	0.67	0.68	0.62	0.69	0.66	
Lipid analysis									
 UFA:SFA2	4.6	4.4	4.4	4.2	1.3	1.2	1.2	1.1	
 Free fatty acid, %	9.0	19.1	29.8	43.5	10.6	22.2	34.1	47.9	
1 Analyzed at least in duplicate according to the methods of the AOAC International (2005).

2 Ratio of unsaturated to saturated fatty acid.

Morphological and Mechanical Tibia Properties

Morphological (length, weight, diameter, cortical thickness) and mechanical properties of the tibia bones from the different experimental groups analyzed in this trial are summarized in Table 3. Except for the tendencies (P < 0.10) observed for cortical thickness and breaking strength in the interaction between the fat source and the FFA, no significant differences (P > 0.05) were found in these parameters among the experimental groups. The macroscopic properties of the bones were not affected by the type of added fat or its acidity level.Table 3 Effects of fat source and dietary free fatty acid content on morphological and mechanical tibia properties.

Table 3Item	Length, cm	Weight, g	Relative weight, % BW	Diameter, mm	Cortical thickness, mm	Breaking strength, N	Elasticity, mm	
Experimental diet								
 S10	11.7	17.3	0.90	7.8	0.57	173.1	1.32	
 S20	11.6	17.0	0.89	7.5	0.67	157.2	0.99	
 S30	11.6	15.7	0.81	7.5	0.68	150.4	1.25	
 S45	11.7	16.8	0.87	7.7	0.66	165.2	1.39	
 P10	11.8	16.9	0.88	8.0	0.73	101.2	1.26	
 P20	11.7	16.3	0.83	7.5	0.53	177.3	0.97	
 P30	11.9	15.7	0.83	7.8	0.61	161.0	1.24	
 P45	11.6	16.6	0.87	7.7	0.58	122.2	0.79	
Fat source								
 Soybean	11.6	16.6	0.86	7.7	0.65	161.7	1.25	
 Palm	11.8	16.3	0.85	7.7	0.61	140.5	1.07	
FFA1content, %								
 10	11.7	17.1	0.89	7.9	0.65	140.4	1.29	
 20	11.7	16.5	0.85	7.5	0.60	167.3	0.98	
 30	11.8	15.7	0.82	7.7	0.65	155.7	1.24	
 45	11.6	16.7	0.87	7.7	0.62	143.7	1.09	
S.E.M	0.16	1.04	0.02	0.14	0.05	16.52	0.22	
Effects, P-values								
 Fat source	0.323	0.725	0.771	0.540	0.392	0.133	0.377	
 FFA content	0.912	0.708	0.752	0.131	0.822	0.452	0.649	
 Fat source x FFA	0.759	0.996	0.969	0.897	0.067	0.074	0.628	
Number of replicates analyzed: for each experimental diet n = 6, for each fat source n = 24, and for each FFA level n = 12.

1 Free fatty acid.

Tibia Mineralization

Table 4 summarizes the results concerning bone mineralization, quantified as ash and mineral content, and determined for each experimental group. Regarding bone ash content (as a % of the tibia weight), no influence of the FFA level and degree of fat saturation was found (P > 0.05). However, a tendency (P = 0.085) in the interaction fat source x FFA was observed. The type of supplemental fat source (degree of saturation) had a significant effect on Ca and P content (both as a % of the tibiae and ash weight), which was higher in the tibiae of the hens fed soybean diets than those fed palm diets (P < 0.001). In addition, increasing the dietary FFA level linearly reduced (P < 0.05) the P content (%) of the tibiae.Table 4 Effects of fat source and dietary free fatty acid content on tibia ash and mineral content.

Table 4Item	Ash, %	Ca, % tibiae	Ca, % ash	P, % tibiae	P, % ash	
Experimental diet						
 S10	60.9	35.7	58.7	16.1	26.4	
 S20	60.0	35.9	59.9	16.2	27.1	
 S30	59.9	35.7	59.7	16.0	26.8	
 S45	59.9	35.4	59.2	15.9	26.6	
 P10	58.3	34.6	59.4	15.5	26.7	
 P20	59.8	34.8	58.3	15.6	26.1	
 P30	60.4	34.3	56.8	15.4	25.5	
 P45	59.9	34.1	56.9	15.2	25.5	
Fat source						
 Soybean	60.2	35.7	59.4	16.1	26.7	
 Palm	59.6	34.5	57.9	15.5	26.0	
FFA1content, %						
 10	59.6	35.2	59.1	15.8ab	26.6	
 20	59.9	35.4	59.1	15.9a	26.6	
 30	60.1	35.0	58.2	15.7ab	26.2	
 45	59.9	34.8	58.1	15.6b	26.0	
S.E.M	0.64	0.25	0.72	0.11	0.34	
Effects, P-values						
 Fat source	0.225	< 0.001	0.005	< 0.001	0.002	
 FFA content	0.898	0.101	0.352	0.033	0.249	
 Fat source x FFA	0.085	0.886	0.071	0.959	0.117	
Linear contrast,2P-values						
 Overall	-	-	-	0.016	-	
Number of replicates analyzed: for each experimental diet n = 6, for each fat source n = 24, and for each FFA level n = 12.

1 Free fatty acid.

2 Linear responses to dietary free fatty acid content.

a-b Means within each variable with more than 2 levels (experimental diet or free fatty acid content) not sharing a common superscript differ according to Tukey's test (P < 0.05).

Concerning the chemical composition parameters (Table 5), in the case of cortical bone (CB), the degree of mineralization (% of mineral and PO4/Amide I ratio) was not affected by the dietary treatments (no statistical differences were observed for CB parameters among the experimental groups) (P > 0.05). In contrast, in the case of medullary bone (MB), a significant interaction (P < 0.05) between the fat source and the FFA content was found in the % of mineral. As the dietary FFA content increased in palm diets, the mineral content in MB decreased, showing a significant linear effect (P < 0.05). However, no differences were observed regarding the degree of mineralization of the MB (PO4/Amide I ratio).Table 5 Effects of fat source and dietary free fatty acid content on tibia material properties.

Table 5	Cortical bone	Medullary bone	
Item	Mineral, %	PO4/Amide I	Mineral, %	PO4/Amide I	
Experimental diet					
 S10	66.9	4.1	37.1ab	1.32	
 S20	65.8	3.9	38.8ab	0.95	
 S30	67.0	4.8	47.8a	1.32	
 S45	66.0	3.7	28.7ab	1.06	
 P10	67.6	4.2	45.2ab	1.54	
 P20	66.2	4.2	34.2ab	1.81	
 P30	65.8	3.8	19.2b	0.84	
 P45	67.0	3.7	28.7ab	1.01	
 Fat source					
 Soybean	66.4	4.1	38.1	1.16	
 Palm	66.7	4.0	31.8	1.30	
FFA1content, %					
 10	67.2	4.2	41.2	1.43	
 20	66.0	4.0	36.5	1.38	
 30	66.4	4.3	33.5	1.08	
 45	66.5	3.7	28.7	1.03	
S.E.M	0.46	0.28	5.38	0.30	
Effects, P-values					
 Fat source	0.557	0.485	0.248	0.585	
 FFA content	0.148	0.291	0.161	0.577	
 Fat source x FFA	0.237	0.293	0.032	0.298	
Linear contrast,2P-values					
 Overall	-	-	-	-	
 Soybean	-	-	0.549	-	
 Palm	-	-	0.035	-	
Number of replicates analyzed: for each experimental diet n = 6, for each fat source n = 24, and for each FFA level n = 12.

1 Free fatty acid.

2 Linear responses to dietary free fatty acid content.

a-b Means within each variable with more than 2 levels (experimental diet or free fatty acid content) not sharing a common superscript differ according to Tukey's test (P < 0.05).

DISCUSSION

A broad variety of lipid sources are routinely incorporated into poultry feed, primarily to increase its energy content. In addition, supplementing diets with fats and oils also offers other benefits. These include supplying essential fatty acids (FA), fat-soluble vitamins and carotenoids, while also improving egg size, palatability, and nutrient absorption (Mateos and Sell, 1981; Ravindran et al., 2016; Palomar et al., 2020). In recent years, there has been a growing focus on acid oils (AO) and fatty acid distillates (FAD) as valuable lipid sources for poultry diets (Wiseman et al., 1991; Blanch et al., 1996; Jimenez-Moya et al., 2021; Varona et al., 2021). These fat by-products differ from crude oils in their molecular structure, with FA in AO and FAD predominantly existing in a free form (FFA) rather than as triacylglycerols (TAG), as typically found in conventional fats (Varona et al., 2021).

On the other hand, inorganic sources of Ca and P are also commonly included in poultry diets. Indeed, the amount, origin, and particle size of these minerals are critical factors in formulating diets for laying hens. Careful adjustment of these nutrients is essential to meet the specific requirements for egg production and bone health, as emphasized by various authors (Nys and Guyot, 2011; Rodríguez-Navarro et al., 2015; Molnár et al., 2018). Therefore, understanding and optimizing these minerals is crucial for sustaining optimal performance and skeletal integrity in laying hens.

The digestion and absorption of dietary lipids are complex processes that can be influenced by the presence of added Ca. Pancreatic lipase hydrolyzes TAG into 2 molecules of FFA and one molecule of 2-monoacylglicerol (MAG), as the end products (Krogdahl, 1985; Ravindran et al., 2016). Under the pH conditions of the intestinal lumen, both the FFA released from TAG and those already present in the fat source tend to form insoluble Ca soaps by binding with Ca ions at a 1:2 molar ratio of Ca to FFA (Gacs and Barltrop, 1977; Leeson and Summers, 2005). This reduces the bioavailability of FFA to form mixed micelles, thereby lowering the absorption rates of FA. In this regard, it has been widely reported that Ca primarily affects the absorption of long-chain SFA and has minimal impact on short-chain, medium-chain, or UFA (Devraj et al., 2013; Ye et al., 2013; Tancharoenrat and Ravindran, 2014; Stroebinger et al., 2021). In fact, Graham and Sackman (1983) observed a negative correlation between the solubility of Ca soaps and acid concentration [H+], carbon chain length, and FA saturation.

Hence, incorporating AO and FAD into laying hen diets, which require elevated Ca levels, might not only compromise FA digestibility but also hinder adequate Ca absorption (Whitehead et al., 1971; Graham and Sackman, 1983; Atteh and Leeson, 1985a; Stroebinger et al., 2021). In our previous studies (Palomar et al., 2023a), significant differences were observed in Ca digestibility: hens fed soybean diets showed higher digestibility coefficients compared to those fed palm diets. Moreover, increasing the FFA content in both fat sources resulted in a linear reduction in Ca digestibility. Thus, both the percentage of FFA and the amount of SFA decrease the absorption of Ca. However, it is important to note that eggshell quality, including eggshell weight, Ca content, thickness, and breaking strength, remained unaffected by the dietary treatments (Palomar et al., 2023b).

As Ca metabolism is crucial in bone-related disorders (Whitehead, 2004; Gloux et al., 2019; Dunn et al., 2021), analyzing bone quality parameters becomes essential when there is concern about compromised calcium absorption. The tibia is frequently used as an indicator of skeletal development and mineralization in both laying hens and chickens (Skinner and Waldroup, 1995; Whitehead, 2004; Shim et al., 2012) due to its fast growth rate and high mineralization within avian skeletal structure. Additionally, this long bone contains large amounts of medullary bone (MB) (Benavides-Reyes et al., 2021; Johnsson et al., 2023).

To meet the substantial mineral demands for eggshell formation (approximately 2.0 g of Ca per egg), laying hens undergo specific physiological adaptations to maintain Ca balance. In addition to increasing Ca solubilization and absorption capacity upon reaching sexual maturity, layers have endogenous Ca reserves in the MB to ensure Ca availability in the uterus during eggshell formation at night (Whitehead, 2004; Nys and Guyot, 2011; Rodríguez-Navarro et al., 2015; Nys and Le Roy, 2018). In contemporary eggshell laying strains, the intense Ca demand throughout the entire laying cycle induces bone resorption and the release of minerals contained in cortical bone (CB), which provides structural support to laying hens (Toscano et al., 2020; Alfonso-Carrillo et al., 2021; Gautron et al., 2021; De Juan et al., 2023).

In this study, the degree of CB mineralization did not differ between the experimental treatments. Conversely, the replacement of palm oil (PO) with palm fatty acid distillates (PFAD) led to a linear decrease in the percentage of mineralization of the MB. This finding aligns with the lower Ca digestibility coefficients observed in palm diets (0.44) compared to soybean diets (0.52) in our previous research. Notably, the lowest coefficient value was found in the P45 dietary treatment (0.36), which had a low UFA:SFA ratio and the highest dietary FFA content (Palomar et al., 2023a). Greater amounts of mineralized MB in soybean diets may serve to protect CB from resorption. In this context, some authors have suggested that better mineralized MB in the tibia correlates with better mechanical properties (Fleming et al., 1994; Dunn et al., 2021).

Moving on the mineral content of the entire tibia, while the percentage of ash in bone did not differ between the experimental treatments in this study, hens fed soybean diets presented higher Ca content in their tibiae compared to those fed palm diets. However, there was no effect of acidity level or interaction between fat source and dietary FFA content. This finding is consistent with Atteh and Leeson (1984), who observed a significant reduction in bone ash and bone Ca content of broiler chickens fed diets supplemented with palmitic acid (C16:0), the primary FA found in PO and PFAD (Palomar et al., 2023a). Furthermore, they reported that Ca soap formation was more pronounced in SFA than in UFA. In contrast, when Atteh and Leeson (1985b) studied the response of laying hens to different FA varying in the UFA:SFA ratio (palmitic acid, oleic acid, or a blend of both at 80 g/kg of inclusion) in the presence of varying calcium levels (3.0, 3.6, or 4.2%), they reported no significant effect of dietary treatments on bone mineral content (ash and Ca).

Overall, the results obtained in this study concerning the composition of the entire bone, as well as CB and MB separately, suggest that the degree of saturation of lipids exerts a greater impact on Ca metabolism than their molecular structure (FFA content). This is consistent with Bornstein and Lipstein (1968), who studied the effect of 2 dietary calcium levels (3.1 or 4.9%) on the utilization of diets containing 60 g/kg of soybean acid oil (SAO) (approximately 73% FFA), and they found no differences in bone mineralization between groups.

Examining the biomechanical properties and morphology of long bones is also an indirect way to assess the gradual loss of Ca reserves and structural bone (Whitehead, 2004; Toscano et al., 2020; Dunn et al., 2021). In this experiment, although differences were observed among treatments in total tibia mineral content and in the degree of MB mineralization, the main morphological and mechanical properties examined remained unaffected.

The ability of hens to maintain adequate mineral balance for optimal bone health and shell strength is compromised during the laying period (Sinclair-Black et al., 2023). In addition to the high demand for egg production, Ca absorption efficiency decreases with age (Whitehead, 2004). It is often stated that the loss of structural bone and the development of avian osteoporosis and other skeletal problems are major concerns at the end of the laying cycle (60–70 wk) (Gloux et al., 2019; Alfonso-Carrillo et al., 2021). In this regard, Jiang et al. (2013) examined the impacts of dietary energy (19, 70, or 100 g/kg of soybean oil [SO]) and Ca levels (2.6, 3.7, or 4.4%) on eggshell quality and bone metabolism in laying hens at 19 wk of age for 60 d. Despite their young age (28 wk at the end of the trial), increasing the added fat content had adverse effects on bone quality (hens fed 70 or 100 g/kg SO had lower bone weight, bone density, and bone strength compared to those fed 19 g/kg), although it did not affect eggshell quality (thickness and breaking strength). It appears that in the presence of unfavorable conditions for proper intestinal Ca absorption, hens may experience secondary effects on their skeletal health, irrespective of age. In our study, the birds were fed the experimental diets until an early stage of the laying cycle (34 wk of age), and there were no differences in bone morphology and functionality.

Among the current trends in the egg industry, there is a notable emphasis on reducing environmental impact through the adoption of sustainable practices, such as utilizing alternative raw materials and extending the laying cycles of hens (100 wk or longer) (Abín et al., 2018; Alfonso-Carrillo et al., 2021). Additionally, to meet customer demand, there is an increasing interest in housing birds in cage-free alternative systems, such as aviaries and floor pens, which provide animals with more freedom of movement. Regarding this matter, it has been reported that not only age but also housing conditions have a fundamental and complex influence on the bone quality of layer hens (Fleming et al. 2006; Rodríguez-Navarro et al. 2018). For example, Johnsson et al. (2023) observed enhanced bone strength and mineralization in commercial flocks housed in floor pens compared to layers kept in enriched cages. Thus, allowing for more exercise stimulates bone formation and increases mineralization of the MB (Shipov et al. 2010; Rodríguez-Navarro et al. 2018).

Our previous results and those presented here indicate that, despite differences being observed in Ca digestibility and tibia composition, the Ca content in the eggshell remained consistent across the experimental treatments. It appears that hens prioritize Ca deposition for eggshell formation over the allocation of mineral reserves to the bones. As described by several authors (Deeming, 2002; D'Alba et al., 2021; Sinclair-Black et al., 2023), this is an evolutionary adaptation that prioritizes the animal's reproductive state to ensure species continuity.

Eggshell morphology is essential for animal survival as it mediates the interaction between the embryo and its environment (Sinclair-Black et al., 2023). Therefore, female birds ensure a continuous supply of Ca via the bloodstream for proper eggshell mineralization (Whitehead, 2004; Nys and Le Roy, 2018). Eggshell components and shape indeed vary widely among avian taxa due to their crucial role in birds' adaptation to diverse environments. Thickness, porosity, and the degree of calcification determine the eggshell's function as protective barrier, safeguarding the egg against physical damage such as fractures and preventing the entry of microorganisms (Deeming, 2002; D'Alba et al., 2016).

In conclusion, results from this study highlight that bone mineralization in laying hens was more affected by the degree of saturation of the added fat source than by the dietary FFA content. Altogether, these findings support the inclusion of alternative fat sources rich in FFA, such as AO and FAD, in laying hen diets, particularly during the early stages of laying. To ensure effective implementation in later stages, a comprehensive study of the long-term effects of feeding laying hens with AO and FAD in non-cage systems is essential. This is especially relevant now as the industry aims to improve lay persistency over extended cycles in alternative housing conditions, aiming to promote sustainable egg production.

DISCLOSURES

The authors declare no conflicts of interest.

ACKNOWLEDGMENTS

This research was carried out with the financial support of the Generalitat Valenciana and the European Social Fund (GV/188/2018 ), and a pre-doctoral research grant from the Generalitat Valenciana and the European Social Fund (ACIF/2019/201 ). The authors wish to thank Greg Hunt for his help in proofreading the manuscript and resolving language-related issues.
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