Egg quality, fatty acids profile, and hatchability of broiler breeders fed diets supplemented with Conjugated Linoleic Acid (CLA)

Qualidade dos ovos, perfil de ácidos graxos da gema e eclodibilidade de matrizes suplementadas com Ácido Linoleico Conjugado (CLA)

ABSTRACT

Conjugated linoleic acid (CLA) refers to a group of positional and geometric isomers of linoleic acid. Hens fed a diet supplemented with CLA exhibit increased levels of saturated fatty acids, which may influence the quality characteristics of eggs and the offspring lipid metabolism. The present study, aimed to evaluate the effect of the dietary inclusion of CLA for broiler breeders on the residual yolk sac fatty acid profile, egg quality, and hatchability. Two 58-week-old Cobb500^® broiler breeder commercial flocks were fed diets supplemented with 0% or 0.025% CLA (trans-10, cis-12). After 26 days, 300 eggs from each treatment group were collected, and among these eggs, 30 were subjected to egg quality assessment and egg yolk fatty acid profile analysis. The remaining 270 eggs were subjected to incubation analysis. The quality of the chicks was evaluated at hatch. The residual yolk sac content and weight at hatch were also evaluated. Unhatched eggs were subjected to residual analysis. The supplementation of the breeders’ diet with 0.025% CLA did not influence the incubation parameters and the weight of the chicks at hatch. However, the egg yolk and residual yolk sac fatty acid profile were altered and CLA accumulation was increased in the residual yolk sac at hatch in this treatment group. In conclusion, CLA supplementation in the diet of breeders did not influence the incubation parameters and the weight of the chicks, although it did alter the profiles of omega-6, palmitoleic, linoleic, and arachidonic acids in both egg yolk and residual yolk sac.

Index terms:
Embryonic development; lipid metabolism; breeder nutrition.

RESUMO

O ácido linoleico conjugado (CLA) compreende o grupo de isômeros posicionais e geométricos do ácido linoleico. Na dieta de matrizes, aumenta a proporção de ácidos graxos saturados dos ovos e pode ser uma importante ferramenta para o estudo dos efeitos da suplementação materna de CLA sobre o metabolismo lipídico da progênie. Objetivou-se avaliar o efeito da inclusão de CLA na dieta de matrizes de frango de corte sobre o perfil de ácidos graxos da gema, a qualidade dos ovos e a incubação. Dois lotes comerciais de matrizes Cobb500^®, com 58 semanas de idade, foram suplementados com 0% ou 0,025% de CLA (trans-10, cis-12). Após 26 dias, 300 ovos por tratamento foram coletados, dois quais 30 foram utilizados para as análises de qualidade e perfil de ácidos graxos da gema, enquanto os ovos restantes foram submetidos à incubação. A qualidade das aves foi avaliada à eclosão, e o peso e conteúdo do saco vitelínico residual foram avaliados à eclosão. Ovos não eclodidos foram submetidos a análise residual. A suplementação das matrizes não influenciou os parâmetros de incubação e peso dos pintos à eclosão. Contudo, modificou o perfil de ácidos graxos da gema e do saco vitelínico, levando ao acúmulo de CLA no saco vitelínico após a eclosão. Conclui-se que a suplementação de 0,025% de CLA na dieta de matrizes não altera parâmetros de incubação e o peso da progênie à eclosão, mas modifica os perfis de ômega-6 e ácidos palmitoleico, linoleico e araquidônico tanto na gema quando no saco vitelínico residual.

Termos para indexação:
Desenvolvimento embrionário; metabolismo lipídico; nutrição da matriz.

Introduction

Conjugated linoleic acid (CLA) refers to a group of positional and geometric isomers of linoleic acid. The term CLA is used for collectively describing all the known linoleic acid isomers, which, rather than being separated by an equivalent organic radical, have double bonds with a simple carbon between them (Schmid et al., 2006). CLA provides numerous health benefits to both humans and animals. Chicks supplemented with CLA, for instance, during the post-hatch phase exhibit improved humoral and cellular immunity (Martins et al., 2023). In addition, studies have reported the effects of CLA on the performance, meat and egg quality, and lipid profile of yolks in birds (Kennedy et al., 2010).

CLA may reduce lipogenesis in adipose tissues, as confirmed by previous in vitro and in vivo studies (Go et al., 2013). Diets containing CLA reportedly reduce the activity of stearoyl-CoA desaturase (Lee, Pariza, & Ntambi, 1998), thereby increasing the proportion of saturated fatty acids compared to that of unsaturated fatty acids in the yolk, which may then influence the quality characteristics of eggs (Hur et al., 2003).

Another possible and critical consequence of an altered proportion of saturated and unsaturated fatty acids is embryonic death (Aydin, & Cook, 2004). The yolk sac is the only energy source available for the embryo during embryonic development, providing fat-soluble vitamins, essential fatty acids, and phospholipids, which are used for tissue formation. (Maiorka, Dahlke, & Morgulis, 2006; Wojnarowicz & Olkowski, 2009). Therefore, changes in the yolk sac content could be harmful to the developing embryo as well (Fu et al., 2020).

The yolk sac is internalized into the abdominal cavity between the 19th and 20th day of incubation and plays important nutritional and immunological functions to the neonate chick (Araújo et al., 2020; Santos et al., 2022a). The supplementation of CLA in the breeders’ and chicks’ diet may also modify the composition of the yolk sac and affect the content of other nutrients present in eggs and consequently, improve these nutritional and immunological functions. An example of this effects can occur in the yolk’s mineral content that can change the structure of the egg membranes that allow the exchange of nutrients between the albumen and yolk (Ahn et al., 1999; Shang et al., 2004; Santos et al., 2022b).

CLA is efficiently transferred to the egg yolk when hens are fed diets with CLA supplementation (Shang et al., 2005). Therefore, a CLA-modified egg is a useful tool to study the effects of maternal dietary CLA on offspring lipid metabolism. Cherian, Ai, and Goeger (2005) reported that maternal CLA supplementation (1% and 2%) reduced the liver triacylglycerol content, carcass total fat, and hatchability in newly hatched chicks.

Nonetheless, studies exploring the impact of CLA supplementation in the diet of broiler breeders, ranging from the investigation of egg parameters to hatch results, are scarce in the literature. It is, however, important to determine if CLA supplementation positively influences egg and chick quality, considering the specific changes in the yolk and residual yolk fatty acids profiles, while being safe for the embryo. The present study, therefore, aimed to study the effects of supplementing breeders’ diet with 0.025% CLA (trans-10, cis-12) on the yolk and residual yolk sac fatty acid profiles, egg quality, and hatch results.

Material and Methods

The study was conducted in accordance with the institutional committee on the use of animals (CEUA UFG Protocol 001/2015). The experiments were conducted at a broiler breeder’s farm in the city of Formosa, GO, Brazil (15°32′13′′ S, 47°20′9 W) and in an experimental hatchery in the city of Goiania, GO, Brazil (16°40′ S, 49°15′ W).

Broiler breeders and diet treatments

The eggs to be used in the present study were collected from a 58-week-old Cobb500^® broiler breeder commercial flock. A total of 24,800 hens and 2,480 roosters were distributed in two distinct breeder houses, each with a stocking density of 8 birds/m². The breeders were fed corn- and soybean meal-based diets containing [or not containing] 0.042% of a commercial product (Lutalin^® BASF), according to the manufacturer’s recommendations to ensure a 0.025% supplementation of CLA (trans-10, cis-12) [or not] for 26 days (Table 1). The diets were formulated according to the nutritional levels proposed by Cobb-Vantress (2018). Fresh diets were prepared each week, and ethoxyquin (0.015% of feed) was added to all diets to prevent fat oxidation.

Table 1:
Composition of the diets fed to breeders (g, as-fed basis).

Ingredient	Control	CLA
Corn	68.17	68.12
Soybean meal 460 g/kg	17.41	17.42
Limestone	7.87	7.87
Caulim	4.00	4.00
Meat and bone meal 410g/kg	1.50	1.50
NaCl	0.22	0.22
Dicalcim phosphate	0.20	0.20
Sodium bicarbonate	0.15	0.15
Mineral supplementA	0.15	0.15
Vitamin supplementB	0.10	0.10
Coline	0.08	0.08
DL-methionine	0.08	0.08
L-threonine	0.03	0.03
AnthioxidantC	0.01	0.01
CLAD	-	0.04
EnzimeE	0.003	0.003
Calculated analysis (digestible)
Metabolizable Energy (Kcal/kg)	2.736	2.734
Crude protein (%)	13.90	13.90
Linoleic acid (%)	2.50	2.57
dig Lysine (%)	0.60	0.60
dig Methionine and cystine (%)	0.47	0.46
dig Threonine (%)	0.50	0.50
Calcium - Ca (%)	3.29	3.29
Available phosphorum (%)	0.20	0.20
Sodium - Na (%)	0.34	0.34

• ^AMineral supplement - guaranteed levels per kilogram of the product: Manganese 80,000 mg, Zinc 70,000 mg, Iron 40,000 mg, Copper 8,000 mg, and Iodine 1,000 mg. ^BVitamin supplement - guaranteed levels per kilogram of product: Vitamin E 110,000 mg, Vitamin A 14,000 IU, Vitamin D3 3,000 IU, Vitamin K3 6,000 mg, Vitamin B1 3,000 mg, Vitamin B2 12,000 mg, Vitamin B6 6,000 mg, Vitamin B12 30 mg, Niacin 60,000 mg, Pantothenic acid 20,000 mg, Folic acid 4,000 mg, and Biotin 300 mg. ^CEthoxiquim 66.6%, BHA 99%, and citric acid 99.5%. ^DCLA (Lutalin®). ^EPytase enzyme (Natuphos® 10,000).

Birds, facilities, and handling procedures

The broiler breeder flock was housed inside open-sided houses with concrete floors covered with wood shavings at a depth of 10 cm. The litter was stirred or turned to maintain its condition. The wet litter spots were replaced with fresh new litter every day. The photoperiod of 16L:8D was maintained throughout the experimental period. All management practices were standardized for the two treatments.

Feed analysis

In each treatment, 600 g of feed was collected directly from the breeder feeders at three different time points. The fatty acid profiles of all diet groups were determined using the methods described by the Association of Official Analytical Chemists (Association of Official Analytical Chemists - AOAC, 1995) inside a commercial laboratory using high-performance liquid chromatography (HPLC), with the total fat extraction based on hydrolysis performed according to AOAC (2010).

Experimental treatments and design and the collection of eggs

On the 26^th day of feeding the breeders, 600 hatching eggs per breeder house were selected in a single day from the nests of both control and CLA diet groups. These eggs were then assigned to the following two treatments:

Eggs from the breeders fed a diet supplemented with CLA;

Eggs from breeders fed a diet not supplemented with CLA.

A completely randomized experimental design was adopted for the experiments, and each egg was considered an experimental unit and a replicate.

In each group, 300 eggs were selected, as experimental units, considering the integrity of the shell, elliptical shape, and average weight (70.30 g ± 4.83 g). The eggs were placed in trays and then transported to the experimental hatchery at the Goiania city the same day.

Egg quality

Egg quality was evaluated at the end of the experimental period by randomly selecting 30 eggs from each treatment (totaling 60 eggs) using a completely randomized design. Each egg was considered a replicate. Only the eggs laid on the day of collection were selected. The eggs were then evaluated for yolk percentage, albumen percentage, eggshell percentage, eggshell thickness, and Haugh unit (HU). The percentage of egg components was calculated as described by Carvalho et al. (2018). Eggshell thickness was evaluated using an IP40 digital outside micrometer (Digimess, Mooca, Brazil) with a readability of 0.001 mm. Measurements were performed at three distinct eggshell regions (apical, equatorial, and basal regions), and the average of the values obtained for these three regions was used as the final value that was expressed in mm. HU was calculated based on the egg weight and albumen height data using an HU-measuring device (model S-8400, Ames, Massachusetts) and the following equation: HU = 100 log10 (H − 1.7 W^0.37 + 7.56), where H = albumen height and W = egg weight (Haugh, 1937).

Fatty acid profile of egg yolk

The fatty acids in the egg yolk were quantified using gas chromatography, as described by Ding et al. (2017). A total of 30 yolks, already used for evaluating egg quality parameters, were analyzed from each treatment group. The egg yolks were grouped into six pools of five units, and each pool was considered a replicate. Samples were freeze-dried and stored until analysis. The fatty acid methyl esters were separated using a GC-9A gas chromatograph equipped with a silica capillary column and a flame ionization detector. Nitrogen was used as the carrier gas at a flow rate of 35 mL/min. The pressure of hydrogen and air was set to 0.5 kg/cm. The injector and detector were maintained at a temperature of 250 °C. The fatty acid methyl esters were identified by comparing their retention times with those of the standards. The fatty acid composition was then expressed as the percentage of total fatty acids. It is possible that the trans-8, cis-10 CLA isomer was present in the CLA source; however, its peak overlapped with that of cis-9, trans-11 CLA, due to which it could not be detected in the present study.

Incubation

A total of 528 eggs were subjected to the assessments of hatchability and residual analysis, and 264 eggs per treatment were, therefore, considered experimental units. In the incubation period, the eggs were distributed using a randomized block design, with each block comprising two setters (P > 0.05). The eggs were distributed in eight trays and then stored in a room at 17 °C and 75% relative humidity (RH) for two days. Each tray contained 66 eggs (33 eggs per treatment). The eggs were preheated for 6 h until the temperature reached 26.0 to 37.7 °C, which was maintained. In each setter, the eggs were distributed in four trays. Thereafter, the trays were placed inside two rotating single-stage incubators (Gaiolas Almeida Commercial, Goiânia, Brazil) set to 37.8 °C, 60.0% RH, and 45 rotations per hour, until 17 days of embryo development (DE17). At 18 days of embryo development (DE18), all eggs were candled, and the eggs with viable embryos were weighed and placed individually in air-permeable fabric bags for treatment control. Afterward, the trays were placed in the setter at 36.7 °C and 65.0% RH for 520 h of incubation. All newly hatched chicks were weighed at hatch.

The hatchability was evaluated for each tray and estimated relative to the number of fertile eggs. The number of fertile eggs was calculated using the results of the residual analysis (85.0% [control] and 85.7% [CLA]). Hatching was monitored during incubation from 470 h to 520 h. All unhatched eggs remaining at the end of the incubation (520 h) were removed from the experimental trays and then evaluated for the residual analysis as described by Plano and Di Matteo (2001).

Fatty acid profile of yolk sac

At hatch, five chicks per treatment were selected randomly for residual yolk sac collection. The newly hatched chicks were weighed and euthanized using cervical dislocation to retrieve the residual yolk sacs with vitelline membrane. The yolk sacs were weighed, and the relative weight was calculated using the following equation: relative weight = (absolute weight × 100)/chick weight. A randomized block design was adopted when evaluating the residual yolk sac weight and chick body weight, with each setter considered a block and five replications considered for the newly hatched chicks. All samples were freeze-dried and stored until analysis. The fatty acids in the residual yolk sac were quantified using gas chromatography as described by Ding et al. (2017).

Statistical analysis

All result data analyzed were first evaluated for normality using the Shapiro-Wilk test. The data from the residual analysis were evaluated based on frequency dispersion using Fisher’s exact test (P < 0.05). The remaining variables were analyzed using analysis of variance (ANOVA). All results were evaluated statistically at a significance level of 0.05. R software Version 3.4.4 (R Core Team, 2016) was used for all analyses. The statistical model used was an Equation (1) as it follows.

$${\text{Y}}_{\text{ij}}\text{=}\mu +{\text{T}}_{\text{i}}+{\epsilon }_{\text{ij}}$$ (1)

where, Y_ij = variable studded, μ = the constant inherent to all experimental units, T_i = effect of treatment (Control and CLA), and ε_ij j = random error.

Results and Discussion

Feed analysis revealed that CLA supplementation increased the levels of monounsaturated fatty acids (P = 0.040) and omega-9 (P = 0.039) in the diet (Table 2). The levels of polyunsaturated fatty acids, unsaturated fatty acids, and saturated fatty acids, on the other hand, remained unaffected (P > 0.05). The saturated to unsaturated fatty acids ratio, omega-3, omega-6, and total fat also remained unaltered (P > 0.05). The levels of palmitic (P = 0.034) and oleic acid (P = 0.033) were higher in the diets supplemented with CLA than control. No differences (P > 0.05) were noted in the levels of other fatty acids after CLA supplementation.

Table 2:
Effects of the inclusion of conjugated linoleic acid (CLA) in the diet of breeders on feed composition.

Composition (%)	Treatments		P-value	SEM
	Control	CLA
Monounsaturated fatty acids	1.90 b	2.12 a	0.040	0.40
Polyunsaturated fatty acids	2.83	2.91	0.366	0.19
Unsaturated fatty acids	4.73	5.03	0.111	1.02
Saturated fatty acids	1.23	1.35	0.077	0.60
Saturated: Unsaturated fatty acids	0.28	0.28	0.373	0.10
Omega-3	0.10	0.09	0.116	0.02
Omega-6	2.72	2.81	0.331	0.34
Omega-9	1.88 b	2.10 a	0.039	0.07
Fat total	5.96	6.40	0.089	1.04
Fatty acid (%)	Control	CLA	P-value	SEM
Mirystic (C14:0)	0.02	0.02	0.373	0.04
Mirystoleic (C14:1)	0.003	0.003	1.000	0.001
Pentadecanoic (C15:0)	0.006	0.003	0.518	0.007
Palmitic (C16:0)	0.83 b	0.93 a	0.034	0.27
Palmitoleic (C16:1)	0.02	0.02	1.000	0.004
Margaric (C17:0)	0.01	0.01	0.373	0.009
Steaic (C18:0)	0.28	0.30	0.260	0.03
Oleic (C18:1n9c)	1.86 b	2.08 a	0.033	0.04
Linoleic (C18:2n6c)	2.72	2.80	0.348	0.04
Alpha-Linolenic (C18:3n3)	0.10	0.09	0.116	0.02
Arachid (C20:0)	0.02	0.03	0.373	0.01
Cis-11-Eicosenoic (C20:1n9)	0.01	0.01	1.000	0.002

• ^a-b Means within the same line and with different letters are different according to the t-test of the analysis of variance (P < 0.05).

In the present study, the dietary supplementation of 0.025% CLA had no significant effect (P > 0.05) on the egg quality of Cobb breeder hens (Table 3). High-dose supplementation of CLA in the diets of breeders may disrupt the homeostasis of lipid metabolism in the liver, leading to a decrease in egg quality (Kim et al., 2007). CLA supplementation may, therefore, alter the lipid profile of the yolk and yolk sac without affecting the quality of the eggs. Previous studies have demonstrated that a diet containing CLA might decrease egg production and quality when the CLA supplementation level exceeds 2% (Kim et al., 2007; Leone, Worzalla, & Cook, 2010).

Table 3:
Effects of including conjugated linoleic acid (CLA) in the diets of breeders on egg quality.

Items	Treatments		P-value	SEM
	Control	CLA
Egg weight (g)	70.68	72.93	0.082	0.87
Eggshell thickness (mm)	0.382	0.373	0.081	0.01
Yolk weight (%)	30.24	31.00	0.151	0.18
Albumen weight (%)	61.27	60.64	0.265	0.27
Eggshell weight (%)	8.46	8.37	0.649	0.15
Haugh unit	78.57	79.78	0.071	2.09

The egg yolks obtained from the breeders fed diets supplemented with CLA exhibited significantly high percentages of margaric (P = 0.007), linoleic (P = 0.009), gamma-linolenic (P = 0.007), and arachidonic acids (P = 0.01) along with high levels of omega-6 (P = 0.005). On the other hand, high percentages of palmitoleic (P < 0.001) and oleic acids (P = 0.005) and high content of monounsaturated (P = 0.002), unsaturated (P = 0.001), and total fats (P = 0.011) (Table 4) were observed in the egg yolks obtained from the breeders fed diets not supplemented with CLA. The percentages of other fatty acids evaluated did not differ between the treatments (P > 0.05).

Table 4:
Effects of the inclusion of conjugated linoleic acid (CLA) in the diets of breeders on egg yolk fatty acid profile.

1Composition (%)	Treatments		P-value	SEM
	Control	CLA
Monounsaturated fatty acids	27.53 a	26.72 b	0.002	0.52
Polyunsaturated fatty acids	10.35	10.42	0.605	1.47
Unsaturated fatty acids	37.87 a	37.14 b	0.001	0.29
Saturated fatty acids	21.73	21.81	0.511	0.68
Saturated: Unsaturated fatty acids	0.57	0.58	0.101	1.00
Omega-3	0.59	0.47	0.480	36.78
Omega-6	9.67 b	9.86 a	0.005	0.45
Omega-9	25.53	25.27	0.509	1.74
Fat total	59.60 a	58.95 b	0.011	0.30
1Fatty acid (%)	Control	CLA	P-value	SEM
Lauric (C12:0)	0.006	0.01	0.373	0.002
Myristic (C14:0)	0.26	0.27	0.373	0.02
Myristoleic (C14:1)	0.06	0.05	0.116	0.01
Pentadecanoic (C15:0)	0.04	0.04	1.000	0.003
Palmitic (C16:0)	16.02	15.94	0.414	0.82
Palmitoleic (C16:1)	1.94 a	1.72 b	<0.001	0.01
Margaric (C17:0)	0.13 b	0.15 a	0.007	0.01
Stearic (C18:0)	5.24	5.39	0.021	0.02
Elaidic (C18:1n9t)	0.06	0.06	0.373	0.00
Oleic (C18:1n9c)	25.32 a	24.74 b	0.005	0.01
Linoleic (C18:2n6c)	7.92 b	8.07 a	0.009	0.002
Gamma-Linolenic (C18:3n6)	0.10 b	0.11 a	0.007	0.003
Alpha-Linolenic (C18:3n3)	0.16	0.16	1.000	0.00
Arachid (C20:0)	0.02	0.02	1.000	0.01
Cis-11-Eicosenoic (C20:1n9)	0.14	0.14	0.116	0.001
Cis-11,14 -Eicosenoic (C20:2)	0.09	0.08	0.116	0.006
Cis-8,11,14 - Eicosenoic (C20:3n6)	0.16	0.15	0.116	0.002
Arachidonic (C20:4n6)	1.48 b	1.52 a	0.001	0.03
Cis-11,14,17-Eicosatrienoic	0.00	0.003	0.373	0.001

• ^a-b Means within the same line and with different letters are different according to the F test of the analysis of variance (P < 0.05). 1The analysis of six pools of five eggs per treatment, after freeze drying.

Further, CLA supplementation had no significant effect (P > 0.05) on fertility, hatchability, or chick body weight (Table 5). Hatchability in both treatment groups was similar to that documented in the Cobb breeder management guide (Cobb-Vantress, 2018). Similar collection and storage conditions may have contributed to these similar results regarding hatchability.

Table 5:
Effects of including conjugated linoleic acid (CLA) in the diets of breeders on hatching results.

Items	Treatments		P-Value	SEM
	Control	CLA
1Fertility (%)	85.0	85.7	0.857	0.43
2Hatchability (%)	72.50	72.08	0.932	0.61
Chick body weight (g)	51.28	51.22	0.825	0.67

• ¹Number of eggs: Control - 264; CLA - 264. ²Number of eggs: Control - 224; CLA - 226.

Moreover, no effect of CLA supplementation (P > 0.05) was revealed in the residual analysis (Table 6). No contamination or embryos with malformations or incorrect positioning inside the eggs were observed in either of the treatment groups.

Table 6:
Results of residual analysis conducted to reveal the effects of including conjugated linoleic acid (CLA) in the diets of breeders.

Items (%)	Treatments		P-value
	Control	CLA
Infertile	17.39	16.95	1.000
1Phase I (0-4 d)	10.04	9.00	0.868
1Phase II (5-17 d)	11.65	11.65	1.000
1Phase III (18-21 d)	0.43	0.87	1.000
Pipped alive	2.67	3.13	1.000
Contaminated	0.00	0.87	0.498
Abnormality	0.00	0.00	1.000
Cracked eggs	1.32	1.32	1.000
Incorrect position	0.00	0.00	1.000
Total mortality	26.11	26.84	0.930

• ¹Embryonic mortality registered during the mentioned phase.

The findings of the present study indicated that the newly hatched chick’s weight was not affected by CLA supplementation in the diet. Breeders’ nutrition is responsible for chick quality at hatch (Pappas et al., 2006; Yalçin et al., 2008; Santos et al., 2022b). The size of the newly hatched chick is affected by several factors, including the nutritional levels of the breeders, the nutritional levels of eggs, and incubation conditions (van der Wagt et al., 2020; Santos et al., 2022b). The initial immune status of the chicks is another factor determining initial performance, and CLA reportedly influences immunity (Martins et al., 2023). CLA may also alter the composition of egg yolk (Bautista-Ortega, Goeger, & Cherian, 2009), facilitating nutrient availability to the developing embryo.

The yolk contains lipids, proteins, vitamins, minerals, and other essential micronutrients, serving as the main source of nutrients for the developing embryo (Santos et al., 2022b). The inclusion of CLA in the diet of breeders reportedly reduces the activity of stearoyl-CoA desaturase (Lee, Pariza, & Ntambi, 1998; Fu et al., 2022) and alters the contents of saturated and unsaturated fatty acids in the yolk, leading to embryonic death (Aydin & Cook, 2004). However, the percentage of CLA (0.025%) supplemented in the present study did not cause these changes to the extent of impairing embryonic development. The findings of the present study suggest that fatty acids were assimilated in the yolk by the embryo. Fu et al. (2022) reported that CLA supplementation in breeders increased the levels of CLA in chick liver, which improved the oxidative status of chicks, leading to the regulation of the hepatic lipid metabolism.

The fatty acid profiles of the chick residual yolk sacs at hatch were also obtained in the present study. The breeders fed diets supplemented with CLA presented higher percentages of palmitoleic (P = 0.004), stearic (P = 0.002), and linoleic acids (P = 0.001) compared to the breeders fed diets not supplemented with CLA, with a relatively high composition of saturated fat (P = 0.075) and a relatively high ratio of saturated to unsaturated fats (P = 0.025) (Table 7). In contrast, the levels of myristic (P = 0.066), Cis-11,14-eicosadienoic (P = 0.007), and arachidonic acids (P = 0.010) were reduced upon CLA supplementation. In addition, omega-3 (P < 0.001), omega-6 (P = 0.001), and polyunsaturated fats were reduced upon CLA supplementation (P = 0.001).

When comparing yolk and residual yolk sac fatty acid profiles between different treatment groups, fatty acids were detected in the residual yolk sac during the final stage of incubation. The presence and rapid consumption of lipids in the yolk sac during the last stages of incubation indicated their importance in embryo development (Ding & Lilburn, 1996).

Table 7:
Effects of the inclusion of conjugated linoleic acid (CLA) in the diets of breeders on residual yolk sac fatty acid profile.

1Composition (%)	Treatments		P-value	SEM
	Control	CLA
Monounsaturated fatty acids	20.14	20.48	0.599	1.51
Polyunsaturated fatty acids	7.31 a	6.52 b	0.001	0.62
Unsaturated fatty acids	27.45	26.90	0.329	1.25
Saturated fatty acids	15.06 b	15.76 a	0.075	0.05
Saturated:Unsaturated fatty acids	0.55 b	0.58 a	0.025	0.007
Omega-3	0.25 a	0.19 b	<0.001	0.002
Omega-6	6.98 a	6.26 b	0.001	0.08
Omega-9	19.03	19.11	0.853	0.09
Fat total	42.51	42.67	0.834	1.01
1Fatty acid	Treatments		P-value	SEM
	Control	CLA
Caproic (C6:0)	0.00	0.003	0.373	0.001
Capric (C10:0)	0.003	0.00	0.373	0.001
Lauric (C12:0)	0.01	0.01	1.000	0.001
Myristic (C14:0)	0.18 a	0.004 b	0.066	0.02
Myristoleic (C14:1)	0.04	0.04	1.000	0.00
Pentadecanoic (C15:0)	0.03	0.03	1.000	0.00
Palmitic (C16:0)	10.92	11.34	0.144	0.26
Palmitoleic (C16:1)	1.07 b	1.23 a	0.004	0.005
Margaric (C17:0)	0.11	0.10	0.116	0.001
Stearic (C18:0)	3.76 b	4.04 a	0.002	0.12
Elaidic (C18:1n9t)	0.03	0.03	0.518	0.001
Oleic (C18:1n9c)	18.89	18.95	0.872	0.09
Linoleic (C18:2n6c)	5.28 b	5.95 a	0.001	0.003
Gama- Linolenic (C18:3n6)	0.08	0.08	1.000	0.001
Alpha- Linolenic (C18:3n3)	0.09 b	0.11 a	0.001	0.001
Araquidic (C20:0)	0.02	0.02	1.000	0.0
Cis-11-Eicosenoic (C20:1n9)	0.11	0.11	1.000	0.001
Cis-11,14- Eicosadienoic (C20:2)	0.07 a	0.06 b	0.007	0.001
Cis-8,11,14-Eicosatrienoic (C20:3n6)	0.11	0.11	1.000	0.00
Arachidonic (C20:4n6)	0.84 a	0.79 b	0.010	0.05
Behenic (C22:0)	0.01	0.01	1.000	0.004
Tricosanoic (C23:0)	0.006	0.003	0.518	0.003
Lignoceric (C24:0)	0.01	0.01	1.000	0.001
Nervonic (C24:1n9)	0.006	0.006	1.000	0.000
Docosahexaenoic (C22:6n3)	0.13 a	0.10 b	0.002	0.01

• ^a-b Means within the same line and with different letters are different according to the F-test of the analysis of variance (P < 0.05). ¹The analysis of five residual yolk sacs per treatment, after freeze-drying.

The supplementation of breeder diets with CLA resulted in increased levels of linoleic acid in the yolk. Moreover, this linoleic acid in the residual yolk was also detected in the hatched chick. Since birds are not capable of synthesizing linoleic acid, its presence in the yolk and yolk sac is essential and its increased availability is beneficial for the metabolism and quality of the chicks.

In addition, CLA supplementation altered the levels of omega-6, palmitoleic, and arachidonic acids in both the yolk and the residual yolk sac. The increase in the levels of arachidonic acid in the yolk upon CLA supplementation implied a greater availability of this fatty acid from the beginning of embryonic formation. Considering the role of this fatty acid in the synthesis of eicosanoids, its greater availability could have a direct influence on the immunity of the newly hatched chick.

However, while increased levels of omega-6 were noted in the yolk upon CLA supplementation, the residual yolk sac had reduced amounts of omega-3 and omega-6. Both fatty acids influence immune function, and omega-3 is important for the development of cell membranes.

Stearoyl-CoA desaturase is an enzyme that catalyzes the step of introducing double bonds in the formation reaction of unsaturated fatty acids (Moran Jr., 2007). Since CLA reduced the activity of this enzyme (Latour et al., 2000), a significant increase in embryonic mortality was expected upon CLA supplementation. However, CLA supplementation in the present study was not sufficient to cause changes that could interfere with the development of the embryo, rendering this level of CLA supplementation safe for the diets of broiler breeders. Further, CLA supplementation reportedly reduces hepatic triglyceride accumulation (Andreoli et al., 2010). It may also decrease and inactivate the expression of the essential components of the lipogenic process in the yolk sac membrane (Fu et al., 2020). According to the results of the present study, it was speculated that the presence of linoleic acid in the residual yolk sac might generate the same results in the progeny.

Few studies have investigated the effects of CLA supplementation in the diets of broiler breeders on hatchery results. In the present study, supplementation with 0.025% CLA (cis-12, trans-10) could alter the yolk and residual yolk sac fatty acid profile without impairing hatchability and chick quality. These results differ from those reported by Cherian, Ai, and Goeger (2005), who noted a 50% reduction in the hatchability of eggs from the broiler breeders fed diets supplemented with 1% CLA. In separate studies, Muma et al. (2006) and Aydin and Cook (2005) observed 100% embryonic death when the breeders were fed diets supplemented with 0.5% CLA. Accordingly, it is recommended that the inclusion of 0.025% CLA in the breeder diet could be considered safe for egg hatching.

Conclusions

Supplementation of 0.025% CLA in the diet of breeders did not affect the incubation parameters and the weight of the chicks at hatching. CLA supplementation altered profiles of omega-6, palmitoleic, linoleic, and arachidonic acids in both the yolk and the residual yolk sac. These findings could aid in developing a novel pre-starter feeding program aimed at improving chick immunity and influencing its lipid metabolism, which could, in turn, decrease the accumulation of abdominal fat in broilers at slaughter.

Aknowledgements

To BASF and Asa Alimentos, for material support of this research. We also acknowledge the Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq) and the Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES) for providing fellowships.

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• Editor de seção:
  Renato Paiva

Publication Dates

• Publication in this collection
  11 Oct 2024
• Date of issue
  2024

History

• Received
  09 May 2024
• Accepted
  16 Sept 2024