Effects of dietary conjugated linoleic acid in broiler breeders and egg storage time on the fatty acid profile, lipid oxidation and internal egg quality
Priscila S. Silvério
A * , Cristiane B. de Lima A , Frederico L. da Silva A , Márcio A. Mendonça A , Candice B. G. S. Tanure A , José Henrique Stringhini B and Aline M. C. Racanicci A
A Faculty of Agronomy and Veterinary Medicine, University of Brasilia (UnB), Campus Darcy Ribeiro, Brasília, DF, Brazil.
B Department of Animal Science, Federal University of Goiás (UFG), Goiânia, GO, Brazil.
Animal Production Science 63(12) 1208-1214 https://doi.org/10.1071/AN22241
Submitted: 7 July 2022 Accepted: 2 May 2023 Published: 2 June 2023
© 2023 The Author(s) (or their employer(s)). Published by CSIRO Publishing. This is an open access article distributed under the Creative Commons Attribution 4.0 International License (CC BY)
Abstract
Context: The need for the storage of fertile eggs is a reality in the poultry industry. At the same time, prolonged storage periods decrease the quality of egg components that are essential for embryo development, and can compromise hatchability and chick quality; thus, the high content of unsaturated fatty acids in embryo tissues increase the susceptibility to peroxidation.
Aims: The objective of this study was to evaluate the addition of cis-9, trans-11, trans-10 and cis-12 isomers of conjugated linoleic acid (CLA) to the broiler-breeder diet and the storage time on the internal egg quality, composition and lipid oxidation.
Methods: In total, 22 000 Cobb female broiler breeders of 58 weeks of age were fed with diets containing 0 or 0.024% CLA and fertile eggs were stored 3, 6 or 9 days prior to incubation. In total, 6912 hatching eggs were used in a completely randomised experimental design in a 2 × 3 factorial arrangement (CLA inclusion × egg storage time). At the end of each storage period, 30 eggs per dietary treatment were sampled to analyse yolk and albumen height, percentage and pH, yolk:albumen ratio, yolk diameter and index, Haugh unit (HU), yolk lipid oxidation, acidity and fatty acid profile.
Key results: The progression of storage negatively affected the internal quality of the eggs; however, inclusion of CLA minimised these effects up to Day 6, especially for yolk diameter, HU, height and albumen pH. The total lipid content was not affected by the dietary treatments; however, CLA inclusion resulted in a higher proportion of stearic acid and a lower concentration of linoleic acid in yolks.
Conclusions: The changes observed in fatty acid profile of the eggs may have favoured the reduction of lipid oxidation, as shown by the decrease in the acidity index and thiobarbituric acid-reactive substance (TBARS) values at shorter storage periods.
Implications: The dietary addition of CLA to broiler breeders may be used to preserve the egg internal quality during a short-term storage period.
Keywords: conjugated linoleic acid, egg storage, Haugh unit, lipid profile, poultry nutrition, poultry production, TBARS, yolk acidity index.
Introduction
Conjugated linoleic acid (CLA) is a group of positional geometric isomers of linoleic acid with cis-9, trans-11, trans-10 and cis-12, considered as the most biologically relevant (Aydin and Cook 2009), found mainly in foods derived from ruminants (EFSA Panel on Additives and Products or Substances used in Animal Feed (FEEDAP) 2016), or can be chemically synthesised through the isomerisation of linoleic acid (Banni 2002).
Mainly known for its antioxidant activity (Stangle 2000; Du et al. 2001; Joo et al. 2002; Ko et al. 2004), and anticancer (Basu et al. 2000; Kilian et al. 2002) and body fat-reducing (Leung and Liu 2000; Park and Pariza 2007) effects, CLA can be an alternative for poultry industry to increase broiler productivity and the quality of day-old chicks.
It is important to consider that the addition of CLA to the breeder’s diet may modify the size of the eggs, yolk and albumen (Suksombat et al. 2006), change the fatty acid profile of the yolks (Suksombat et al. 2006; Aydin and Cook 2009) and thereby affect hatchability, causing an increase in embryo mortality (Muma et al. 2006).
Likewise, the storage of fertile eggs in the poultry industry is a reality. However, prolonged storage periods decrease the quality of egg components that are essential to embryo development (Barbosa et al. 2008) and can compromise hatchability (Macari et al. 2013) and chick quality (Reijrink et al. 2010). Thus, the high content of unsaturated fatty acids in embryo tissues increase the susceptibility to peroxidation (Ko et al. 2004); therefore, it is important to search for antioxidant alternatives for long-term-stored eggs. It is well known that long storage periods can lead to lower hatchability (Fasenko 2007), slower embryo growth and increased mortality (Fasenko et al. 2001; Fasenko 2007).
The aim of this study was to evaluate the effects of dietary addition of CLA to broiler breeders associated with storage time on egg quality, composition and lipid oxidation.
Materials and methods
All experimental procedures were approved by the Ethics Committee at Brasília University (CEUA-UnB number 62528/2015), and followed Ethical Principles for the Use of Experimental Animals of the Brazilian Society of Science in Laboratory Animals (SBCAL/COBEA).
In total, 22 000 Cobb broiler breeders in the laying phase (58 weeks of age) were distributed equally into two dietary treatments (with CLA and without CLA) in two different sheds. Breeders were fed ad libitum with a diet (Table 1) formulated according to Rostagno et al. (2011) and supplemented (or not) with 0.024% CLA, which corresponded to the inclusion of 0.042% of the commercial product Lutalin during 29 days. The commercial product used was composed of 56% of methyl esters of CLA (C18:2), 28% of the cis-9, trans-11 isomers and 28% of the trans-10, cis-12 isomer, according to information from the manufacturer (BASF).
| Item | With CLA (%) | No CLA (%) |
|---|---|---|
| Ingredients | ||
| Corn, 7.88% CP | 68.179 | 68.128 |
| Soybean meal, 45.5% CP | 17.416 | 17.425 |
| Limestone, 38% Ca | 7.876 | 7.876 |
| Kaolin | 4.000 | 4.000 |
| Meat and bone meal, 43% CP | 1.500 | 1.500 |
| Salt | 0.223 | 0.223 |
| Dicalcium phosphate | 0.200 | 0.200 |
| Sodium bicarbonate | 0.150 | 0.150 |
| Mineral premixA | 0.150 | 0.150 |
| Vitamin premixB | 0.100 | 0.100 |
| Choline 60% | 0.082 | 0.082 |
| DL-Methionine 88% | 0.080 | 0.080 |
| L-Threonine 98% | 0.030 | 0.030 |
| AntioxidantsC | 0.016 | 0.016 |
| CLAD | – | 0.042 |
| PhytaseE | 0.003 | 0.003 |
| Nutrient composition | ||
| Metabolisable energy (kcal/kg) | 2736 | 2734 |
| Crude protein (%) | 13.90 | 13.90 |
| Digestible lysine (%) | 0.60 | 0.60 |
| Digestible methionine + cysteine (%) | 0.47 | 0.46 |
| Digestible threonine (%) | 0.50 | 0.50 |
| Calcium (%) | 3.29 | 3.29 |
| Available phosphorus (%) | 0.20 | 0.20 |
| Na (%) | 0.34 | 0.34 |
AMineral premix: manganese 80 000 mg, zinc 70 000 mg, iron 40 000 mg, copper 8000 mg, iodine 1000 mg.
BVitamin premix: vitamin A, 14 000 IU; vitamin D3, 3000 IU; vitamin E, 110 000 mg; vitamin K3, 6000 mg; vitamin B1, 3000 mg; vitamin B2, 12 000 mg; vitamin B6, 6000 mg; vitamin B12, 30 mg, pantothenic acid, 20 000 mg; nicotinic acid, 60 000 mg; folic acid, 4000 mg; biotin 300 mg.
CEthoxyquin 66.6%, BHA 99% and citric acid 99.5%.
DCLA (Lutalin).
EPhytase (Natuphos10 000 FTU).
Freshly collected eggs (3546 eggs) from each dietary treatment (with or without CLA), selected and classified by weight between 63 and 72 g (total of 7092 eggs for the entire experiment), were divided into three groups to be stored at the egg room (average temperature and relative humidity were 19–21°C and 76% respectively) during 3, 6 or 9 days. Each group contained 1182 eggs and included randomly distributed trays, using a 2 × 3 factorial design (with or without CLA in the broiler breeder diet × 3 storage periods, namely, 3, 6 and 9 days).
Internal egg quality
At the end of each storage period, a sample of 30 eggs from each treatment (each egg was considered a repetition) was collected for quality measurements in the laboratory. The eggs were weighed individually with a scale (GEHAKA, model BK3000, São Paulo/SP, Brazil) and opened on glass boards to assess the height of the albumen and the yolk by using a tripod micrometer, as follows: albumen and yolk height (AH, YH), yolk diameter (YD), yolk and albumen pH (YpH, ApH), yolk weight (YW) for the yolk index (YI) and Haugh unit (HU), considering the following: HU = 100 log(h – 1.7 w0.37 + 7.6), where h = height of the albumen (mm) and w = weight of the egg (g) (Brant and Shrader 1958). The yolks were manually separated and weighed individually. Albumen weight (AW) was calculated as follows: egg weight – (yolk weight + shell weight). For the yolk index (YI), the formula used was: YI = h/d, where h = yolk height (mm) and d = yolk diameter (mm). The percentages of yolk (Y%) and albumen (A%) were calculated from their respective weights, divided by the egg weight, and multiplied by 100. The yolk:albumen ratio (Y:A%) was calculated according to the formula Y:A% = (yolk weight/albumen weight) × 100.
The albumen and yolk pH were evaluated in triplicate in a pool of five eggs by using a portable digital pH meter (Testo, model T 205, Lenzkirch, Germany). Then, albumen samples were frozen and kept in a domestic freezer (−14°C), the yolk samples were frozen for 24 h and freeze-dried (LIOTOP, Model L101, São Carlos/SP, Brazil) to minimise lipid oxidation damage. After freeze-drying, the samples were ground in a knife mill equipped with a cooling system (TECNAL, TE 631, Piracicaba/SP, Brazil) and stored until analysis.
Lipid oxidation in yolk
The assessment of lipid oxidation used the acidity index (AI) and the quantification of secondary lipid oxidation compounds by using TBARS. The acidity index (Ca 5a-40 method; Association of Official Analytical Chemists (AOAC) 1990) was performed in duplicate in a pool of five eggs; lipids extracted from yolks according to Folch et al. (1957) adapted by Bligh and Dyer (1959) and expressed in mg of NaOH/g of lipids. TBARS were evaluated in duplicate by using the method described by Sørensen and Jørgensen (1996), expressed in μmol of malonaldehydes (MDA)/kg of yolk.
Lipids and fatty acid profile in yolk
The total lipids (TL, % of raw matter) were evaluated using the AOAC methodology (Association of Official Analytical Chemists (AOAC) 1990), with six replicates per treatment, in a pool of five eggs in ground, freeze-dried yolk samples.
The fatty acid profile was determined in the same lipid samples extracted as mentioned for acidity index determination, followed by methylation (Christie 1989). The quantification of esterified fatty acids used a gas chromatograph (Shimadzu, CG-2014, Kyoto, Japan) with detector MS-QP2010 Plus and autoinjector AOC-5000. The separation of fatty acids was undertaken using a 60 m (length), 0.25 mm ID (internal diameter), 0.25 μm (film thickness) column from J & W Scientific (122-2362 DB-23). Helium was used as the carrier gas, with a continuous flow of 0.40 mL/min in the column. The injected volume was 1 μL (Split mode) and the detector temperature was 260°C. The heating conditions of the column were as follows: 140°C for 5 min, then increasing by 2°C every minute until 240°C, totalling 56 min of the chromatographic run. The identification of fatty acids was undertaken by comparing the peaks with the retention time of the standard fatty acid Supelco 37 component FAME mix (Supelco Analyticals™ from Merck Group, Darmstadt, Germany) and the results were expressed as a percentage of the area of each fatty acid, in relation to the total area.
Statistical analyses
The results were compared using a mixed model, with treatments being fixed effects and storage periods random effects, using PROC MIXED procedure from Statistical Analysis Systems (ver. 9.3; SAS Institute Inc 1989). The averages were compared by the Tukey test, with 5% significance.
Results and discussion
The yolk weight (YW) was not affected by the addition of CLA or by the storage time in the egg room (Table 2). Likewise, there was no effect of inclusion of CLA on yolk height (YH), which was negatively affected by storage time, as expected, and previously described (Oliveira and Oliveira 2013). Although storage time negatively affected the yolk index (YI), the addition of CLA preserved YI, as described by Shinn et al. (2015).
| Factor | YW | YD | YH | YI | Y% | Y:A% | YpH | AW | AH | A% | HU | ApH |
|---|---|---|---|---|---|---|---|---|---|---|---|---|
| With CLA | 22.04 | 45.66B | 18.81 | 0.41A | 32.34 | 54.75 | 5.74 | 40.42 | 5.23A | 59.28 | 65.68A | 9.03B |
| No CLA | 21.65 | 46.11A | 18.46 | 0.40B | 31.81 | 53.45 | 5.75 | 40.78 | 4.86B | 59.73 | 61.70B | 9.10A |
| 3 days | 21.71 | 44.80C | 19.90A | 0.44A | 31.53B | 52.62B | 5.72B | 41.50A | 5.61A | 60.15A | 69.25A | 8.87C |
| 6 days | 21.96 | 46.08B | 18.86B | 0.41B | 31.99A | 53.64B | 5.75A | 41.12A | 5.49A | 59.85A | 68.53A | 9.12B |
| 9 days | 21.87 | 46.78A | 17.16C | 0.37C | 32.70A | 56.03A | 5.78A | 39.18B | 4.01B | 58.51B | 53.30B | 9.20A |
| P-value | ||||||||||||
| CLA | 0.0949 | 0.0134 | 0.0829 | 0.0151 | 0.0839 | 0.1075 | 0.4336 | 0.4267 | 0.0185 | 0.1675 | 0.0227 | 0.0051 |
| Storage | 0.6662 | <0.0001 | <0.0001 | <0.0001 | 0.0080 | 0.0021 | 0.0001 | <0.0001 | <0.0001 | 0.0001 | <0.0001 | <00001 |
| CLA × storage time | 0.0968 | 0.0382 | 0.5045 | 0.9789 | 0.3869 | 0.2862 | 0.9022 | 0.4210 | 0.4349 | 0.2351 | 0.4569 | 0.0087 |
| CV (%) | 7.18 | 4.15 | 9.44 | 11.07 | 6.57 | 10.23 | 0.91 | 7.73 | 25.37 | 3.85 | 21.66 | 2.05 |
| Interactions | |||||
|---|---|---|---|---|---|
| Yolk diameter (YD) | |||||
| Dietary treatment | Storage time (days) | P-value | CV (%) | ||
| 3 | 6 | 9 | |||
| With CLA | 44.48c | 45.63bB | 46.87a | <0.0001 | 0.13 |
| No CLA | 45.12b | 46.52aA | 46.68a | <0.0001 | 0.13 |
| Albumen pH (ApH) | |||||
|---|---|---|---|---|---|
| Dietary treatment | Storage time (days) | P-value | CV (%) | ||
| 3 | 6 | 9 | |||
| With CLA | 8.86c | 9.03bB | 9.19a | <0.0001 | 0.02 |
| No CLA | 8.88b | 9.21aA | 9.20a | <0.0001 | 0.02 |
Statistically significant differences by Tukey’s test (at P = 0.05) in the same column are indicated by different uppercase letters. Statistically significant differences by Tukey’s test (at P = 0.05) in the same row are indicated by different lowercase letters.
The addition of CLA did not affect the yolk percentage (Y%) and the yolk:albumen ratio (Y:A%); however, the increasing storage time raised these values, which can be explained by the decrease of the albumen weight (AW) in this period, since YW was not affected by treatments. This decrease of the albumen weight (AW) may have been caused by the difference in osmotic pressure that is responsible for the water movement from the albumen to the yolk during storage; thus, Y% increases and A% decreases, resulting in yolk enlargement and reduced viscosity (Macari et al. 2013).
The YpH was not affected by the dietary use of CLA. However, the storage caused an increase in YpH, as previously shown by Ganeco et al. (2012). However, Shinn et al. (2015) observed pH changes in eggs from breeders fed CLA only when stored up to 30 days, and it seems that higher changes in egg pH were associated with higher concentrations of CLA in the layer diets.
The storage time affected the yolk diameter (YD), as can be seen in Table 2, with YD increasing with the storage time, reaching the highest values at 9 days of storage (Table 2), since YD increases in liquefied yolk (Macari et al. 2013). In contrast, treatment with CLA preserved the YD until Day 6; however, this effect disappeared after 9 days of storage due to the antioxidant effect of dietary CLA described previously by Stangle (2000), Du et al. (2001), Joo et al. (2002) and Ko et al. (2004). The inclusion of CLA did not affect albumen weight (AW), which was reduced after 9 days of storage (Table 2) and can be explained by the increase in pH due to the loss of carbonic gas through the pores of the eggshell, causing liquefied albumen and decreased height (Karoui et al. 2006). Contrary to previous studies (Suksombat et al. 2006; Cherian et al. 2007), the dietary inclusion of CLA for breeders showed a positive effect on AH, preserving albumen quality, when compared with the control group. In turn, increasing storage time reduced AH, as expected. The albumen percentage (A%) in the eggs from breeders fed CLA was not affected, but decreased during storage, which may reflect the results found for AW and AH.
The average HU found in this study was lower than expected for fertile eggs (>80, according to Macari et al. 2013), which can be explained by the age of the breeders used in the experiment (between 58 and 61 weeks), since older breeders usually show lower albumen height and poor internal egg quality (Oliveira and Oliveira 2013). In contrast, the dietary CLA resulted in higher HU values than in the control (65.68 vs 61.70), a positive effect indicating that CLA was able to maintain the internal quality of the eggs up to 6 days. As expected, HU values were reduced as storage time increased, especially at Day 9.
Table 2 also shows that the albumen pH was negatively affected by the storage time in the egg room, since higher values were found at 9 days of storage. It is well known that ApH naturally increases after oviposition, as a result of carbon dioxide loss through the eggshell and plays an important role in the embryo development; lower ApH may negatively affect hatchability (Decuypere et al. 2001). Also, the addition of CLA to the breeder diet resulted in lower ApH values and preserved internal quality of eggs stored for 6 days.
Yolk lipid content and oxidation
The dietary supplementation of breeder diet with CLA showed no effect on the content of total lipids (TL) in yolks; however, it was significantly reduced as the storage time increased (Table 3). This is not unusual in stored eggs and can be explained by the increase in osmotic pressure gradient between albumen and yolk, resulting in movement of water from the albumen to the yolk and dilution of the yolk components, as a consequence of the liquefaction process (Macari et al. 2013). In addition, this process can ultimately cause damage to embryo viability, as described previously (Deeming 2002), since lipids from the yolk are their main source of energy during development.
| Factor | TL | TBARS | AI | |||
|---|---|---|---|---|---|---|
| With CLA | 30.71 | 0.41B | 1.90 | |||
| No CLA | 30.61 | 0.51A | 1.90 | |||
| 3 days | 33.17A | 0.42B | 1.84 | |||
| 6 days | 29.97B | 0.76A | 1.98 | |||
| 9 days | 28.84C | 0.19C | 1.83 | |||
| P-value | ||||||
| CLA | 0.7937 | 0.0082 | 0.9950 | |||
| Storage | <0.0001 | <0.0001 | 0.1276 | |||
| CLA × Storage | 0.5527 | 0.0129 | 0.0053 | |||
| CV | 6.51 | 58.71 | 15.96 | |||
| Interactions | ||||||
|---|---|---|---|---|---|---|
| TBARS | ||||||
| Dietary treatment | Storage time (days) | P-value | CV (%) | |||
| 3 | 6 | 9 | ||||
| With CLA | 0.29bB | 0.76a | 0.17b | <0.0001 | 0.02 | |
| No CLA | 0.55bA | 0.76a | 0.21c | <0.0001 | 0.02 | |
| Acidity index (AI) | ||||||
|---|---|---|---|---|---|---|
| Dietary treatment | Storage time (days) | P-value | CV (%) | |||
| 3 | 6 | 9 | ||||
| With CLA | 1.90 | 1.83B | 1.93 | <0.0001 | 0.05 | |
| No CLA | 1.79b | 2.14aA | 1.73b | <0.0001 | 0.05 | |
Statistically significant differences by Tukey’s test (at P = 0.05) in the same column are indicated by different uppercase letters; statistically significant differences by Tukey’s test (at P = 0.05) in the same row are indicated by different lowercase letters.
Although the storage time in the egg room increased the lipid oxidation in the yolk up to Day 6 (Table 3), TBARS values decreased on Day 9 of storage. This may suggest that secondary compounds of lipid oxidation (malonaldehydes, MDA) reacted with other components of the yolk, such as amino acids, sugar and proteins, or even were transformed to MDA dimers or trimers, diminishing reactivity with thiobarbituric acid (Barriuso et al. 2013). This may also be associated with the reduced content of total lipids found in 9 days stored eggs.
At the same time, the dietary use of CLA in breeder diets seems to protect the yolk lipids from lipid oxidation, since TBARS values were lower than in the control (0.41 vs 0.51), which suggests an antioxidant effect of CLA, as previously described by Hayat et al. (2010). The CLA supplementation has been associated with an increase in superoxide dismutase and glutathione peroxide activity, neutralisation of free radicals and reduced damage to tissues and membranes, as well as formation of secondary products of lipid oxidation, such as malonaldehydes (Kim et al. 2005; Jiang et al. 2014). In our study, this antioxidant effect was clearly seen at the early stages of storage, particularly on Day 3 (Table 3), but faded as the storage time at the egg room increased.
The dietary CLA reduced acidity index (AI) in eggs after 6 days of storage, when compared with the control (not supplemented; Table 3); however, no differences were detected on Days 3 and 9 of storage and these results were not related to TBARS. Since AI values in eggs from CLA treatment remained unchanged throughout the storage, in contrast to control, it could indicate better preservation of lipids from rancidity.
Yolk fatty acid profile
The percentages of palmitic, stearic, oleic and linoleic acids were evaluated. In this study, the concentration of palmitic acid (C16:0) in the yolks was not affected by dietary CLA or storage time in the egg room (Table 4), as described previously by Keum et al. (2018). However, dietary CLA increased the percentage of stearic acid (C18:0), as reported by other authors (Shinn et al. 2015; Liu et al. 2017; Keum et al. 2018). Some authors have associated this increase in the fatty acid saturation of the egg yolks with the inhibition of the stearoyl-CoA desaturase enzyme (Δ9-desaturase) in the liver (Park et al. 2000); this enzyme is known for the desaturation of Δ9-cis into several unsaturated fatty acids (Cohen et al. 2002).
| Factor | C16:0 | C18:0 | C18:1 | C18:2 |
|---|---|---|---|---|
| With CLA | 28.34 | 9.40A | 43.41 | 11.72B |
| No CLA | 27.84 | 8.37B | 42.63 | 13.72A |
| 3 days | 28.18 | 9.06 | 43.41A | 12.66 |
| 6 days | 28.18 | 8.95 | 42.34B | 12.43 |
| 9 days | 27.92 | 8.64 | 43.31A | 13.07 |
| P-value | ||||
| CLA | 0.3174 | 0.0019 | 0.0510 | 0.0004 |
| Storage | 0.8998 | 0.3946 | 0.0506 | 0.4975 |
| CLA × Storage | 0.8940 | 0.2547 | 0.2170 | 0.1667 |
| CV | 2.69 | 7.88 | 2.14 | 9.92 |
Statistically significant differences by Tukey’s test (at P = 0.05) in the same column are indicated by different letters.
In contrast to Shinn et al. (2015), the dietary CLA did not affect oleic acid (C18:1) concentration in yolk. However, the concentration of oleic acid was reduced after 6 days of storage at the egg room, as reported by Cherian et al. (2007).
The proportion of linoleic acid (C18:2) in yolks was reduced by the dietary addition of CLA, and no effect of the storage time was observed. The results were similar to those found by Suksombat et al. (2006) and Shinn et al. (2015), that showed a significant decrease on the polyunsaturated fatty acids in the yolks from CLA dietary treatment.
Although the concentration of the supplemented CLA in the breeder diet was low in this study, it modified the fatty acid composition of the yolks (Table 4), such as the increased of saturated fatty acids (C18:0) and decreased polyunsaturated fatty acids (C18:2), as reported previously by Liu et al. (2017) and Keum et al. (2018). This may be related to the lower progression of lipid oxidation in yolks found in TBARS and AI analysis (Table 3), and may also reduce embryo survival (Muma et al. 2006; Aydin and Cook 2009).
Conclusions
The dietary supplementation of the broiler breeder diet with CLA minimised the negative effects in the internal quality of the yolk and albumen and preserved the lipids from oxidation at the early stages of storage; however, it modified the fatty acid composition of the yolks.
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