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Currently, nutritional programs for new hybrids of laying hens are based on maximizing genetic expression, mainly to maintain a profitable egg production up to 100 weeks of age (Martínez et al. 2021a). With this objective, progress has been made in obtaining various natural and synthetic additives, which promote or modify, and both, the immune response, antioxidant capacity, absorption of nutrients and productive response of hens, as in the case of probiotics, prebiotics, phytobiotics, organic acids, bioactive peptides and L-carnitine (Adedokun and Olojede 2019). According to Ali et al. (2021), additives have been commonly used in the nutrition of apparently healthy laying hens and in the face of different challenges to contribute to the normal development of physiological functions and to compensate for their deficiencies.
Human and animal nutrition have recommended the oral use of L-carnitine, which is a water-soluble, bipolar ionic compound, synthesized in vivo from lysine and methionine (McCann et al. 2021). This compound (L-carnitine) increases the transport of long-chain fatty acids through the inner membrane of mitochondria for β oxidation and to eliminate toxic accumulations of fatty acids in the mitochondria (García-Flores et al. 2021), which causes increased cellular energy production (ATP). Although L-carnitine is endogenously biosynthesized in certain tissues such as the liver, kidneys and brain (Koeth et al. 2019), its absorption and use are exacerbated under conditions of maximum production and stress, regulated by nuclear receptors sensitive to specific nutrients (Ringseis et al. 2012).
The use of L-carnitine has been extensively studied in poultry (Adabi et al. 2011). Ringseis et al. (2018) mentioned that L-carnitine has a non-antibiotic growth promoting effect, stimulates immunity, reduces oxidative stress due to its antioxidant effect and improves semen quality (Xu et al. 2003 and Khan 2011). Specifically, in laying hens, Celik et al. (2004) and Kita et al. (2005) reported that L-carnitine improves internal egg quality and production, with better emphasis on albumen. However, other authors found no positive response in laying hens, when using up to 500 mg/kg of L-carnitine in different feeding schemes (Corduk and Sarica 2008 and Daskiran et al. 2009). Few studies have been carried out to elucidate the influence of L-carnitine on the productive response of new hybrids of laying hens with other nutritional requirements and with higher persistence in laying intensity (Hy-Line Brown 2020).
The objective of this study was to evaluate the effect of the inclusion of L-carnitine on egg quality and productivity of 85-week-old Hy-Line Brown® laying hens.
Materials and Methods
Experimental location. The study was developed at the Poultry Research and Teaching Center of the Escuela Agrícola Panamericana Zamorano, located in Valle del Yegüare, San Antonio de Oriente municipality, Francisco Morazán department, km 32 from the Tegucigalpa to Danlí highway. This location has a height of 800 m.a.s.l., mean annual temperature is 27 ºC and mean annual rainfall is 1.100 mm.
Animals, experimental design and treatments. A total of 200 Hy-Line Brown® laying hens, 85 weeks old, were randomly distributed into two treatments, with 20 repetitions each, and five animals per repetition during 10 experimental weeks. Dietary treatments consisted of a control diet and the inclusion of 24 mg/kg of L-carnitine, according to the manufacturer's recommendations (AVIGAN, Honduras). Table 1 shows the ingredients and nutritional contributions of the experimental diets.
Table 1 Ingredients and nutritional contributions for Hy-Line Brown® laying hens
| Treatments | ||
|---|---|---|
| Ingredients, % | Control | L-carnitine |
| Corn meal | 62.70 | 62.676 |
| Soybean meal | 24.63 | 24.63 |
| Choline chloride | 0.05 | 0.05 |
| African palm raw oil | 0.30 | 0.30 |
| Mineral and vitamin premix¹ | 0.20 | 0.20 |
| Salt | 0.35 | 0.35 |
| Biofos® | 1.67 | 1.67 |
| Thin calcium carbonate | 3.34 | 3.34 |
| Coarse calcium carbonate | 6.21 | 6.21 |
| Mycotoxin sequestrant | 0.12 | 0.12 |
| DL-Methionine | 0.25 | 0.25 |
| L-Lysine | 0.10 | 0.10 |
| L-Threonine | 0.08 | 0.08 |
| L-carnitine | 0.00 | 0.024 |
| Metabolizable energy, MJ/kg | 11.30 | 11.30 |
| Crude protein, % | 15.52 | 15.52 |
| Ca, % | 4.24 | 4.24 |
| Available P, % | 0.34 | 0.34 |
| Lysine, % | 0.72 | 0.72 |
| Methionine + cystine, % | 0.65 | 0.65 |
| Threonine, % | 0.51 | 0.51 |
1 Mineral and vitamin premix: vit. A, 1,000 IU/kg; vit. D3, 2,000 IU/kg; vit. E, 30 IU/kg; vit. K3, 2.0 mg/kg; vit. B1, 1.0 mg/kg; vit. B2, 6.0 mg/kg; vit. B6, 3.5 mg/kg; vit. B12, 18 mg/kg; niacin, 60 mg/kg; pantothenic acid, 10 mg/kg; biotin, 10 mg/kg; folic acid, 0.75 mg/kg; choline, 250 mg/kg; iron, 50 mg/kg; copper, 10 mg/kg; zinc, 70 mg/kg; manganese, 70 mg/kg; selenium, 0.30 mg/kg; iodine, 1.0 mg/kg
Experimental conditions. The laying hens were housed in a 400 m2 commercial shed, in 61×36 cm cages, with ceiling fans and an artificial lighting system. Water was offered ad libitum in two nipple drinkers per cage and feed intake was restricted to 115 g/d/hen in linear feeders. Sixteen hours of light were provided each day, and no therapeutic veterinary care was used during the experimental stage. To achieve adequate adaptation to the new diets, a 7-day pre-experimental feeding phase was used, recommended by Abd El-Hack et al. (2015).
Productive performance. To determine egg weight, 50 eggs were collected weekly per each treatment, between 8:30 a.m. and 9:30 a.m. Eggs were weighed on an OHAUS® digital technical balance (New Jersey, USA), with an accuracy of ± 0.1 g. Feed intake was determined three times a week, according to the offer and reject method (Martínez et al. 2021a). For laying intensity, total egg production/week/treatment was considered. One egg/d/hen was assumed, nested as 100 %. Mass conversion was calculated from the formula:
Egg external and internal quality. At week 90 and 95, 30 eggs were collected per each experimental treatment. All were collected at the same time and transferred to the egg quality laboratory of the Poultry Research and Teaching Center of Zamorano Pan-American Agricultural School. Egg quality was analyzed on the same day of collection using an automatic TSS EggQuality analyzer (York, England) and Eggware v4x program. Resistance to rupture of the eggshell (middle pole) was measured with a QC-SPA® resistance analyzer (York, England).
For analyzing egg shell thickness (EST) (middle pole), a QC-SPA® micrometer screw (York, England) was used, with a precision of ± 0.001 mm. For internal egg quality and albumen height (AH), a QHC® height analyzer (York, England) was used with a precision of ± 0.01 mm. Haugh unit was calculated with the following formula:
where:
HU is Haugh unit
AH is albumen height
EW is egg weight
Yolk color (YC) was evaluated using a CCC® electronic colorimeter (York, England), which considers the Roche scale of 15 colors.
Statistical analysis. Data was analyzed by T Students test for two independent samples. Yolk color was determined by the non-parametric U-Mann Whitney test. Values of P <0.05 were taken to indicate significant differences. The SPSS 23.0.1.2014 program (SPSS Inc., Chicago, IL, USA) was used for statistical analyzes. In addition, dirty eggs were determined by comparison of proportions, using the COMPRAPRO 1.0® program (Font et al. 2007).
Results and Discussion
Table 2 shows the effect of L-carnitine on the main productive indicators of laying hens from 85 to 95 weeks of age. The inclusion of L-carnitine did not change significantly (P> 0.05) laying intensity, feed intake, mass conversion and percentage of dirty eggs. However, this experimental treatment (L-carnitine) increased egg weight in relation to control diet (P <0.05).
Table 2 Effect of the inclusion of L-carnitine on productive indicators of laying hens (85-95 weeks old)
| Experimental treatments | ||||
|---|---|---|---|---|
| Indicators | Control | L-carnitine | SE ± | P value |
| Laying intensity, % | 80.18 | 81.94 | 2.76 | 0.532 |
| Feed intake, g/hen | 102.72 | 103.43 | 0.411 | 0.100 |
| Egg weight, g | 63.35 | 65.68 | 0.653 | 0.002 |
| Mass conversion, kg/kg | 2.03 | 1.93 | 0.077 | 0.219 |
| Dirty eggs, % | 1.24 | 1.18 | 0.060 | 0.091 |
The inclusion of L-carnitine promoted egg weight by 3.68 % in relation to control treatment (table 2). Yalçin et al. (2005) found that the inclusion of L-carnitine in the diet of laying quail increased egg weight, without changes for its production. Furthermore, Suchý et al. (2008) reported that L-carnitine improved egg weight and production of the common pheasant (Phasianus colchicus). However, studies by Yalçin et al. (2006), Corduk et al. (2008) and Rezaei et al. (2008) do not refer noticeable changes in egg weight, when they used L-carnitine in diets of laying poultry.
It is known that L-carnitine increases β-oxidation of fatty acids and adenosine triphosphate (ATP) production and, in turn, the cellular energy yield (McCann et al. 2021). Apparently, this mitochondrial biochemical effect caused an increase of egg weight (Rizk et al. 2019), especially at this productive age (85-95 weeks), in which the animal decreases productivity and increases egg weight, due to the decrease in sex hormones and the correlation among bird weight, oviduct weight and egg weight, respectively (Hy-Line Brown 2020). Also, energy efficiency has a direct relationship with egg weight (Ewonetu and Kasaye 2018). In this sense, Barzegar et al. (2020) informed that a more efficient energy utilization improved egg weight in Hy-Line Brown laying hens.
The dietary use of L-carnitine did not change egg production (table 2). Similar results were found by Corduk and Sarica (2008), when they used up to 500 mg/kg of L-carnitine in diets with different sources of fatty acids and metabolizable energy in laying hens. However, Kazemi-Fard et al. (2015) reported that the dietary use of 100 and 150 mg/kg of L-carnitine increased egg production by 7.81 and 10.74 % in laying hens, respectively, because this additive (L-carnitine) improves lipolysis and liver protection. Likewise, Neuman et al. (2002) reported that L-carnitine supplementation increased mitochondrial energy production and, in turn, laying intensity of this birds. Apparently, the use of 24 mg/kg of L-carnitine (recommended by the manufacturer) and the experimental age (85-95 weeks) of laying hens influenced on the fact that no changes were found in egg production (table 2).
The optimal experimental conditions could also have a direct influence on these results (egg production) (table 2), since the use of L-carnitine in the diet or in drinking water has been recommended under stress conditions (Liu et al. 2021). According to Çetin and Güçlü (2020), supplementation with 200 mg/kg of L-carnitine in hens with high densities increased the activity of endogenous antioxidant enzymes and decreased serum level of malondialdehyde and nitrous oxide. Likewise, Güçlü et al. (2011) reported that L-carnitine reduced lipid peroxidation in laying hens fed high Cu concentrations, although without improvements for productivity. Apparently, this experiment allowed animals to express their maximum productive potential, considering that egg production (75 vs. 81.94 %) for these experimental weeks was higher than that indicated in the Hy-Line Brown manual (2020).
Feed intake also remained unchanged among treatments (table 2). There is no scientific evidence that this additive (L-carnitine) promotes feed intake. Yalçin et al. (2005), Yalçın et al. (2006) and Rezaei et al. (2008) found no changes in feed intake, when evaluating L-carnitine in laying hens and quail. Likewise, Corduk and Sarica (2008), when studying the dietary effect of 500 mg/kg of L-carnitine on the productivity of laying hens fed saturated and unsaturated diets, found that feed intake and mass conversion had no significant changes among treatments. Although egg weight increased in this experiment (table 2), mass conversion was not different among treatments (table 2). However, Kazemi et al. (2015) reported that higher productivity improved egg mass.
At week 90, the inclusion of L-carnitine did not modify (P> 0.05) albumen height, Haugh unit, eggshell strength and yolk color. However, L-carnitine increased (P <0.05) eggshell thickness with respect to control treatment. At week 95, the dietary use of L-carnitine did not modify any indicator of egg external and internal quality (table 3).
Table 3 Effect of L-carnitine inclusion on egg external and internal quality of laying hens
| Experimental treatments | ||||
|---|---|---|---|---|
| Egg quality | Control | L-carnitine | SE ± | P Value |
| Albumen height, mm | 11.41 | 11.42 | 0.346 | 0.992 |
| Haugh unit | 103.82 | 104.29 | 1.550 | 0.761 |
| Eggshell strength, kgF/cm2 | 3548.63 | 3546.73 | 17.690 | 0.617 |
| Eggshell thickness, mm | 0.29 | 0.32 | 0.008 | 0.010 |
| Yolk color | 3.00 (0.354) | 3.00 (0.712) | 0.482 | |
| Albumen height, mm | 10.78 | 11.06 | 0.345 | 0.410 |
| Haugh unit | 101.96 | 102.39 | 1.477 | 0.769 |
| Eggshell strength, kgF/cm2 | 3906.63 | 4010.03 | 19.661 | 0.597 |
| Eggshell thickness, mm | 0.39 | 0.38 | 0.018 | 0.056 |
| Yolk color | 3.00 (0.615) | 3.00 (0.581) | 0.417 | |
Apparently, the use of 24 mg/kg of L-carnitine in the diets of the animals under study (85-95 weeks) did not influence on protein synthesis in albumen and, therefore, on the height of this edible structure (albumen) and Haugh unit (table 3), since these indicators (albumen height and Haugh unit) are highly correlated and are considered for evaluating egg freshness and its storage time on the shelf (Martínez et al. 2021b).
Similar results were found by Yalçın et al. (2006), when they used 100 mg/kg of L-carnitine in diets for laying hens, 22-40 weeks of age. Likewise, Daşkıran et al. (2009) found no notable changes in egg internal quality, when using 150 mg/kg of L-carnitine in laying hens between 62 and 72 weeks of age. However, Rabie et al. (1997) and Kita et al. (2005) reported that supplementation with 50 and 500 mg/kg of L-carnitine in laying hens improved albumen quality and Haugh unit, due to the increase of the metabolic rate for egg formation in the magnum and the stimulation of ovomucin secretion, respectively. Also, Ghods-Alavi et al. (2017) demonstrated that the inclusion of 100 mg/kg of L-carnitine, in laying hens, increased albumen height and Haugh unit, probably due to a better albumen/yolk ratio and a decrease of harmful lipids in egg yolk (mainly cholesterol). According to Rabie et al. (1997), results of the use of L-carnitine in laying hens are influenced by the concentration of the additive, chemical composition of feed ingredients, age, health status and experimental conditions.
It was interesting that the inclusion of L-carnitine increased eggshell thickness of laying hens at 90 weeks of age (table 3). Corduk and Sarica (2008) reported improvements in shell thickness, when they used 500 mg/kg of L-carnitine. According to Förster et al. (2021), L-carnitine can modulate cell antioxidant activity against different pro-oxidant factors.
Abad et al. (2021) reported that antioxidant products reduce oxidative stress in the gastrointestinal tract (GIT), which translates into a better productive response and egg quality. Thus, this product (L-carnitine) could improve calcium absorption in the intestinal lumen and incorporate a greater quantity of this mineral (calcite) in the uterus for the eggshell formation. According to Barret et al. (2019) the deterioration of shell quality is an indicator related to the stress of laying hens. However, further research is necessary to confirm this hypothesis, because no notable changes were found among treatments at week 95 (table 3).
Despite the results in shell thickness, L-carnitine did not influence on eggshell strength in both experimental weeks (table 3). Martínez et al. (2021b) did not find a positive correlation between eggshell thickness and strength in white eggs. Yalçın et al. (2006), Parizadian et al. (2011) and Ghods-Alavi et al. (2017) reported similar results, when they evaluated the use of L-carnitine in laying birds. It should be noted that the data found for eggshell strength at week 95 (4,010.53 kgF/cm2) is higher than those stipulated by the genetic line (3,940 kgF/cm2; Hy-Line Brown 2020), which shows that shell quality was not affected by the dietary use of L-carnitine.
Also, a higher yolk pigmentation has been related to the reduction of oxidative stress in the GIT and with higher absorption of carotenoid pigments (Galamatis et al. 2021). The use of 24 mg/kg of L-carnitine did not modify yolk pigmentation under these experimental conditions (table 3). In both experimental groups, a similar content of yellow corn, rich in zeaxanthin, was recorded (table 1) as the main pigment of the diet (Calvo-Brenes and O'Hare 2020). These results coincide with those of Celik et al. (2004) and Daşkıran et al. (2009), who concluded that L-carnitine did not change yolk quality (weight and color pigmentation) in laying hens.
The inclusion of 24 mg/kg of L-carnitine in diets for Hy-Line Brown laying hens (85-95 weeks of age) increases egg weight and shell thickness at week 90, without significant changes for other productive and quality indicators of the egg at week 95 of age.
