The effects of supplementation with AURA on the concentration of selected fatty acids (i.e., those fatty acids that differed significantly between treatments or were of interest overall) in chicken breast, thigh, kidney, liver or skin with adhering fat are shown in Table 5. The full fatty acid profile determined for each tissue/organ is provided in Supplementary Table S1. The highest DHA concentrations were detected in the liver followed by the skin with adhering fat, thigh and kidney, with the breast meat having the lowest DHA content of all the sampled tissues. In all cases, the DHA content of the tissues/organs of supplemented birds were significantly higher than the unsupplemented control group. For breast and thigh samples both 1% groups had significantly more DHA than all other treatments with similar levels of enrichment observed for the 1%WL and 1%FP groups. The groups supplemented with 0.5% AURA were enriched to a similar degree with no difference observed between the 0.5%WL and 0.5%FP treatments. In the liver, both 1% treatments were again the most enriched with the duration of supplementation not effecting the level of enrichment. In the kidney, the 1%WL and both FP treatments were enriched to a similar degree, with the 0.5%WL group having significantly less DHA than the 1%WL group. In the skin the 1%WL treatment had significantly more DHA than each of the other treatments, with all remaining supplemented groups enriched to a similar degree. A quadratic relationship between DHA intake and tissue/organ DHA content was observed for the thigh, kidney and liver samples, while a linear relationship was observed for breast and skin samples (Figure 1). The efficiency of DHA transfer from the feed to the breast and thigh was estimated and indicated that feeding for the final 21 days resulted in a more efficient transfer of DHA from the feed to the tissues for both breast and thigh (Figure 2).

Means that do not share a superscript differ significantly. 1 Eicosapentaenoic acid; 2 Docosapentaenoic acid; 3 Docosahexaenoic acid; 4 Total Omega 3 = {C18:3n3+C20:3n3+C20:5n3+C22:5n3+C22:6n3}; 5 Total Omega 6 = {C18:2n6cis+C18:3n6+C20:2n6+C20:3n6+C20:4n6+C22:4n6}. a,b,c Means within a row that do not share a superscript differ significantly.

**Figure 1.**  The r2 value, significance of the relationship (*p*) and model equation are shown for each graph in panel **F**.

The concentration of DPA did not differ significantly between any of the treatment groups for any of the tissues/organs sampled. The highest levels of EPA enrichment were observed in the skin with adhering fat, followed by the kidney, liver, thigh and breast. For each tissue/organ sampled the highest EPA concentration was detected in the 1%WL group, with this group having more EPA than the control group in each case. The 1%FP group also had significantly more EPA than the control group in each case with the exception of the breast tissue samples. The 1%FP group was enriched to a similar degree as the 1%WL group in each tissue with the exception of the skin, in which the 1%WL group had significantly more EPA (Table 5). These differences contributed to significant differences between the groups in terms of their total n-3 PUFA concentration, with groups receiving the 1% AURA treatments generally enriched to a greater degree. For each tissue the C22:4n-6 concentration detected in the supplemented groups was generally significantly lower than the control, however, the same trend was not observed for the total n-6 concentrations, which did not differ between the treatment and control groups. The n-6/n-3 ratio for both 1% treatments were significantly lower than the control group in every tissue/organ sample. Both 0.5% AURA treatments also had significantly lower n-6/n-3 ratios in every tissue/organ samples with the exception of the skin.

**Figure 2.** Mean efficiency of DHA transfer (± 95% C.I.) from feed to the breast (**A**) or thigh (**B**) tissues. Transfer efficiency was estimated for one bird per pen (n = 7 per treatment) as follows; (mg DHA in feed × average daily feed intake x 42 days) ÷ (mg DHA/g breast or thigh × breast or thigh weight) × 100. a,b Columns that do not share a letter differ significantly (*p* < 0.001).

#### **4. Discussion**

In our previous studies, the addition of AURA was found to have no effect on any of the broiler productivity parameters measured [16,17]. In contrast, the 1%WL group in the current study was found to have a lower average weight than the control group by day 42, likely due to a significantly lower average daily feed intake between day 21 and 42. For the whole 42 day period, feed intake did not differ between the groups with no differences in feed conversion ratio, European Production Efficiency Factor, dressing % or breast weight observed. In our previous study, investigating supplementation at up to 5% of the diet, no differences in productivity were observed between the groups [16] which may suggest that the differences observed in the current study were not as a result of the inclusion of AURA. Some other n-3 PUFA rich ingredients have been shown to negatively impact broiler productivity, with the anti-nutritional properties of linseed limiting its inclusion in broiler diets [3]. In contrast, some authors have shown improvements in weight gain, feed intake and FCR when providing omega-3 rich *Schizochytrium* [10,27] or AURA [28]. The effects on the 1%WL group could be considered minor and considering there was no differences between the two groups supplemented for the fattening period only, supplementation with AURA is unlikely to reduce the productivity of broilers in practice.

It has previously been demonstrated that supplementing the diets of broilers with AURA is an effective method that can increase the concentration of DHA in broiler tissues [16,17]. When comparing the results of our previous studies, feeding 0.5% AURA for 42 days resulted in a similar level of tissue enrichment as feeding 1% AURA for 21 days [16,17]. Interestingly, the cost of supplementation (with an approximate AURA cost of €7 per kg) for 42 days at a level of 0.5% of the diet (€0.18 per bird) was lower than the cost of providing AURA at a level of 1% of the diet for 21 days (€0.26 per bird). These studies were carried out in the same research facility, over a two-year period using similar diets, however, differences in the level of enrichment observed between the studies could have been caused by factors other than the duration of feeding. As such, it was important to test the effect of AURA feeding duration on tissue DHA enrichment in a single experiment. Here we found that feeding a lower amount of AURA over a longer period did not lead to similar levels of enrichment as feeding higher levels over a shorter period. Furthermore, we found no effect of duration of feeding (i.e., 21 or 42 days) on the level of enrichment observed at each dose. The cost of supplementing the birds for 21 days at 1% of the diet in the current study would be an estimated €0.23 per bird. Feeding at a rate of 0.5% for the whole life of the bird would be less expensive (€0.16 per bird), but the degree of enrichment achieved remained significantly below that of birds supplemented at a rate of 1% for a shorter duration. Feeding for the final 21-day fattening period only is further supported by the significantly higher transfer efficiencies observed for the FP treatments when compared to the WL treatments. These results indicate that the most efficient feeding strategy is to feed for the final 21-day fattening period only.

The level of enrichment observed in the breast samples of birds supplemented with AURA at a rate of 0.5% fell within the range of values reported from our previous studies (23–35 mg DHA/100 g breast) when broilers were supplemented at the same rate. The DHA content of the thigh samples from the current study (47 and 54 mg DHA/100 g thigh following 21 and 42 days, respectively) exceeded the range of 24–43 mg DHA/100 g thigh reported from our previous studies. A similar trend was observed for the 1% AURA treatments, with the DHA concentration of breast samples from the current study (50 and 55 mg DHA/100 g breast following 21 or 42 days, respectively) and those from the thigh samples (80 and 86 mg/100 g thigh after 21 or 42 days respectively) exceeding the range reported from the breast (36–48 mg DHA/100 g breast) and thigh (42–50 mg DHA/100 g thigh) from our previous trials [16,17]. The fat content of the tissues in the current study was generally higher than those of the tissues from previous studies, increasing their capacity for DHA enrichment. However, the degree of fat deposition has been shown to be unaffected by increasing levels of n-3 PUFA with other nutritional and genetic factors likely responsible for the differences observed [29], between the overall levels of fat detected in the different studies.

Zuidhof et al. [30] investigated the effect of the duration of dietary supplementation of broilers using ground full-fat flaxseed as a source of n-3 PUFA and found that feeding for 24.1 days was the most cost effective duration that achieved an adequate level of tissue enrichment. Kanakari et al. [31] also used flaxseed to supplement broiler diets and showed that feeding for 2–4 weeks prior to slaughter would be sufficient for broilers to accumulate the same amount of n-3 LCPUFA as being fed for six weeks, which is similar to the findings of the current study. When supplementing the diets of broilers with fish oil accompanied by either flaxseed, lard or rapeseed, Konieczka et al., [32] found that feeding for a duration of three weeks before slaughter resulted in significantly higher EPA + DHA concentrations than feeding for one or two weeks only. Our previous work in laying hens indicated that supplementation at a level of 1% of the diet increased the DHA content of eggs for the first 24 days, with no significant increase in the concentration of egg DHA after that point [18]. Overall, these results, using a variety of different n-3 PUFA sources, indicate that feeding for periods longer than 2–4 weeks is not a cost-effective use of the supplemental ingredient.

For a food to be labelled "a source of omega-3" in the European Union (EU) it must contain at least 40 mg EPA+DHA/100 g, while to be labelled "high in omega-3" it must contain 80 mg EPA + DHA/100 g [33]. For the breast meat, both the 0.5%WL and the 0.5%FP groups fell just short of this target with 35 and 33 mg EPA + DHA/100g detected respectively. The thigh samples however, did meet these criteria with 59 mg and 51 mg EPA + DHA/100 g detected in the 0.5%WL and 0.5%FP groups respectively. The breast samples of the 1%WL and 1%FP groups met the criteria to be considered a source of omega-3, with 60 and 54 mg EPA+DHA/100 g detected respectively, while the thigh samples could be considered high in omega-3 with 94 and 87 mg EPA+DHA/100 g detected respectively. Based on these results, the broilers would need to be supplemented at a rate of 1% of the diet so that both the breast and thigh meat could be labelled as "a source of omega-3" in the EU.

No EPA was detected in any of the diets provided, but despite this, an increased inclusion of AURA generally led to increased tissue concentrations of EPA. Similar results have been obtained in our previous trials [16,17], with the increase likely due to the retro-conversion of DHA to EPA [34]. In contrast, no differences were observed between the treatments in terms of the tissue n-3 DPA concentration. This is in keeping with the results of our previous study, when supplementing broilers with 0, 0.25, 0.5 and 1 % AURA, resulted in no differences in n-3 DPA concentrations between treatments in breast tissue samples, while in the thigh samples, the 0.5% treatment had significantly less n-3 DPA than the control group with no differences observed between the other groups [17]. Small but significant increases in breast n-3 DPA were observed when birds were supplemented with AURA at a rate of 2.5% and 5% of the diet with 10.19 and 10.52 mg n-3 DPA/100 g detected respectively, while at 0 and 0.5% of the diet 9.02 and 9.02 mg n-3 DPA/100 g were detected respectively. It does not appear that this increase in tissue n-3 DPA is due to some form of retro-conversion from DHA. It could more likely be a result of the elongation of EPA, which was present in higher concentrations in the 2.5 and 5% treatments. The elongation of EPA to n-3 DPA is more efficient than the elongation of EPA to DHA [35]. In humans, supplementary DHA from an algal source (containing no EPA) was found to significantly increase blood EPA concentrations while reducing the concentration of n-3 DPA indicating that DHA supplementation could negatively influence the concentration of n-3 DPA, possibly as result of competition at the level of fatty acid esterification [36]. These findings indicate that supplementary DHA-rich AURA, when provided at a level of 0.5–1% of the diet can also increase the tissue concentrations of the beneficial fatty acid EPA, despite it being absent from the experimental diets.

The significant increases in both EPA and DHA contributed to the higher total n-3 PUFA concentrations detected in many of the tissue/organ samples. The concentration of C20:4n6 was significantly lower in most of the tissue/organ samples from supplemented birds, however, this did not translate into lower total n-6 PUFA concentrations in any of the tissues/organs. Importantly, supplementation led to lower n-6/n-3 ratios, in every treatment and in every tissue/organ with the exception of the WL skin with adhering fat treatments. Reductions in a person's overall n-6/n-3 ratio have been shown to improve the health outcomes of patients suffering from a variety of illnesses, including cardio-vascular diseases and some forms of cancer, with the effective ratio based on the type of illness in question [37]. Consuming this AURA enriched broiler meat could help to reduce the overall n-6/n-3 ratio of a person's diet.

No attempt was made to assess the effect of supplementation on the consumer acceptability of the meat of supplemented birds. However, supplementing broiler diets with marine protists at a level of 2.8% of the diet was previously found to have no impact on consumer acceptability [14]. As the inclusion rates observed in this trial were below this, it is unlikely that the consumer acceptability would be affected. However, it would be beneficial to determine the consumer acceptability of these enriched meats when fresh and following a period of storage. Recent work investigating the frozen storage stability of DHA found significant reductions in the tissue DHA content after a period of 12 or 24 weeks [26]. Chicken meat for human consumption is unlikely to be stored for this extended period of time and over this duration the DHA concentration of breast meat decreased by 35%, 35%, 29.4% and 27.8% in broilers supplemented with AURA at a rate of 0%, 0.5%, 1.5% and 2.5% of the diet. As such, it would be beneficial to establish the effects of storage on the stability of DHA and the consumer acceptability of the AURA enriched broiler meat over a shorter time period.

#### **5. Conclusions**

These results indicate that providing AURA as a dietary supplement for broilers for the final 21-day fattening period can improve the nutritional quality of broiler meat by significantly increasing the DHA and EPA concentrations of tissues and organs, as well as lowering the n-6/n-3 ratio without significantly impacting productivity.

**Supplementary Materials:** The following are available online at http://www.mdpi.com/2076-2615/9/6/327/s1. Table S1: The full fatty acid profile of breast, thigh, liver, kidney and skin (with adhering fat), taken from birds fed diets supplemented with A. limacinum at a rate of 0, 0.5 or 1% for their whole life (WL; day 0–42) or for the fattening period only (FP; day 21–42).

**Author Contributions:** Conceptualization and methodology, C.A.M., M.M. and G.F.; supervision, M.M and G.F.; project administration, C.A.M. and M.M.; data curation, M.M. and J.D.K.; formal analysis, M.M., J.D.K. and G.F.; writing—original draft preparation, M.M. and J.D.K.; writing—review and editing, C.A.M., J.D.K., M.M., and G.F.

**Funding:** The research was funded by Alltech SARL (France).

**Acknowledgments:** The authors would like to express their gratitude to the following: Rebecca Timmons (Alltech Inc., USA) for her technical input on *Aurantiochytrium limacinum* and Tuoying Ao (Alltech Inc., USA) for his knowledge of the application of *A. limacinum* in broiler nutrition.

**Conflicts of Interest:** The authors C.A.M. and J.D.K. are employees of Alltech which produces and markets ALL-G-RICH®, the commercial product used in this study

#### **References**

© 2019 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (http://creativecommons.org/licenses/by/4.0/).

#### *Article*

### **E**ff**ect of Dietary Modulation of Selenium Form and Level on Performance, Tissue Retention, Quality of Frozen Stored Meat and Gene Expression of Antioxidant Status in Ross Broiler Chickens**

**Doaa Ibrahim 1,\*, Asmaa T.Y. Kishawy 1,\*, Safaa I. Khater 2, Ahmed Hamed Arisha 3, Haiam A. Mohammed 3, Ahmed Shaban Abdelaziz 4, Ghada I. Abd El-Rahman 5 and Mohamed Tharwat Elabbasy 6,7**

Received: 23 April 2019; Accepted: 5 June 2019; Published: 11 June 2019

**Simple Summary:** Although the importance of usage of selenium as essential trace element in poultry production has been proven, the best source and level has not been fully addressed yet. Three different dietary selenium forms with three different levels were chosen to be added in broiler diet. Met-Se or nano-Se up to 0.6 mg/kg increased their performance and was more efficiently retained in the body than SeS. Frozen stored meat quality was improved in a dose-dependent manner especially with both Met-Se and nano-Se. Nano-Se was more potent than Met-Se, which in turn was more potent than inorganic Se against oxidative stress, which improved the quality of meat under frozen conditions.

**Abstract:** This study compares between different selenium forms (sodium selenite; SeS, selenomethionine; Met-Se or nano-Se) and levels on growth performance, Se retention, antioxidative potential of fresh and frozen meat, and genes related to oxidative stress in Ross broilers. Birds (n = 450) were randomly divided into nine experimental groups with five replicates in each and were fed diets supplemented with 0.3, 0.45, and 0.6 mg Se/kg as (SeS, Met-Se), or nano-Se. For overall growth performance, dietary inclusion of Met-Se or nano-Se significantly increased (*p* < 0.05) body weight gain and improved the feed conversion ratio of Ross broiler chicks at the level of 0.45 and 0.6 mg/kg when compared with the group fed the same level of SeS. Se sources and levels significantly affected (*p* < 0.05) its concentrations in breast muscle, liver, and serum. Moreover, Se retention in muscle was higher (*p* < 0.05) after feeding of broiler chicks on a diet supplemented with Met-Se or nano-Se compared to the SeS group, especially at 0.6 mg/kg. Additionally, higher dietary levels from Met-Se or nano-Se significantly reduced oxidative changes in breast and thigh meat in the fresh state and after a four-week storage period and increased muscular pH after 24 h of slaughter. Also, broiler's meat in the Met-Se and nano-Se groups showed cooking loss and lower drip compared to the SeS group (*p* < 0.05). In the liver, the mRNA expression levels of glutathione peroxidase, superoxide dismutase, and catalase were elevated by increasing dietary Se levels from Met-Se and nano-Se groups up to

0.6 mg/kg when compared with SeS. Therefore, dietary supplementation with 0.6 mg/kg Met-Se and nano-Se improved growth performance and were more efficiently retained than with SeS. Both sources of selenium (Met-Se and nano-Se) downregulated the oxidation processes of meat during the first four weeks of frozen storage, especially in thigh meat, compared with an inorganic source. Finally, dietary supplementation of Met-Se and nano-Se produced acceptable Se levels in chicken meat offered for consumers.

**Keywords:** broilers; selenium sources-levels; selenium retention; antioxidant capacity; frozen meat

#### **1. Introduction**

Selenium (Se) is an important trace nutrient for the maintenance, growth, and animals and humans health [1]. It improves the nutritive value and of meat quality [2]. As feed additives, Se can enhance growth productivity in broiler chickens [3]. Selenium is an important constituent of at least 25 selenoproteins involved in various physiological processes, including immune function, reproduction, and the maintenance of antioxidant defenses to avoid tissue damage. Selenium deficiency results in a number of disorders and injuries in poultry, such as skeletal myodegeneration, exudative diathesis (ED), muscular hemorrhages, atrophy of pancreas, decreased production of eggs, liver injury, reduced hatchability, and inhibited growth of bursal and thymic [4], and increase susceptibility of humans to certain degenerative diseases, such as cancer [5]. The fortification of poultry meat with Se represents a viable strategy for increasing human intake of Se. The national research council (NRC) [6] recommendations established a low selenium level (0.15 mg/kg) for the supplementation of broilers. This level is not adequate to avoid production losses resulted from selenium deficiency disorders [7]; consequently, there is a need to increase dietary selenium levels. Moreover, Se bioavailability not only depends on its physical form but also on dietary concentration and the levels of other trace elements. Excess levels of Se can be toxic when provided above the biological requirement. Thus, meeting Se requirements and optimizing performance is an important step in modern poultry production. Practically, selenium can be added for poultry's diet in the form of inorganic Se, organic Se, and most recently, nano-Se. The inorganic form of selenium (Se selenite) is primarily and commonly used for dietary supplementation, and exhibits a very narrow border between its dietary requirement and its toxicity [8]. Recently, it has been recognized that organic Se has a higher rate of tissue retention and bioavailability thus lower toxicity than inorganic Se, so it is preferable to inorganic Se in broilers [9]. In addition, organic Se is deposited more efficiently in breast muscle than inorganic forms [10]. With the development of nanotechnology, nano-Se has attracted widespread research interest due to its high catalytic efficiency and higher adsorbing capacity, and has exhibited strong absorption efficiencies and lower toxicity than inorganic Se [11]. Moreover, recent studies found that nano-Se has a higher effectiveness in controlling selenoenzymes and displays less toxicity than selenium-selenite [12]. Moreover, supplementation of dietary Se could also enhance oxide dismutase (SOD), glutathione peroxidase, (GPx) and catalase (CAT) activities, and reduce oxidative stress and lipid peroxidation biomarkers, consequently reducing oxidative stress in broilers [13]. Moreover, Se plays a key role in the signaling of redox via removal of hydrogen peroxide and lipid hydroperoxides using glutathione as an ultimate electron donor [14]. These antioxidant properties of Se have also been shown to continue in postmortem muscle tissue and prevent lipid oxidation [15]. For this reason, many dietary regimes in animal nutrition have been established to produce Se-enriched meat in order to increase human Se consumption [16]. The type and level of available Se is important to meet broilers' dietary requirements and optimize their production without producing any hazardous effects on broilers or human health. Definitive comparative studies to fully exploit the benefits of dietary supplementation with different available Se sources and levels in Ross broilers remains poorly investigated. Thus, the aim of the present study was to compare the bioavailability of different levels and sources of Se on performance,

Se retention, lipid oxidative stability of meat, meat quality, and mRNA expression of some selected genes related to antioxidant capacity in Ross broiler chickens.

#### **2. Materials and Methods**

#### *2.1. Selenium Sources*

Sodium selenite (SS) and selenomethionine (Met-Se) were obtained from Sigma-Aldrich Chemical Co., St. Louis, MO, USA and Sel-Plex; Alltech Inc., Nicholasville, KY, USA, respectively. nano-Se powder was prepared according to [17] where 4 mL of 25 mM GSH containing 15 mg of bovine serum albumin were mixed with one ml of 25 mM sodium selenite. The resulting red suspension was dialyzed against double-distilled water for 96 h. Every 24 h, the water was replaced to isolate the oxidized GSH from the nano-Se. Then, nano-Se and bovine serum albumin were lyophilized. The phase characterization and morphology of nanoparticles were analyzed by means of X-ray diffraction (XRD) using EMPYREAN diffractometer and JEM-200CX transmission electron microscopy (TEM) working at 30 kV as shown in (Figure 1). XRD patterns corresponding to the (100), (101), (110), (102), (111), (201), (003), (202), (210), and (211) planes of the formed nano-Se were observed at 20 angles of 23.6◦, 29.9◦, 41.4◦, 43.8◦, 45.4◦, 51.8◦, 55.9◦, 61.8◦, 65.3◦, and 68.3◦, respectively [18]. The nano-Se was spherical in shape with average size 42 ± 1.4 nm (total count of 100 NPs).

**Figure 1.** Characterization of nano-Se (**A**,**B**): (**A**) Morphology of the formed nano-Se pictured by transmission electron microscopy (TEM) and; (**B**) X-ray diffraction (XRD) pattern of the nano-Se.

#### *2.2. Birds and Experimental Procedures*

Four hundred and fifty, one-day-old, Ross broiler chicks (Ross 308) were individually weighed and divided to nine dietary groups, each group consisting of five replicates of ten chicks each per floor pen. Broiler chicks were fed diets containing inorganic Se (sodium selenite; SeS), organic Se, (selenomethionine, Met-Se), or nano-Se, each at three levels 0.3, 0.45, or 0.6 mg/kg Se (as fed). The basal diet was formulated to meet nutrient requirements of Ross broilers according to [19] except Se (Table 1). Diets were fed from 1 to 38 d including starter (1–11 d), grower (12–22 d), and finisher (23–38 d) diets. All chicks were given ad libitum access to feed and water. The environmental temperature was kept at 32 ◦C for the 1st week and then gradually decreased until reached 23 ◦C. All the experimental procedures were performed at the Institute of Nutrition and Clinical Nutrition and Poultry Farm following the Faculty of Veterinary Medicine guidelines and in accordance with the protocols approved by Institutional Animal Care and Use committee at Zagazig University (Approval no: ZU-IACUC/2/F/123/2018).

The proximate analysis of feed ingredients was done according to the standard method of [20]. For Se analysis in feed, one gram of feed was heated for 5 h in a furnace at 550 ◦C for ashing. Mixture from 3 N HCl (10 mL) and an ashed sample was heated until the solution became clear. After cooling, the sample was filtered and diluted to 50 mL with 0.1 N HCl. For analysis of selenium, lanthanum 185.4 L 50 gm/kg was added to 6 mL of the sample solution. Then, analysis was achieved by a spectrometer at a wavelength of 400 nm [20].

**Table 1.** The ingredients and nutrient levels of the basal diet (on dry matter basis).

\* Provided for each kilogram of diet: Vitamin A, 10,000 IU; vitamin E, 7200 IU; vitamin D3, 3000 IU; vitamin K, 2 mg; vitamin B1, 2640 mg; vitamin B6, 1200 mg; calcium pantothenate, 10 mg; nicotinamide, 50 mg; biotin, 40 mg; choline chloride, 500 mg; folic acid, 0.5 mg; cobalamin, 0.01 mg; calcium, 9000 mg; manganese, 120 mg phosphorus, 2100 mg; sodium, 3700 mg; iron, 110 mg; copper, 10 mg; zinc, 100 mg; iodine 1.1 mg. b Calculated values except selenium.

#### *2.3. Growth Parameter Measurement*

Live body weight (LBW) and feed intake of broiler chicks/pen were estimated individually at 21 and 38 d of age to calculate live body weight, body weight gain (BWG), total feed intake, feed conversion ratio (FCR) and relative growth rate (RGR).

#### *2.4. Sampling and Analytical Procedures*

At the end of the feeding trail, tissues samples (liver and breast meat) were collected from five birds/replicates that were slaughtered (slaughtering house under supervision of Institutional Animal Care and Use Committee at Zagazig University Faculty of Veterinary Medicine) and handled and kept at −20 ◦C until analysis of selenium content and meat quality tests. Blood samples were collected with or without anticoagulant, then plasma and serum were kept at −20 ◦C until the analysis of selenium content and chemical analysis was performed.

#### 2.4.1. Tissue Retention of Selenium

Briefly, liver and breast muscle were weighed (0.1 g) and mixed with of HNO3 (8 mL) then digested by microwave. After that, deionized water was added to produce a 10 mL volume. The selenium content was determined following the procedure of [21] by atomic absorption spectrophotometer (Shimadzu Ltd., Shimane Shimadzu, Japan).

#### 2.4.2. Selenium Content in Serum Constituents

Selenium content was measured in serum by atomic absorption spectrophotometer (AA6501, Shimadzu Ltd., Japan). Plasma samples were used for measuring of aspartate amino transferase (AST), alanine glutamyl transferase (ALT), and creatinine calorimetrically by diagnostic kits (MAK055, MAK052, and C4255, respectively) manufactured by Sigma-Aldrich.

#### 2.4.3. Laboratory Analysis for Meat Quality

#### Meat pH and Drip and Cooking Loss in Meat Samples

Breast meat was used to determine postmortem pH (t = 0.5 and 24 h) by pH meter. Drip loss was estimated according to [22] (percent; proportional weight loss of a sample suspended for 72 h in a closed plastic bag under refrigerated conditions at 4 ◦C). After storage at −20 ◦C, cooking loss was determined (percent; weight loss proportionate of a sample after cooking for 40 min in a water bath at 70 ◦C followed by cooling).

#### Preparation of Samples for Total Antioxidant Capacity

Six hours after slaughter and handling, breast meat was cut into cubes of approximately 3 cm square); visible connective tissues and fat were removed. These muscle cubes mixed with distilled water then homogenized and centrifuged and used for measuring total antioxidative markers as free radical scavenging assay using 2,2 -azino-bis-3-ethylbenzothiazoline-6-sulfonic acid (ABTS) thiobarbituric acid reactive substances (TBARS) assay, and 2,2-diphenyl-1-picrylhydrazyl (DPPH) assay, and ferric reducing/ antioxidant (FRAP) assay.

Thiobarbituric acid-reactive assay: Oxidation was evaluated on the first day and after one week from storage by the thiobarbituric acid assay described by [23]. Perchloric acid (27 mL, 3.83% v/v) was added to of meat sample (5 g) then homogenized for 1 min and filtered by filter paper, then 2 mL thiobarbituric acid was added to supernatants and incubated in a water bath (100 ◦C) for 20 min. Subsequently, immediate cooling to room temperature and centrifugation for 15 min was performed, then the absorbance was read by the spectrophotometer at 532 nm. The results were then calculated according to the standard curve and values were expressed as mg malondialdehyde (MDA)/kg meat.

ABTS assay: The total antioxidant capacity of chicken breast and thigh meat was analyzed by Trolox-equivalent antioxidant capacity (TAC) assay [24]. Briefly, the reaction between 14 mM ABTS [2,2-azinobis-(3-ethylbenzothiazoline-6-sulfonic acid)] with an equal volume of 4.9 mM potassium persulfate was catalyzed to stimulate the formation ABTS+ radical cation formation, then incubated in the dark at room temperature for 12–16 h. After that, 10 μL of meat homogenate was added to the ABTS+ solution (1.0 Ml) and mixed thoroughly and after 60 s absorbance was read at 734 nm.

DPPH assay: The scavenging activity of the muscle samples was analyzed by 1,1-diphenyl-2 picrylhydrazyl radical (DPPH) [25]. Briefly, the meat samples were homogenized in distilled water and then centrifuged. The supernatant was mixed with ethanol and DPPH radical solution and incubated in a dark room for 10 min. Next, the absorbance measurement was read at 517 nm. The ability to scavenge the DPPH radical was expressed as μM per g of wet muscle tissue.

FRAP assay: Ferric reducing antioxidant power (FRAP) assay [26] was carried out on meat homogenates. The meat samples were homogenized in potassium phosphate buffer, centrifuged, and the supernatant was collected. Then, supernatant (1 mL) was collected and added to FRAP buffer (3 mL) containing 10 mM 2,4,6-Tris(2-pyridyl)-s-triazine) in 40 mM HCl, and 20 mM Fe2Cl3 was added to 300 mM acetate buffer. Immediately after mixing, the absorbance was measured at 593 nm. A standard curve was prepared with FeCl2. The antioxidant power of the samples was expressed as μM of Fe2+ per 1 g wet muscle tissue.

#### *2.5. RNA Extraction, Reverse Transcription, and Quantitative Real-Time PCR.*

At the end of the feeding trial (day 38), three birds per group were randomly selected, marked and injected with tert-butyl hydroperoxide, 0.2 mmol/kg body weight, intraperitoneally purchased from Sigma-Aldrich Chemical Co (St. Louis, MO, USA, CAS Number 75- 91-2) to induce the oxidative stress. Birds were slaughtered, and liver samples were collected 48 h post-injection. From liver tissue, the total RNA was extracted by RNeasy Mini Kit; Qiagen, Cat. No. 74104. according to the manufacturer regulation. The extracted RNA was quantified using the NanoDrop® ND-1000 Spectrophotometer (NanoDrop Technologies, Wilmington, NC, USA). The first strand cDNA was synthesized using kits of RevertAidTM H Minus (Fermentas Life Science, Pittsburgh, PA, USA). One μL of this cDNA was blended with 12.5 μL of 2× SYBR® Green PCR mix with ROX from BioRad, 5.5 μL of RNase free water, and 0.5 μL (10 pmol/μL) of each forward and reverse primer for the selected genes were added. The primers' sequences of catalase, glutathione peroxidase, and superoxide dismutase genes involved in antioxidant function were designed as previously described in [27]. The real-time PCR amplification was carried out with Rotor-Gene Q2 plex (Qiagen Inc., Valencia, CA, USA) with the following conditions; initial denaturation at 95 ◦C for 10 min and 40 cycles at 95 ◦C for 15 s and 60 ◦C for 1 min. Relative fold changes in the expression of target genes measured in triplicate were estimated by the comparative 2−ΔΔCt method with the GAPDH gene as an internal control to normalize target gene expression levels [28].

#### *2.6. Statistical Analysis*

Data were submitted to a 2-way ANOVA, using PASW statistics 18 (SPSS Inc., Chicago, IL, USA) to clarify the effects of dietary Se sources, its levels, and their interaction. Gene expression data were statistically analyzed using one-way ANOVA and relevant figures were generated by Graphpad Prism 7 (GraphPad Software Inc., San Diego, CA, USA). Tukey's test was used to separate the means when the treatment difference was significant (*p* < 0.05). All data were expressed as the mean ± SEM. Statistical significance was considered at *p* ≤ 0.05.

#### **3. Results and Discussion**

#### *3.1. Growth Performance*

The effects of different dietary treatments on overall growth performance parameters (1–38 days) are presented in Table 2. The present study showed that the interaction between different dietary sources and levels of Se had significant (*p* < 0.05) effect on the body weight and gain of broilers at 38 days. The groups supplemented with selenomethionine (Se-Met) and nano-Se showed a significant increase (*p* < 0.05) in body weight and gain of Ross broiler chicks when compared with selenite selenium (SeS). Moreover, variety of levels and sources of Se played an important role in our study as when Se- Met or nano-Se were added to diets, body weight and gain increased as dietary Se levels increased, while higher levels in the SeS group at Se concentration of 0.3–0.45 mg/kg diet caused declines as dietary SeS levels increased. Different sources and levels of Se had no effect on feed intake (*p* < 0.05). Feed conversion ratio (FCR) of broilers was affected by the Se sources and levels as they play an important role in improving FCR. The FCR was improved by dietary supplementation of Se-Met or nano-Se, while the best FCR was established with a level of 0.6 mg Se/kg diet from Se-Met

or nano-Se, followed by groups supplemented with 0.3–0.45 mg/kg from Se-Met or nano-Se. Our data demonstrated that the application of dietary Se-Met or nano-Se up to 0.6 mg/kg resulted in the maximum growth rate of broiler chicks, while the same dose from SeS tended to reduce the growth performance of broilers chicks. These results proved that such selected dietary Se-Met or nano-Se levels had higher bioavailability than inorganic forms of Se. These results are in agreement with [29] who found a decline in body weight gain and feed utilization as supplemental inorganic Se increased, while for nano-Se, average daily gain, FCR, and survival ratio reached their highest levels at an Se concentration of 0.15–1.20 mg/kg. [13] Showed that feeding of broilers on 0.3 and 0.5 mg/kg from nano-Se significantly improved FCR and increased tissue selenium content. Our findings were also in agreement with those of [30], who described no differences in feed intake among broilers fed diets supplemented with either organic or inorganic forms of Se. Broilers chicks groups fed on 0.2 mg/kg diet from organic selenium or nano-Se had a similar growth rate as compared to the group supplemented with the same level of Se-selenite [31]. Our findings were also in agreement with those of [32] who reported that increased selenium levels had improved average daily gain in the same time there was no differences on average daily gain between nano-selenium and organic selenium in broiler chickens. The function of Se on growth rate may relate to its role in the selenoprotein P and selenoenzymes type I iodothyronine deiodinase expression, which have critical roles in the synthesis of thyroid hormones and Se transport [33]. Moreover, our results of increased growth performance with selenium methionine and nano-Se could possibly due to an increased thyroid hormone regulating the body's energy metabolism and increased digestibility of protein [34]. The results of this study suggest that different Se sources and levels may be necessary to optimize the performance of broilers, and that the form of organic Se may be of importance.

**Table 2.** Effects of dietary sources and levels of Se (mg/kg) on growth performance of broilers over 38 days.

SeS = sodium selenite; Met-Se = selenomethionine; nano-Se = nano-selenium; BW = body weight; BWG = body weight gain; FI = feed intake; FCR = feed conversion ratio, RGR = relative growth rate. a,b,c Means within a row carrying different superscript letters denote significant differences (*p* < 0.05).

#### *3.2. Selenium Retention in Serum, Muscle, and Liver*

In the present study, the different Se sources and levels had significantly affected (*p* < 0.05) Se concentrations in serum, liver, and breast muscle of broilers (Table 3). The groups fed on a diet supplemented with nano-Se and Met-Se showed higher (*p* < 0.05) serum, liver, and breast muscle Se concentrations when compared with those fed diets supplemented with SeS, indicating that nano-Se and Se-Met were better retained in the body than SeS, although the effect of Se-Met was more prominent for tissue Se retention than nano-Se at the same lowered level. Accumulation of minerals in tissues is considered an indicator for mineral utilization [35]. The concept of increasing Se content in human foods by altering dietary Se sources and level given to livestock is now of interest to nutritionists [36]. Wang et al. [37] stated that transport and uptake of selenium by broiler intestinal cells were higher in nano-Se than that of SeS. The difference in retention of Se between Se yeast and SeS or nano-Se may be clarified by the probable metabolic pathways and absorption process for Se from different Se sources [29]. The safe limit of Se in human food has been established at 2.0 mg/kg for the United States [38]. This level agreed with our results that up to 0.6 mg/kg of Se in broiler diets precipitates less than 1 mg/kg in meat with all sources of Se. Selenium uptake from Se-selenite occurs by passive diffusion contributing the poor availability of Se-selenite [39], and up to 50–75% of consumed Se-selenite

is lost through urine. Another limitation of adding selenite to feed is the short period storage of Se in the animal's body [40]. Our results of Se retention in tissue in accordance with those of [41], who demonstrated that broiler chicks fed on dietary organic Se had higher (*p* < 0.05) Se content in breast muscle and liver than those fed diets fortified by SeS. [36] also proved that the contents of Se in liver and muscles were affected by dietary Se supplementation, and retention of Se was increased when organic Se was supplemented as compared with inorganic Se. Cai et al. [13] stated that increasing dietary nano-Se increased the concentration of selenium in liver and muscle tissue (*p* < 0.01). An explanation for increased tissue content from nano-Se may be attributable to improved intestinal absorption of nano-Se due to smaller particle size and larger surface area [42]. SeS and nano-Se, on the other hand, are changed to the transitional selenide and then employed for synthesis of selenoprotein or methylated and after that excreted. However, Met-Se contains a large amount of selenomethionine. When recognized as a Se species, it can be altered to selenocysteine through the trans-selenation pathway and then lysed to selenide. So Met-Se might be simply utilized in the tissue than SeS or nano-Se [43]. Another property of Met-Se involves the chemical similarity between Met-Se and Met, which permits the body to use them interchangeably in protein synthesis as Met-tRNA cannot distinguish between Met and Met-Se, which makes it possible to build Se reserves in the body [2].

#### *3.3. The E*ff*ect of Di*ff*erent Levels and Sources of Se on Selected Serum Parameters*

The activity of liver enzymes including ALT and AST were not significantly affected by the interaction between different levels and sources of Se. The same trend was recorded for serum creatinine values (Table 3). Selim et al. [44] stated that activity of liver enzymes including ALT and AST were not significantly affected by addition of Zn-Se-Meth, P-Nano-Se, or L-nano-Se in broiler diets. Moreover, increasing the supplemental Se level from 0.3 to 0.45 ppm in broiler diets could not cause any significant difference in plasma creatinine level. In previous studies, [44] found that liver enzymes were not affected by adding different forms of Se (inorganic, organic, or nano) at levels up to 0.3 mg Se/kg diet.

means ± standard error.

#### *3.4. Antioxidant Potential of Di*ff*erent Sources and Levels of Se*

These data showed that the expression pattern of selected antioxidant-related genes (glutathione peroxidase, GPx, super oxide dismutase, SOD and catalase, CAT) in relation to different Se levels and sources was addressed in (Figure 2). The expression of GPx mRNA significantly increased in groups fed nano-Se at 0.6 mg/kg diet followed by groups supplemented by 0.3 and 0.45 mg/kg diet from Met-Se and nano-Se when compared with SeS with the same levels. The highest expression of SOD was observed in groups supplemented with 0.45 and 0.6 mg/kg diet from nano-Se followed by the group supplemented by a 0.3 mg/kg diet from nano-Se and groups supplemented by 0.45 and 0.6 mg/kg diet from Met-Se, when compared with SeS supplemented group. The mRNA expression of catalase significantly increased with an increasing level of nano-Se and Met-Se when compared with SeS. The antioxidant enzymes such as SOD, CAT, and GPx [45], and non-enzymatic constituents such as glutathione (GSH) [46], play an important role for keeping the animal health, and physiological antioxidant systems. Selenium is a cofactor in several selenoproteins and the antioxidant selenoenzymes as glutathione peroxidase (GPx), thus its functional role is associated with the Se concentration in tissues [47]. Xiao et al. [48] demonstrated that the supplementation of Se in the maternal diet significantly (*p* < 0.05) enhanced the activity of GPx, T-SOD, and CAT in heat stress treated chick embryos when compared with the basal diet, as the levels of GPx1 mRNA were significantly (*p* < 0.05) elevated by adding Se. This may be clarified by higher Se retention in maternal Met-Se treatment [49], which aids in the production of more selenoproteins to preserve chick embryos with a higher antioxidant level. Under heat stress, [35] reported that the addition of organic Se significantly improved GPx activities as compared with broilers fed with inorganic Se. [50] established that the highest GPx activity and lowest MDA content in blood and testis were attained in the treatment of 0.5 mg/kg, as the GPx enzymes were involved in scavenging toxic H2O2 [51]. In animal research, the activity of GPx enzymes and their expression genes in tissues were correlated with the concentration of Se added to feed [47]. This finding is also in accordance with [52], who described that Se deficiency caused the reduction of GPx mRNA levels in four GPx genes found in chicken livers. The superoxide dismutase (SOD) and CAT are important antioxidant enzymes for poultry. The superoxide anion is transformed to H2O2 by SOD [53], and CAT changes H2O2 into water [54], although Se is not a component of SOD and CAT. Our results also indicated that nano-Se and Met-Se increased the mRNA expression of these genes. Yuan. [49] showed that in broiler breeding experiments, hepatic GPx1 and TrxR1 mRNA levels in Met-Se groups were higher (*p* < 0.05) than that in the SeS group.

**Figure 2.** Effects of dietary Se source and level on the relative antioxidant enzymes expression (**A**–**C**). (**A**) Glutathione peroxidase (GPx); (**B**) super oxide dismutase (SOD); and (**C**) catalase (CAT) in the liver of broiler chickens at 40 days. SeS = sodium selenite, Met-Se = selenomethionine, nano-Se = nano-selenium. a,b,c,d Different superscript letters denote significant difference (*p* < 0.05). Values are means ± standard error.

#### *3.5. E*ff*ect of Di*ff*erent Se Sources and Levels on Meat Quality*

The role of diets supplemented with different Se sources and levels on breast meat quality in broiler chickens are shown in Table 4. Compared with SeS, dietary Met-Se and nano-Se inclusion in broiler diet improved meat quality, especially as Se levels increased from 0.45 to 0.6 mg/kg.

#### *3.6. Post-Mortem pH of Meat, Cooking Loss and Drip Loss*

Breast meat from groups that received an increased level of Met-Se and nano-Se exhibited increased (*p* < 0.05) pH 0.5 and 24 h later when compared with the SeS groups. In addition, birds in the Met-Se and nano-Se groups, specially at high levels, had lesser drip and cooking loss group (*p* < 0.05) compared to those in the SeS groups. The presence of Se in animal diets are a key influence on meat water retention, with the form and level regulating the variation in meat drip loss [55]. The results of our study agreed with [56], who found that the drip loss was lower and water-holding capacity was higher in pigs fed with organic selenium. It has been reported by some authors that the mechanism by which antioxidants modify the water-holding capacity and drip loss of meat can be attributed to their ability to maintain muscle membranes' integrity post-mortem [57], while others have suggested that proteolysis and protein oxidation acts as an essential factor for determining the moisture retention of meat [58]. Lambert et al. [59] reported that the accumulation of a large amount lactic acid in the muscles, combined with a cessation of blood circulation which induces cellular hypoxia and results in a decreased pH after slaughter, changed the permeability of cell membrane and decreased the water-holding capacity. But our study demonstrated increased water-holding capacity of breast meat in broilers fed on Met-Se and nano-Se. This may be explained by the metabolic conversion of glucose to lactic acid in post-mortem muscle being delayed with organic Se or nano-Se supplementation, thus improving the water-holding capacity of meat and decreasing drip loss [60]. It has been reported elsewhere that 0.3 mg/kg Met-Se supplementation resulted in an increase in the pH of the breast meat of broilers [61] and in geese [62] as compared with 0.3 mg/kg SS supplementation. Other studies demonstrated that water-holding capacity is affected by organic Se supplementation [63] and nano-Se [64]. Cai et al. [13] proved that application of nano-Se increases the ability of broiler muscle proteins to attract water, thus reducing drip loss percentage. The present study further indicates that the role of Met-Se and nano-Se on the biochemistry of muscle tissue is more prominent than with SeS.

#### *3.7. Thiobarbituric acid Reactive Substances (TBRAS) Content of Meat as a Marker for Lipid Oxidation*

Frozen storage of all analyzed meat significantly increased (*p* < 0.05) the TBRAS values in breast and thigh meat, with the lowest values for TBRAS recorded in breast meat, which could be related to the total lipid content. With increasing dietary level of organic Se and nano-Se, the TBRAS values decreased in breast and thigh meat when compared with SeS supplementation (Table 4). Exposure to different physiochemical or pathological conditions has recently been shown to be one of the main predisposing agents controlling free radical formation in the body [65]. On the other hand, chicken meat enriched with polyunsaturated fatty acids (PUFA) augmenting the meat susceptibility to oxidation progressions [66]. Bakhshalinejad [32] reported that oxidation resistance of broiler meat was higher in case of supplementation of organic of Se and the higher concentration of Se the higher glutathione peroxidase activity, total antioxidant capacity and malondialdehyde formation. Oxidation of lipids produces free radicals, leading to mutagenesis, carcinogenesis, and aging of the cell [67]. The antioxidant role of Se has also been shown to continue post-mortem in muscle tissue, where it is reported Se reduced oxidization of lipids in meat and had an effect on its quality [15]. Providing Se-enriched meat for human consumption by manipulating animal feed therefore also protects the quality of meat [68]. In this respect, [69] showed that inclusion of Se in poultry diets provides Se-enriched meat and protects the meat from oxidation after slaughter, increasing the stability of the meat against various storage conditions which accelerate the oxidation processes that destroy membrane lipids, consequently reducing the meat's nutritional value [70]. Similarly, higher protection of muscle samples against lipid oxidation have been demonstrated by Se yeast with broilers [61] and turkey meat [71]. In addition, the breeders' diet supplemented with Se also provides antioxidant protection of lipid rich tissues, which was detected by lower TBARS values after slaughter [72]. Calvo et al. [73] found that birds supplemented with organic Se had lower malondialdehyde (MDA) concentrations in muscle samples than the SeS group with the same storage time. In agreement with our results on muscle pH, it has been reported that the pH reduction could accelerate lipid oxidation due to the enhanced autoxidation of hemoglobin at reduced pH [74]. With decreasing muscle pH, higher TBARS values have been reported [75].

#### *3.8. Total Antioxidant Capacity of Meat*

The presence of antioxidants in poultry meat is a powerful factor influencing its quality. Once antioxidant defense systems are debilitated, dysfunction of all body cells and tissues may occur. Thus to keep body functions optimal, antioxidant levels are important [76]. As Se plays major role in protecting cells against oxidative stress, measuring the antioxidant biomarkers is a beneficial tool for evaluating the Se antioxidative role. In the present study ABTS, DPPH, and FRAP assays were used to estimate antioxidant capacities, as theses assay reflect the antioxidant properties of meat [77].

DPPH Assay: Thigh meat was characterized by significantly higher DPPH free radical scavenging ability than breast meat. The supplementation of nano-Se and organic Se at higher levels (0.6 mg/kg) into the Ross broiler diet increased the ability of meat to scavenge free radical DPPH and this capacity increased with the storage period (Table 4). Using specific sources from selenium in poultry diet increases the meat's ability to scavenge the free radical DPPH, due to Se antioxidative functions. During frozen storage, the removal ability of DPPH augmented in all examined samples of chicken meat, demonstrating that Se is stable in the meat [78].

ABTS Assay: The ability of breast and thigh meat to scavenge free radical ABTS were affected by dietary inclusion of Met-Se and nano-Se up to 0.6 mg/kg. During frozen storage, the ability of the meat parts to remove the free radical ABTS tended to increase, reaching the highest values after four weeks of storage (Table 4). These results agree with [78], who stated a higher antioxidative potential of chickens breast to remove free radicals tended to increase during frozen storage, reaching the highest values after storage period of 90 days. This can be accompanied by moisture loss as a result of evaporation, besides alterations in proteins structure and lipids due to oxidation progressions. Also, implementation of Met-Se and nano-Se to chicken diets significantly rises the breast's ability and thigh tissues to scavenge the synthetic free radical ABTS when compared with SeS.

FRAP Assay: In general, the capacity of the thigh myofibrillar protein to reduce Fe3+ to Fe2+ was higher than in breast myofibrillar protein. In the first three hours, dietary inclusion of 0.45 and 0.6 mg/kg diet of Met-Se and nano-Se had the same reducing capacity of Fe3+ to Fe2+, while after four weeks the reducing capacity of Fe3+ to Fe2+ was more prominent in breast meat and thigh meat for groups supplemented with nano-Se. It is well understood that Se is vital for the intra- and extra-cellular antioxidant systems of the body [79]. Selenium is also effective in delaying post-mortem oxidation responses [15]. The association between meat quality and oxidation resistance of muscle is well recognized. Huff-Lonergan et al. [58] described that changes in the antioxidant defense system of animals and muscles would affect water-holding capacity, meat proteolysis and calpain activity, thus quality characteristics of meat. In former studies, the water-holding capacity and chicken muscles color were enhanced by dietary Se addition [80]. Se application to chicken diets causes a significant increase in the iron reduction ability for both sets of the leg and back muscles, which can be associated with the higher Se retention in the lipids-rich parts [78]. Li et al. [81] described how total protein solubility, pH at 45 min, and myofibrillar protein solubility decreased while cooking loss was improved after feeding broiler chickens 0.3 mg/kg of either Met-Se or nano-Se as compared with SeS. Muscle proteins comprise connective tissue, sarcoplasmic and myofibrillar, [82]. Protein solubility resulted from protein denaturation during muscle ageing. In addition, denaturation of muscle protein is associated with antioxidant capacity [83]. When muscle amino acids as cysteine, tryptophan are oxidized, disulfide bonds and carbonyl are produced. At that time, the protein structure is destroyed, which would decrease the solubility of protein [84]. Current study, revealed significant increases in the iron reduction capacity which can be related to higher deposition of Met-Se and nano-Se in breast and thigh of chickens specially when supplemented with higher dose (0.6 mg /kg diet) compared with SeS supplementation, which could be a consequence of improved antioxidant capacity.

SeS = Sodium selenite; Met-Se =

selenomethionine;

 nano-Se =

nano-selenium;

 means within a row carrying different superscript letters denote significant difference (*p* < 0.05).

#### **4. Conclusions**

Our results suggested that in Ross broiler chickens, dietary supplementation of either Met-Se or nano-Se up to 0.6 mg/kg increased their performance and was more efficiently retained in the body than SeS. In addition, under stress the antioxidant resistance of broilers fed selected higher levels of Met-Se or nano-Se was enhanced. Moreover, frozen stored meat quality was improved in a dose-dependent manner with both Met-Se and nano-Se. Nano-Se was more potent than Met-Se, which in turn was more potent than inorganic Se against oxidative stress, which improved the quality of meat under frozen conditions.

**Author Contributions:** Data curation, A.T.Y.K., and A.H.A.; Formal analysis, D.I.; Funding acquisition, A.S.A., G.I., A.E.-R. and M.T.E.; Investigation, H.A.M.; Methodology, D.I., A.T.Y.K., and S.I.K.; Project administration, S.I.K.; Resources, A.T.Y.K.; Software, A.T.Y.K., and A.H.A.; Writing—original draft, D.I.; Writing—review & editing, A.T.Y.K.

**Funding:** This research received no external funding.

**Conflicts of Interest:** The authors declare no conflicts of interest.

#### **References**

© 2019 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (http://creativecommons.org/licenses/by/4.0/).

*Article*

### **E**ff**ect of In Ovo Injection of L-Arginine in Di**ff**erent Chicken Embryonic Development Stages on Post-Hatchability, Immune Response, and Myo-D and Myogenin Proteins**

**Sivakumar Allur Subramaniyan 1, Da Rae Kang 1, Jin Ryong Park 1, Sharif Hasan Siddiqui 1, Palanisamy Ravichandiran 2,3, Dong Jin Yoo 2,3, Chong Sam Na 1,\* and Kwan Seob Shim 1,\***

Received: 8 May 2019; Accepted: 5 June 2019; Published: 14 June 2019

**Simple Summary:** In the current study, we hypothesized that the in ovo injection of L-arginine (L-Arg) at different stages of embryonic development, which would have positive effects on the survival rate, hatching rate, immunoglobulin M (IgM) levels, heat shock proteins (HSPs) such as HSP-47, HSP-60, and HSP-70, and muscle development markers as well: Mainly, myoblast determination protein (myoD) and myogenin in pectoral muscles. As indicated, the in ovo injection of L-Arg resulted in an increased hatch rate and weight, survival rate, higher levels of IgM, and myogenin and MyoD expression in the muscles. At the same time, a decrease in the level of HSP-47, HSP-60, and HSP-70 expressions in the tissues was observed on the 14th day of injection compared to the eighth and 18th day of the injection period. In addition, the in ovo injection of L-Arg decreased the serum glutamate oxaloacetate transaminase (SGOT) and serum glutamate pyruvate transaminase (SGPT) concentration in serum as well micronuclei and nuclear abnormality in the blood on the 14th day of the incubation period. Hence, the 14th day would be a suitable day for the injection of L-Arg to promote the hatching rate and muscle growth of broiler chickens.

**Abstract:** The aim of this study was to evaluate the effect of in ovo injection with different ratios of L-arginine (L-Arg) into Ross broiler eggs at three different embryonic developmental stages (eighth day (d), 14th day, and 18th day) on the survival, hatchability, and body weight (BW) of one-day-old hatched chicks. Additionally, we have analyzed the levels of serum glutamate oxaloacetate transaminase (SGOT) and serum glutamate pyruvate transaminase (SGPT), the protein expression of heat shock proteins (HSPs), and we have also determined micronuclei (MN) and nuclear abnormality (NA). In addition, the genotoxic effect was observed in peripheral blood cells such as the presence of micronuclei and nuclear abnormalities in the experimental groups. The results showed that survival and hatching rates as well as body weight were increased on the 14th day of incubation compared to the eighth and 18th day of incubation at lower concentrations of L-Arg. Moreover, the levels of SGOT and SGPT were also significantly (*p* < 0.05) increased on the 14th day of incubation at the same concentration (100 μg/μL/egg) of injection. In addition, immunoglobulin (IgM) levels were increased on the 14th day of incubation compared to other days. The protein expressions of HSP-47, HSP-60, and HSP-70 in the liver were significantly down-regulated, whereas the expression of myogenin and myoblast determination protein (MyoD) were significantly up-regulated on the 14th day after

incubation when treated with all different doses such as 100 μg, 1000 μg, and 2500 μg/μL/egg, namely 3T1, 3T2, and 3T3, respectively. However, the treatment with low doses of L-Arg down-regulated the expression levels of those proteins on the 14th day of incubation. Histopathology of the liver by hematoxylin and eosin (H&E) staining showed that the majority of liver damage, specifically intracytoplasmic vacuoles, were observed in the 3T1, 3T2, and 3T3 groups. The minimum dose of 100 μg/mL/egg on the 14th day of incubation significantly prevented intracytoplasmic vacuole damages. These results demonstrate that in ovo administration of L-Arg at (100 μg/μL/egg) may be an effective method to increase chick BW, hatch rate, muscle growth-related proteins, and promote the immune response through increasing IgM on the 14th day of the incubation period.

**Keywords:** embryonic development; heat shock proteins; immunoglobulin; intracytoplasmic vacuoles; L-arginine

#### **1. Introduction**

The selection of chickens (*Gallus gallus*) for meat production has led to the generation of inbred strains that show accelerated growth performance, particularly enhanced muscle growth that mostly occurs during embryogenesis [1,2]. During embryogenesis, nutrients and energy are mainly acquired from yolk, which mainly contains lipids and low levels of carbohydrates [3]. Subsequently, the health of the embryo and post-hatch chicken depends on gluconeogenesis from essential amino acids [4,5]. In recent years, researchers have found that the administration of amino acids into fertilized broiler eggs, which is called in ovo feeding, may provide poultry companies with an alternative method to increase the hatchability and muscle growth weight of newly hatched chicks [6,7]. The supplementation of nutrients into fertilized broiler eggs influences embryo development and growth during incubation and the post-hatch growth performance of chicks [7]. The nourishment and supplementation with bioactive substances such as bioactive amino acids, polyphenols, and prebiotics can enhance the immune system, decrease osteoporosis, and decrease the risk of heart diseases [8]. Similarly, previous reports have indicated that the amino acids, carbohydrates, and vitamins that are applied to eggs through in ovo feeding can improve the hatching rate, body weight, survival rate, growth performance, and marketing size [9]. Moreover, an earlier study demonstrated that the in ovo feeding site and time can affect hatchability [10].

During embryonic development, the chorioallantoic membrane develops, which can vascularize on the 12th day of the incubation period. Moreover, the embryo is surrounded by the amniotic fluid, which remains in contact with the embryonic gastrointestinal tract and enables the transport of substances from the air chamber into the intestine [11]. Several genes are associated with cellular interactions and differentiation during the organogenesis of the eye, ear, brain, skin, and tissues such as bones and cartilages; the expression of those genes is either transient or initiated during later stages of embryogenesis [12]. Some authors have indicated that the injection of amino acids into the egg on the first day is sufficient to fully support embryonic development [13,14], leading to increased hatching and breast weight [15]. It has been demonstrated that the injection of sucrose and dextrin into chicken embryos can result in a greater percentage of pectoral muscle weight than the control [5,16]. Recently, it has been reported that chicken embryos injected with L-glutamine on the first day of incubation can increase the fiber area, pectoral muscle mass, and endothelial cell proliferation while stimulating vasculogenesis and angiogenesis [17]. The in ovo administration of amino acids or peptides increases the expression levels of MyoD1 and paired box protein 7 (Pax7), which are necessary for muscle growth during embryogenesis [18].

Standardization of the injection site, needle length, and embryonic age using amino acid (Lys + Met + Cys, Thr + Gly + Ser or Ile + leu + Val) with 11-mm and 24 mm-needles on the seventh and 14th day of incubation has resulted in poor hatchability and poor muscle growth markers [7]. An in ovo injection

of glutamine in conjugation with (silver nanoparticles) Ag NPs on the first day of chicken embryos increased the muscle mass [19], and L-arginine (L-Arg) in one-day-old quail embryos increased the hatchability and growth performance [20]. In our study, we checked different time intervals (eighth day, 14th day, and 18th day) and different doses of L-Ar (100, 1000, and 2500 μg/100 μL/egg) for responses related to the survival rate, hatchability, body weight, and muscle growth-related proteins such as myogenin and MyoD and immunoglobulin M (IgM) levels.

#### **2. Materials and Methods**

#### *2.1. Ethics Statement*

The experimental protocol was approved by the Institutional Animal Care and Use Committee (IACUC) of Chonbuk National University, with the project number 2017R1D1A1B03032217. Animal care and handling are in compliance with the regulations of the IAEC Guidelines for the Euthanasia of Animals: 2015 Edition. The sampling procedures complied with the "Guidelines on Ethical Treatment of Experimental Animals" (2015) No. CBNU 2015048 set by the Ministry of Science and Technology, Korea.

#### *2.2. Chemicals*

L-arginine, hematoxylin and eosin (H&E), and periodic acid-Schiff's (PAS) were purchased from Sigma-Aldrich (Sigma-Aldrich, St. Louis, MO, USA). Chemiluminescent for band detection was purchased from Thermo Scientific (Rockford, IL, USA). Antibodies were purchased from ENZO Life Science (Farmingdale, NY, USA). All the laboratory glassware was acquired from Falcon Lab ware (Becton, Dickinson and Company, Franklin Lakes, NJ, USA).

#### *2.3. Experimental Design and Incubation*

Ross 1040 broiler chicken eggs were obtained from Samhwa-Won Jong, South Korea. On the first day of incubation, eggs were weighed (60 ± 1.36 g) and separated into different groups. Eggs were randomly divided into 13 groups (4 × 20 × 3 = replication × eggs × injection) as described in Table 1. L-Arg was injected at three concentrations (100 μg, 1000 μg, and 2500 μg/100μL/egg) on the eighth, 14th, and 18th day of the incubation period, respectively. A 0.100-mL of L-Arg (1% PBS) was injected. Immediately after the injection, the hole was sealed with liquid paraffin. Then, eggs were placed in an incubator for 20 days under standard conditions (temperature, 37.8 ◦C; humidity, 60%). On the 18th day, eggs were transferred to hatching boxes and promptly placed in a hatcher incubator with humidity maintained at 60% and temperature set at 37 ◦C to hatch chicks.

**Table 1.** Experimental design for dose (L-Arg) and embronic stage (eighth day, 14th day, and 18th day) fixation.

Note: In Ovo Injection and Treatment Groups.

#### *2.4. Survival Rate Measurement*

Embryos' survival rates during the incubation period were measured on the eighth day. Treated eggs were checked to determine the number of live and dead eggs as well as fertilized and non-fertilized ones among the total number of eggs. At 18th day of incubation, after injection, the eggs live eggs were moved to another hatching incubator with their respective experimental group. The survival rate was calculated with the following Equation (1):

$$\text{Survival rate } \%= \frac{\text{No. of live eggs}}{\text{No. of fertilized eggs}} \times 100\tag{1}$$

#### *2.5. Hatching Rate and Body Weight Measurements*

On the 21st day, hatched chicks were moved from the hatcher incubator to hatching boxes to determine hatching rates. These hatched chicks were kept without food and water at 32 ◦C. Then, they were weighed to record their live body weights. The hatching rate was calculated with the following Equation (2):

$$\text{Hatching rate } \%= \frac{\text{No. of chicks matched on 21st day}}{\text{No. of fertilized eggs that were in } ovo \text{ fed}} \times 100\tag{2}$$

#### *2.6. Biochemical Indices*

At the end of the experimental period, the hatched chicks were sacrificed under anesthesia (diethyl ether). Blood was collected from the jugular vein in tubes for serum separation. A small amount of collected blood was immediately smeared on clean grease-free microscope slides and air-dried for micronuclei (MN) and nuclear abnormality (NA). The breast muscle and liver were collected and washed in ice-cold saline for further study. The body was cut opened; muscle and liver samples were excised, washed with ice-cold saline, and then homogenized with 0.1 M of cold phosphate buffer, pH 7.4. Concentrations of serum glutamate pyruvate transaminase (SGPT) and erum glutamate oxaloacetate transaminase (SGOT) in serum were measured using commercial kits (Asan Pharamaceuticals Co., Ltd., Seoul, Korea).

#### *2.7. Micronuclei (MN) and Nuclear Abnormality (NA) Tests Using Periodic Acid Schi*ff*'s (PAS) Staining*

MN and NA were assayed in the liver by standard methods presented elsewhere [21,22]. Blood samples collected from the first day of the hatching period were immediately smeared on clean grease-free microscope slides and air dried. Afterwards, slides were fixed with methanol for 5 min at room temperature, gently rinsed with running tap water for 1 min, and immersed in a periodic acid solution for 5 min at room temperature. Then, these slides were rinsed using DH2O, immersed in PAS Schiff's reagent for 15 min at room temperature, and gently washed with running tap water for 5 min. Finally, counter-staining was performed with a hematoxylin solution for 90 s. Then, slides were rinsed in running tap water for 30 s, air dried, and examined with a light microscope (100×) using immersion oil.

#### *2.8. Measurement of IgM Concentration in Serum*

Serum samples were collected from individual experimental animals to determine serum immunoglobulin (Ig) M levels using chicken IgM ELISA kit (Abcam, Suite B2304, Cambridge, MA 02139-1517, USA) following the manufacture's specification. IgM levels were analyzed based on absorbance values measured at 450 nm.

#### *2.9. Analysis of Heat-Shock Proteins (HSPs) by Western Blot*

Proteins were extracted from 100 mg of muscle samples using radioimmunoprecipitation assay (RIPA) buffer to determine the protein expression levels of HSP-47, HSP-60, HSP-70, myoD, and myogenin in experimental groups. Protein concentrations were determined using a BIO-RAD protein assay kit (BIO-RAD). Extract samples containing 50 μg of protein were solubilized in *Laemmli bu*ff*er,* separated by 12% acrylamide gel, and then transferred to Hybond-P PVDF membranes (GE Healthcare Inc., Amersham, UK) for 60 min at 200 mA. Then, these PVDF membranes were blocked with 5% skimmed milk powder in 0.5 M of Tris-buffered saline (pH 7.4) with 0.05% Tween 20 (TBST) at room temperature for 2 h. Western immunoblotting with HSP-47, HSP-60, HSP-70, Myo-D, and myogenin are primary antibodies (1:2500 dilution) took place overnight. After washing three times with TBST, these membranes were probed with HRP-conjugated secondary antibodies (1:5000 dilutions) for 60 min at room temperature, and then washed three times with TBST (10 min each wash). Protein bands were visualized using a Chemiluminescent assay kit from Thermo Scientific for 1–5 min. Bands were imaged with an iBright™ CL1000 Imaging System (Invitrogen in Thermo Fisher Scientific, Life Technologies Korea LLC, Jeonju-si, Jeollabuk-do, Korea) and quantified using Image J Software. The relative density of the band was normalized to that of β-actin as an internal control.

#### *2.10. In Silico Molecular Docking Studies*

To understand the mechanism of interaction of L-Arg with heat shock protein, crystal structures of GroEL mutant A109C (PDB ID: 5OPW) [23] and human HSP70 substrate binding domain L542Y mutant (PDB ID: 5XIR) [24] were downloaded from the Protein Data Bank. Molecular docking studies were performed using the GLIDE program [25] (Version 8.5, Schrodinger LLC, New York, NY, USA). To analyze docking results and execute the protocol, the Maestro user interface (Version 8.5, Schrodinger LLC, New York, NY, USA) was employed. Validation of the protocol was performed by redocking. The structure of L-Arg was sketched using ACD/chemsketch (Freeware version). The GLIDE grid generation wizard was used to define the docking space. Docking was performed using XP (Extra Precision mode) docking protocol.

#### *2.11. Histopathological Study of the Liver*

Livers were collected after chickens were sacrificed, immediately fixed with 10% neutral buffered formalin (NBF), and processed in an auto processor (Excelsior ES, Thermo Scientific, Waltham, MA, USA). After embedding in paraffin, 5-μm sections were made and subjected to H&E staining. Digital images were obtained using a Leica DM2500 microscope (Leica Microsystems, Wetzlar, Germany) at fixed 100× (200×) magnification.

#### *2.12. Statistical Analysis*

All the values are presented as mean ± SD from 12 determinations from each group and statistically analyzed using Duncan test following ANOVAs with SAS® software, version 9.4, (Institute of INC, North Carolina, USA).

#### **3. Results and Discussion**

#### *3.1. Survival Rate and Hatchability*

Survival rate was significantly (*p* < 0.05) increased in the 2T1 and 3T1 groups than that in other groups. The lowest survival rate was observed in 1T3, 3T2, and 3T3 groups (Figure 1). These results showed that the survival rates differed depending upon the injection period and the concentration of L-Arg. Embryos may utilize in ovo administered amino acids to improve energy status and save muscle protein to improve their enteric development, hatching, and survival rate [26]. In our study, the same mechanism might have occurred; the administration of L-Arg could improve the survival

rate at the minimal concentration (3T1) on the 14th day of the injection period (Figure 1). However, during incubation, an excess of amino acids such as glycine and proline failed to improve embryo development [27]. The same attributes could have been observed in our current study: that a maximum concentration of L-Arg affects the embryonic growth.

**Figure 1.** Effects of in ovo injections at different concentrations of L-arginine (L-Arg) with different developmental embryonic stages on survival rate. Small characters indicate significant differences among experimental groups at *p* < 0.01. Values are presented as mean ± SD from 12 determinations. Data were analyzed using Duncan test following ANOVAs with SAS® software, version 9.4, (Institute of INC, North Carolina, USA).

Different concentrations of L-Arg injected in embryos can influence biological molecules and toxicity during embryogenesis. However, several studies have reported that higher doses of L-Arg become toxic, which can cause significantly increased mortality rates and impaired weight gain, whereas chicks injected with lower concentrations of L-Arg (1.0%) showed better growth performance than those injected with a higher concentration (1.5%) of L-Arg [28]. In a parallel effect revealed in our current study to a lower concentration of L-Arg (2T1) on the 14th day of injection increasing the hatching rate (96.29%), body weight (64.25 g) also increased the survival rate (98.03%) compared to other groups (Figures 1–3). The in ovo injection of L-Arg to late-term embryos can increase the body weight (5% to 6%) compared to controls [5]. In addition, in ovo administration of all 20 different amino acids can increase chick weight by 3.6% and 2.1%, respectively [10]. The in ovo administration of amino acids might have stimulated the utilization of amino acids with a concomitant decrease in the degradation of amino acids by the embryo [29]. In ovo feeding of L-Arg resulted in higher embryo weight due to an increase in muscle mass [30]. The in ovo injection of L-Arg could be utilized by the embryo, resulting in increased muscle mass and a heavy embryo, which can increase the hatching rate [31]. L-Arg may attenuate adverse effects of rearing chickens under cold ambient temperatures or at high altitudes [32]. Furthermore, feeding broiler chickens with a diet that is deficient in L-Arg under cold stress at high altitudes can depress nitric oxide synthesis, decrease feed intake, reduce body weight gain, and increase the right ventricle to total ventricle weight ratio, mortality rate, and ascites mortality [31]. A previous study reported that a lower percentage (1.36%) of L-Arg supplemented to broiler eggs was more easily digestible than a higher percentage of arginine, and it could obtain the highest egg weight [33]. Albeit, a low dose of L-Arg stimulates the secretion of the growth hormone, which could increase the body weight [21]. The same mechanism might occur in our present study; L-Arg appeared to improve the body weight of chicks in group 2T1 (Figure 3). However, the body weights did not significantly vary among the other groups. Hatchability, gut microflora population, immune-related gene expression, and muscle fiber increased as a result of the 12–14-days in ovo injection of various substances such as Raffinose, *Lactococcus lactis*, and *Silybum marianum* extract [34–36]. Hence, a low dose (2T1) of L-Arg injected on the 14th day of the incubation period could improve body weight;

the reason behind this might be that the lower concentration of L-Arg may stimulate the growth hormones in the middle stages of embryonic development when compared to the early (eighth day) and late (18th day) embryonic stages.

**Figure 2.** Effects of in ovo injections at different concentrations of L-Arg with different developmental embryonic stages on hatching rates. Small characters indicate significant differences among experimental groups at *p* < 0.01. Values are presented as mean ± SD from 12 determinations. Data were analyzed using Duncan test following ANOVAs with SAS® software, version 9.4, (Institute of INC, North Carolina, USA).

**Figure 3.** Effects of in ovo injections at different concentrations of L-Arg with different developmental embryonic stages on Chicks weight. Small characters indicate significant differences among experimental groups at *p* < 0.01. Values are presented as mean ± SD from 12 determinations. Data were analyzed using Duncan test following ANOVAs with SAS® software, version 9.4, (Institute of INC, North Carolina, USA).

#### *3.2. Biochemical Indices (SGOT and SGPT)*

Elevated SGOT and SGPT levels indicate improper liver function due to damages of the cell integrity and cell membrane in the liver. Our results revealed that the injection of L-Arg at all doses except the lower dose affected SGOT and SGPT levels on the eighth and 18th-day embryonic stages (Figure 6A). SGOT and SGPT levels were significantly decreased in the 2T1 and 2T2 groups of embryos compared to 2T3, 3C1, 3T1, 3T2 and 3T3. Increased levels of SGOT and SGPT in the blood are conducive to liver function damage [37–39]. In fact, free radicals can attack hepatocytes and release stored SGPT to re-enter the blood serum [40]. A lower concentration of L-Arg supplementation caused a greater percentage reduction in SGOT and SGPT levels in sickle cell anemia subjects [39]. The supplementation of L-Arg to mice in higher concentrations showed that increased SGOT and SGPT levels had been linked to damage to hepatic cells and hemolysis [41]. The cause of liver damage is unclear. Hence, confirming that a higher concentration of L-Arg might have damaged the hepatic cell through the elevation of SGOT and SGPT in the 1T1, 1T2, 1T3, 3T1, 3T2 and 3T3 groups. On the other hand, the 2T1 group of injected chicken embryos could re-back the SGOT and SGPT levels compared to the other groups. Stimulating the action of nitric oxide (NO) production by L-Arg results showed that it improved the degree of the hepatocellular structure by blocking of B-cell lymphoma-2 (Bcl-2) and tumor necrosis factor-α (TNF-α) [42]. In addition, L-Arg at 1 g/day decreased the liver enzymes such as SGOT and SGPT through increasing the nitric oxide (NO) synthesis. NO synthase plays an important role in liver injury through inducible nitric oxide synthase (iNOS) pathways [43]. The same mechanism could be involved in our current study, too. This same mechanism that might have occurred in our study could be that the production of NO reduces necrosis and apoptosis by attenuation of the inflammatory pathway, which in turned prevented the hepatotoxicity. Moreover, it also improved the hepatobiliary function, and the ultrastructure of liver results reduced the SGOT and SGPT levels in L-Arg treatment in the lower dose (2T1) on the 14th day injection of embryos (Figure 4).

#### *3.3. Micronuclei (MN) and Nuclear Abnormality (NA) Tests Using Periodic Acid-Schi*ff*'s (PAS) Staining*

The wide use of different doses of L-Arg at three different incubation periods requires examining the genotoxic activity in peripheral blood by the method of [44]. MN and NA tests were conducted to examine peripheral blood cells in all the groups of experimental chicks (Figure 5). The MN test can measure subcellular processes of chromosomal breaks (clastogenesis) or cell spindle malfunctions (aneugenesis) as well as the formation of mitochondrial disruption and nuclear DNA, which can lead to mitochondria-dependent apoptosis in chicken embryos as an indicator of chromosomal damage [45]. Similar results were obtained in our current experiment: the MN and NA in peripheral blood erythrocytes were observed, which clearly demonstrates the higher genotoxicity of a high dose of L-Arg on the eighth, 14th, and 18th day of the incubation period. Moreover, the 2T1 and 2T2 groups showed a normal architecture of nuclei in peripheral blood cells, which was similar to the control group. Figure 5 shows marked inflammation around the periportal region with microvesicular and macrovesicular fatty infiltration (yellow arrows).

**Figure 5.** Photomicrographs of erythrocytes with normal nuclei in the peripheral blood cells of experimental groups. Micronuclei and nuclear abnormalities such as blebbed nuclei or lobed nuclei are indicated by an arrow ("→").

#### *3.4. Protein Analysis by Western Blot*

Western blot was performed in muscles to determine whether the different doses of L-Arg supplemented at various days of the incubation period may alter the protein levels of the HSP family such as HSP-47, HSP-60, and HSP-70. As shown in Figure 6, the protein expressions of HSP-47, HSP-60, and HSP-70 were significantly (*p* < 0.01) down-regulated in the 2T1 group compared to other groups. Moreover, their levels in the 3T2 and 3T3 groups were significantly (0.01) up-regulated compared to the 2T1 and 2T2 groups, although the protein expressions of HSP-60 and HSP-70 showed no significant difference (*p* < 0.01) among the 1T1, 1T2, 1T3, and 2T1 groups. Moreover, HSP-46, HSP-60, and HSP-70 were down-regulated in the 2T1 group compared to those in the other groups, whereas there was no significant difference in their levels between 1C and 2T1. HSP-70 is a reliable index of stress in chickens, while "3-hydroxyl-3-methyl-glutaryl coenzyme A reductase" has been used as an indicator of stress [46]. Pretreatment with L-Arg markedly reduced the dramatic down-regulation of HSP-60 and HSP-70 in hypoxic rat model. The increased expression of HSP-60 and HSP-70 might be related to their leakages from tissue, which can cause tissue injury due to free radical production [47,48]. Tissue injury might be caused by nitric oxide, a free radical, through the stimulation of endothelial cells and neutrophils that is generated from a higher dose of L-Arg [49]. Hence, the present results may suggest that the increased levels of HSP-47, HSP-60, and HSP70 in high doses of L-Arg may have a major role in tissue injury. The results of the study show that the increase of HSP-60 and 70 may be involved in tissue injury in the 3T1, 3T2, and 3T3 groups due to free radical production. The 2T1 group can prevent tissue injury via the down-regulation of HSP-46, HSP-60, and HSP-70. Moreover, the protein expressions of myogenin and MyoD were significantly up-regulated in the 2T1 group, whereas they were down-regulated in the 3T1, 3T2, and 3T3 groups compared to the other experimental groups.

**Figure 6.** (**A**) Effects of expression levels of L-Arg, heat shock protein (HSP)-47, HSP-60, and HSP-70 as well as myogenin and myoblast determination (MyoD) protein expressions in different stages of chicken embryos at different doses. Small characters indicate significant differences among experimental groups at *p* < 0.01. (**B**) The bar graph represents the quantitative expression of different proteins in all the groups. Data are expressed as the ratio of relative intensity with β-actin. Values are presented as mean ± SD from 12 determinations.

Oxidative stress can cause muscle atrophy by reducing myogenic differentiation markers such as myogenin and MyoD in skeletal muscles [50]. Some growth factors, namely cytokines and oncogenes, suppress the activity of myogenin and MyoD, thus resulting in decreased in the mass of muscle, which is defined as muscle atrophy [51]. A previous study reported that myogenic regulatory factors—mainly MyoD and MRF4—are only expressed later in different embryonic muscle groups as a result of increased muscle mass [52]. L-Arg increased the muscle cell as well as myogenin and MyoD under oxidative stress. Moreover, results from a previous experiment demonstrated that lower doses of

L-Arg could promote HSP70 expression in pig intestine [53]. Nevertheless, our present study has proved that increasing the concentration of L-Arg on the eighth and 18th day of the injection period could up-regulate the expression of HSP-60 and HSP-70; this effect might be through whey protein hydrolysate, which indicates the improper use of a functional food ingredient. Moreover, the L-Arg on the 14th day with (100 μg/100 μL/egg) promoted the muscle mass through the up-regulation of MyoD and myogenin due to their free radical scavenging activity.

#### *3.5. Measurement of IgM Concentration in Serum*

Concentrations of immune response markers such as IgM in all the experimental groups were analyzed. The duration and amount of L-Arg supplementation may influence immune status. Short-term supplementary L-Arg can influence the immunity power, because L-Arg has antioxidant and anti-inflammatory effects [54,55]. It can attenuate inflammatory reactions by suppressing the generation of inflammatory mediators such as inflammatory cytokines and C-reactive protein, which play major roles in the progression of tissue damage and organ dysfunction [56]. The treatment of L-Arg shows improved renal function through improved immune function [57]. Levels of IgM could provide an overall picture of immune function. It has been recently demonstrated that L-Arg can increase the specific immune response against infectious bursal disease (IBD) in chickens [58]. L-Arg provided by treatment has been reported to be the sole precursor of nitric oxide with lots of immune functions and growth performance [59]. These same biological attributes might be present after a low dose of L-Arg injection on the 14th day of the incubation period. It may improve immunity via the generation of IgM and the suppression of inflammatory cytokines and C-reactive protein (Figure 7).

**Figure 7.** L-Arg induces immunoglobulin M (IgM) levels in different stages of chicken embryos at different doses. Small characters indicate significant differences among experimental groups at *p* < 0.01. Values are presented as mean ± SD from 12 determinations. Data were analyzed using Duncan test following ANOVAs with SAS® software, Version 9.4, (Institute of INC, North Carolina, USA).

#### *3.6. Histopathology (H&E) Staining*

Figure 8 shows the histology of liver of all the experimental groups. Sections from the control group exhibited a complete structure and regular shape of liver cells. Sections from the 1T1 and 2T1 groups showed normal hepatocyte gap compared to the 1C and 1C1 groups. Sections from the 1T3, 2T3, 3T1, 3T2, and 3T3 groups appeared with intracytoplasmic vacuoles in hepatocytes around the centrilobular regions. Moreover, hepatocyte tubes were surrounded by inflammatory cells and showed necrosis with nuclear fragmentation in the 3T2 and 3T3 groups. The hepatocyte gap was increased in the 2T3, 3T1, 3T2, and 3T3 groups. The hepatocyte gap appeared in normal architecture in the 1C, 1C1, 2C1, 3C1, 1T1, and 2T2 groups. The degeneration of livers was observed for birds when treated with 167 and 334 mg/L of L-Arg, which had an adverse effects on organs [60]. The liver after treatment with L-Arg (334 mg/L) had congested vascular spaces and periportal mononuclear inflammatory infiltration [61]. The addition of L-Arg to poultry diets is required to avoid harmful influences of excessive free radicals produced during normal metabolism [62]. Dietary L-Arg supplementation plays a key role in enhancing meat quality.  Additionally, supplementation with high doses (50% and 100%) of L-Arg has negative effects on the structure of the liver of Sasso birds proved by H&E staining. However, our current results showed that in ovo injection with low doses (2T1) of L-Arg on the 14th day of egg embryo development did not have any negative effects compared to higher doses of L-Arg on the eighth or 18th day of the incubation period.

**Figure 8.** Histopathology of liver using hematoxylin and eosin straining. Sections from control chicks' hepatic lobule indicate complete structures. The liver cell has a regular shape that is within normal limits. Intracytoplasmic vacuoles are shown in hepatocytes around the centrilobular region in the 1T3, 2T3, 3T1, 3T2, and 3T3 groups. Hepatocyte marked inflammation around the periportal region, with microvesicular and macrovesicular fatty infiltration (yellow arrows) liver cells appearing near necrosis with nuclear fragmentation in the 3T2 and 3T3 groups. The hepatocyte gap was also increased in the 2T3 3T1, 3T2, and 3T3 groups. There was no difference between the control and the 2T1 group.

#### *3.7. In Silico Molecular Docking Studies*

The in silico molecular docking of L-Arg was studied. The entire glide, E model scores, and hydrogen bond interactions are presented in Figure 9. The main aim of the molecular modeling study is to understand the interactions of functional groups present in L-Arg with residues of targeted proteins theoretically. Heat shock protein 70 (HSP70) is one of the main nonglobin proteins, which has a similar structure in almost all living organisms. Between organisms as varied as yeast, chicken, Drosophila, and human, HSP70 is highly conserved. A previous study has described that HSPs are antigenically linked to the chicken HSPs by means of rabbit polyclonal antibodies [25]. In the same way, the similarity of HSPs extends to a DNA sequence. The chicken and human HSP70 genes are 64–72% similar (homologous) in the obtained amino acid sequences [65]. In addition, the HSP70 gene(s) of chicken, Drosophila, mouse, and human are very much conserved with the comparison of the DNA sequence [66,67]. Therefore, in the present study, solution structure for the human HSP70 substrate binding domain L542Y mutant (PDB id: 5XIR) was selected for the in silico molecular modeling study with L-arginine [68].

**Figure 9.** (**A**) 2D docking interaction of L-arginine with the active site of 5XIR. (**B**) 3D docking of L-arginine in the active site of 5XIR. (**C**) Docking packing representation of L-arginine with suitable binding pockets of 5XIR.

As presented in Figure 9, the main aim of the molecular modelling study is to understand interactions of functional groups present in L-Arg with residues of targeted proteins theoretically. In L-Arg, primary and secondary amines have strong hydrogen bonding interactions with protein residues. In brief, the molecular interaction of L-Arg with 5XIR protein and primary and secondary amines established a strong affinity with protein resides such as ASP A 328, ASP A 83, and HIS A 401. In addition, the carboxyl group present in the molecule also exhibits good hydrogen bonding interactions with LYS A 498. In a similar way, L-arginine with 5XIR protein showed good molecular interactions with ASP A 54, PHE A 49, GLU A 25, THR A 26 and GLY A 29 (Figure 9A).

The molecular modelling study revealed that L-Arg was bound onto similar active site cavity in the protein molecule. Superposition of interactions of an active site of L-Arg and amino acid residues of 5XIR protein is evidently portrayed in Figure 9B,C. The molecular docking of L-Arg with 5XIR protein is exposed to seven hydrogen bonding interactions, respectively with corresponding active site of the protein molecule. Besides, L-Arg showed strong hydrogen bonding interactions with good surface molecular interactions due to the presence of primary, secondary amines, and carboxylic acid groups. These relationships among 5XIR protein, and L-Arg might explain the experimental activity of them. Further research is going on in a due course to explore their possible modes of action. It is possible that L-Arg might block the activity of HSPs, and activation of Myogenin and Myo-D as well improved the immunoglobulin levels, thereby regulating the muscle growth (Figure 10).

**Figure 10.** Possible mechanism of high doses and low doses of L-Arg on toxicity and muscle growth in chicken embryo.

#### **4. Conclusions**

In this study, we described a suitable embryonic developmental stage for the accessibility of in ovo injection using L-Arg at different concentrations for the first time. The injection of L-Arg on the 14th day at 100 μg/μL/egg enhanced both hatching and survival rates. It also increased body weight and immune response (IgM). In addition to oxidative stress, a sign of genotoxic effect was also observed in peripheral blood cells in which a presence of micronuclei and nuclear abnormalities such as blebbed nuclei, lobed nuclei, and notched nuclei were observed in the 2T3, 3T1, 3T2, and 3T3 groups. Histology from the control and 2T1 groups showed normal architecture, while injection on the 18th day of incubation and first day of chicks showed liver tissue damage. Overall results demonstrate that the optimum dose is 100 μg/μL/egg, and the optimum injection stage is on the 14th day to improve the immunity, hatching, and survival rate, which can be used for the poultry industry. In ovo injection in early and late embryonic stages could not offer good benefits for survival, hatching rate, or muscle development. If we choose the middle stage of embryonic development for in ovo injection, L-Arg might be able to promote muscle growth and improve the immune power without inducing adverse effects on the liver.

**Author Contributions:** Conceived and designed the experiments: S.A.S., K.S.S. Performed the experiments and measurements of serum biochemical parameters: S.A.S., D.R.K., J.R.P. and S.H.S. Protein analysis by western blot: S.A.S. Histopathology (H&E) staining: S.A.S. In silico molecular docking studies: P.R., D.J.Y and C.S.N.

**Funding:** This research was funded by National Research Foundation of Korea: Project No. NRF-2017R1D1A1B03032217 and 2017R1D1A3B03028490.

**Acknowledgments:** This research was supported by the Basic Science Research Program, National Research Foundation of Korea (NRF), Ministry of Education (Project No. NRF-2017R1D1A1B03032217 and 2017R1D1A3B03028490) and the "Research Funds of Chonbuk National University in 2018". We thank to Choi, Eun-Jin, Center for University Research Facility (CURF) at Chonbuk National University.

**Conflicts of Interest:** The authors declare no conflicts of interest.

#### **References**

1. Berri, C.; Wacrenier, N.; Millet, N.; Le Bihan-Duval, E. Effect of selection for improved body composition on muscle and meat characteristics of broilers from experimental and commercial lines. *Poult. Sci.* **2001**, *80*, 833–838. [CrossRef] [PubMed]

© 2019 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (http://creativecommons.org/licenses/by/4.0/).

*Article*

### **E**ff**ects of Dietary Supplementation of L-Carnitine and Excess Lysine-Methionine on Growth Performance, Carcass Characteristics, and Immunity Markers of Broiler Chicken**

**Seyed Mohammad Ghoreyshi 1, Besma Omri 2, Raja Chalghoumi 2, Mehrdad Bouyeh 1, Alireza Seidavi 1,\*, Mohammad Dadashbeiki 3, Massimo Lucarini 4, Alessandra Durazzo 4, Rene van den Hoven 5 and Antonello Santini 6,\***

Received: 18 March 2019; Accepted: 12 June 2019; Published: 16 June 2019

**Simple Summary:** L-carnitine, lysine, and methionine are amino acids of important nutritional and nutraceutical interest and are used as dietary supplements to improve feed quality characteristics in broiler chicken. This study investigated the effect of different levels of L-carnitine and extra levels of lysine-methionine on growth performance, carcass characteristics, and some immune system markers. The findings of this study showed that the diet with around 30% of lysine-methionine content increased the back thoracic vertebrae and the proventriculus weights. A combination of lysine-methionine (level equal to NRC recommendations) with L-carnitine (15% and 75%) improved the immune response of broiler chickens against Newcastle and Gumboro diseases by stimulating the antibody production.

**Abstract:** L-carnitine as well as lysine and methionine are amino acids of important nutritional and nutraceutical interest and are used in nutritional strategies as dietary supplements to improve feed quality characteristics in animals and broiler chicken in particular. This study investigated the effect of different levels of L-carnitine and extra levels of lysine-methionine on growth performance, carcass characteristics, and some immune system markers. Two hundred seventy male Ross 308 broilers were a fed control diet (C) and eight different diets supplemented with an excess of amino acids. In the experimental diets, identified as D1, D2, D3, D4, D5, D6, D7, and D8, extra L-carnitine, lysine, and methionine were added in excess with respect to the American National Research Council (NRC) recommendations: L-carnitine equal to NRC (D1), control diet supplemented with lysine at 30% in excess of NRC, methionine at 30% in excess of NRC, and L-carnitine equal to NRC (D2), control diet supplemented with lysine equal to NRC, methionine equal to NRC, and L-carnitine at 15% in excess of NRC (D3), control diet supplemented control diet supplemented with lysine at 15% in excess of NRC, methionine at 15% in excess of NRC, and L-carnitine at 15% in excess of NRC (D4), control diet supplemented lysine at 30% in excess of NRC, methionine at 30% in excess of NRC, and L-carnitine at

15% in excess of NRC (D5), control diet supplemented with lysine equal to NRC recommendations, methionine equal to NRC recommendations, and L-carnitine at 75% in excess of NRC (D6), control diet supplemented with lysine at 15% in excess of NRC, methionine at 15% in excess of NRC, and L-carnitine at 75% in excess of NRC (D7); and control diet supplemented with lysine at 30% in excess of NRC, methionine at 30% in excess of NRC, and L-carnitine at 75% in excess of NRC (D8). During the starter and growth phases, feed intake was not affected by dietary treatment (*p* > 0.05). By contrast, body weight and FCR were both affected (*p* < 0.01) during the starter period. During the finisher phase, feed consumption was affected (*p* < 0.05) by dietary treatment. Feed intake of broilers fed on C, D3, D6, and D7 were statistically similar (*p* > 0.05) (1851.90, 1862.00, 1945.10, and 1872.80 g/pen/day, respectively) and were higher (*p* < 0.05) than 1564.40 g/pen/day (D5). With the exception of drumsticks, neck, back thoracic vertebrae, and proventriculus weights, the economical carcass segments were not affected (*p* > 0.05) by the dietary supplementation of amino acids. Duodenum and ileum weights and lengths decreased with amino acid supplementation (*p* < 0.05). IgT and IgG titers against Sheep Red Blood Cells (SRBC) for both primary and secondary responses were not affected by dietary treatments (*p* > 0.05). Dietary amino acids supplementation did not affect IgM titer after the secondary challenge (*p* > 0.05) and had a significant effect (*p* < 0.05) on serum antibody titers in broilers vaccinated against Newcastle disease (NCD) and Gumboro 's disease at the 27th and 30th days, respectively.

**Keywords:** amino acids; dietary supplementation; broiler; growth performance; humoral immunity

#### **1. Introduction**

Nowadays, the growing demand for poultry meat has resulted in pressure on breeders to increase the growth rate of birds, the feed efficiency, the size of breast muscles, and the reduction in abdominal fatness [1]. Therefore, research is being oriented toward improving the techniques of poultry meat production. The improvement in carcass compositions with additives has become a focus on nutrition research. As an example, the addition of amino acids and metabolic intermediates to diets may lower the abdominal fat deposition in poultry. One example is L-carnitine, the biologically active form of carnitine, which is synthesized in the liver, kidney, and brain [2] from the essential amino acids' lysine and methionine, that can be considered as L-carnitine precursors [3,4]. L-carnitine (δ-trimethylamino-β-hydroxybutyrate) is a quaternary hydrosoluble amine with a small molecular weight that occurs naturally in microorganisms, plants, and animals [5]. Its concentration in animals varies according to species [6], tissue type [4,7], nutritional status of the animal [8,9], and the feed quality [10]. Dietary effects of L-carnitine, lysine, and methionine supplementations on the growth performance and body composition of broiler chickens are still poorly understood. Many studies have suggested that the dietary addition of lysine and methionine in excess with respect to the American National Research Council (NRC) [11] recommendations may result in enhanced performances, especially with regard to breast meat yield, body weight gain, and feed conversion ratio [12–21]. Corzo et al. [22] reported how a high dietary density of amino acids can lead to increased breast meat related to an increase in lean muscle tissue. Moreover, Mukhtar et al. [10] reported that a significant improvement in amino acids feed intake improves the average body weight gain and feed conversion ratio. On the other hand, Si et al. [15] concluded that the level of methionine should not be increased if lysine is in excess of its minimum needs.

It has been also reported that the dietary supplementation of lysine and methionine can improve the immunity of broiler chickens against different diseases [23–26]. Moreover, it has been reported that methionine constructively affects the immune system by improving both cellular and humoral responses [27–29]. The mechanisms proposed to explain methionine interference in the immune system is the T cells proliferation, which are sensitive to intracellular glutathione and cysteine levels, compounds that participate in the methionine metabolism [30].

L-carnitine supplementation is used to improve broiler productivity [31], and its bioavailability depends on the composition of the diet. Theoretically, supplementing broiler diets with adequate content of L-carnitine would facilitate the fatty acids β-oxidation and decrease the esterification reactions and triacylglycerols storage in the adipose tissue [3,32,33]. However, the impact of extra supplied L-carnitine may depend on the magnitude of its endogenic biosynthesis from lysine and methionine in the presence of Fe2+ and a number of vitamins (e.g., ascorbate, niacin, and pyridoxine), which are required as cofactors for the enzymes involved in the metabolic pathway of L-carnitine [4,34–36].  Nonetheless, Bouyeh and Gevorgyan [39] and Celik et al. [40] showed that the growth performance of broilers was improved by L-carnitine supplementation. A study by Hosseintabar et al. [33] evaluated the effects of different levels of L-carnitine, lysine, and methionine on the blood concentrations of energy, protein, and lipid metabolites of male broiler chickens and concluded that, compared to a standard diet, the addition of 150 mg/kg of L-carnitine plus 15% lysine and methionine sustained a low plasmatic total cholesterol concentration compared to a standard diet. El-Wahab et al. [41] reported the broilers fed high levels of lysine and methionine with a surplus amount of L-carnitine (350 mg kg−1 to the diet) led to significantly lower cholesterol levels vs. a low L-carnitine intake. To the knowledge of the authors, the effect of simultaneous feed supplementation with L-carnitine and excess lysine-methionine on growth performance, carcass characteristics, and immunity markers of broiler chicken has not been studied before.

There is a need to standardize the dose of lysine-methionine and L-carnitine supplementation in the diet of broiler chickens not only to enhance their growth performance and carcass characteristics but also to improve their immune response. Hence, the main objective of this study has been to evaluate the effect of dietary supplementation of different levels of L-carnitine with or without an excess of lysine-methionine compared to dietary nutrient requirements on broiler chickens' growth performance, humoral immunity markers, and carcass characteristics during a 6-week rearing trial.

#### **2. Materials and Methods**

#### *2.1. Animal Welfare and Ethics*

All procedures related to animals' care and sampling were conducted under the approval of the Institution's Ethic Committee at the Department of Animal Science, Rasht Branch, Islamic Azad University, Rasht (Iran) (protocol N◦ 105/19) before the beginning of the experimental trial.

#### *2.2. Experimental Diets Preparation*

A control diet (C) for broiler chicken based on corn and soybean-meal was prepared according to the dietary nutrient requirements of broilers [1]. The ingredients and chemical composition of the control diet are given in Table 1. Thereafter, eight amino acids-supplemented diets (indicated as D1, D2, D3, D4, D5, D6, D7, and D8) were prepared by mixing the control diet thoroughly with the designated supplements at the required incorporation levels as shown in Table 2.

As stated above, the L-carnitine, lysine, and methionine levels in starter, grower, and finisher feeds for the control diet were determined according to the NRC [11] recommendations. In the experimental diets, D1, D2, D3, D4, D5, D6, D7, and D8, extra L-carnitine, lysine, and methionine (Carniking1, Lonza Ltd., Basel, Switzerland) were added in excess to the NRC [11] recommendations, as shown in Table 2, since NRC [11] recommended diets are suggested for feeding Ross 308 broiler chickens because of fewer phases of feeding periods and lower workloads [42].

**Table 1.** Ingredients and chemical compositions of the control diet.

\* Calcium Pantothenate: 4 mg/g; Niacin: 15 mg/g; Vitamin B6: 13 mg/g; Cu: 3 mg/g; Zn: 15 mg/g; Mn: 20 mg/g; Fe: 10 mg/g; K: 0.3 mg/g; \*\* Vitamin A: 5000 IU/g; Vitamin D3: 500 IU/g; Vitamin E: 3 mg/g; Vitamin K3: 1.5 mg/g; Vitamin B2: 1 mg/g.

\* L-carnitine (American National Research Council (NRC)): Starter period: 17.8 mg/kg; Grower period: 18.1 mg/kg; Finisher period: 22.9 mg/kg; \*\* lysine (NRC): Starter period: 1.41%; Grower period: 1.26%; Finisher period: 1.22%; \*\*\* methionine (NRC): Starter period: 0.61%; Grower period: 0.57%; Finisher period: 0.48%.

#### *2.3. Animals and Experimental Design*

Two hundred seventy 1-day-old, male Ross 308 broiler chicks obtained from a local commercial hatchery were used in this experiment. Chicks were randomly distributed into 27 pens (9 groups × 3 replications, each replication included 10 chicks). Each group was allocated to one of the nine dietary treatments indicated above. Birds were given starter feed from 1 to 21 days, a grower feed from 22 to 35 days, and a finisher feed from 36 to 42 days of age. Feed and water were provided ad libitum throughout the experimental assay. For the growth performance traits, the experimental unit was the pen. For the carcass and immunity traits, the experimental unit was the chicken.

#### *2.4. Growth Performance Monitoring*

Tens of birds per pen were weighed together on the 1st, 21st, and the 35th days of age to determine the live body weight and the weight gain. The feed consumption and feed conversion ratio (FCR) were also calculated for each growing phase as follows: from the 1st to the 21st day, from the 22nd day to the 35th day, and from the 36th to the 42nd day of the 42 days experimental study as described by Bouyeh and Gevorgyan [39].

#### *2.5. Carcass Characteristics Determination*

As shown by Panda et al. [43–45], at the end of the experiment (day 42), three broilers per same treatment (e.g., one broiler per same diet per pens) (*n* = 3) were randomly selected, weighed without prior fasting, and scarified between 9:00 am and 10:00 am by cervical dislocation to evaluate the characteristics of the carcass. After skin removal and total evisceration, the feet were separated from the carcass in the tibio–tarsal joint. Economic carcass and gastrointestinal segments were removed, weighed, and the ratios of each segment to body weight were calculated.

#### *2.6. Humoral Immune Response Measurements*

Non-pathogenic antigens of Sheep Red Blood Cells (SRBC) were used to monitor the humoral immune response of broilers. The SRBC were purchased from a local Iranian supplier. A suspension was prepared by mixing 1 mL of phosphate-buffered saline (PBS) with 10 mL of SRBC. Six birds per same treatment (e.g., two broilers per same diet per pens) (*n* = 6) were subcutaneously injected in the breast with 0.5 mL of SRBC suspension on the 22nd and the 36th days of the experimental trial.

Then, seven days after each sensitization (28 and 42 days, respectively), antibody titers against SRBC were measured by a hemagglutination inhibition (HI) test according to Cunningham [46]. All antibody titers were recorded according to previous studies [47,48].

Birds were also vaccinated against infectious bronchitis (IB) on the 1st and 16th days of age, against Newcastle disease (NCD) on the 8th and 20th days of age, and against Gumboro's disease on the 14th and 23rd days of age. The humoral immune responses of chickens to the IB virus at the 23rd day of age, to the NCD virus at 27th day of age, and to the Gumboro virus at the 30th day of age were measured using the HI and ELISA methods as described by references [47,48]. Blood samples were collected from the brachial vein. Serum was separated by centrifugation (3000× *g* rpm for 15 min), and antibody titers against IB, NCD, and Gumboro virus were measured using commercially available ELISA kits (Bio-check BV, Gouda, Holland) according to the manufacturer's instructions. The absorbance of controls and samples were read at a wavelength of 405 nm using an ELISA reader (Bio-Tek Instruments Inc. ELX 800, Winooski, VT, USA).

#### *2.7. Statistical Analysis*

All data were subjected to an ANOVA statistical analysis with the General Linear Model (GLM) procedure of SAS [49]. The GLM was used according to the following model:

$$\mathcal{Y}\_{\rm ijk} = \mu + \alpha\_{\rm j} + \beta\_{\rm k} + (\alpha\beta)\_{\rm jk} + \varepsilon\_{\rm ijk} \tag{1}$$

where Yijk = the jth observation on the ith treatment, μ = overall mean, αj = the main effect of the L-carnitine level, βK = the main effect of the methionine-lysine level, αβjk = the effect of the interaction of L-carnitine and of methionine-lysine treatments, and εijk = The random error.

#### **3. Results**

#### *3.1. Growth Performance*

Table 3 summarizes the results of the growth performance using the different diets. During the starter and growth phases, feed intake was not affected by dietary treatment (*p* > 0.05). By contrast, body weight and FCR were both affected (*p* < 0.01) during the starter period. In fact, D1, D2, D3, D6, D7, and D8 were associated with the highest live body weight (*p* < 0.01) with mean values of 706.31, 745.50, 671.10, 733.67, 741.00, and 723.27 g/pen/21day, and consequently, the feed conversion ratio was the lowest (*p* < 0.01) during this period compared to the Control Diet (C).

methionine at 30% in excess of NRC, and L-carnitine at 15% in excess of NRC; D6 = control diet supplemented with lysine equal to NRC recommendations, methionine equal to NRC recommendations, and L-carnitine at 75% in excess of NRC; D7 = control diet supplemented with lysine at 15% in excess of NRC, methionine at 15% in excess of NRC, and L-carnitine at 75% in excess of NRC; D8 = control diet supplemented with lysine at 30% in excess of NRC, methionine at 30% in excess of NRC, and L-carnitine at 75% in excess of NRC; SEM = standard error of the mean; \* *p* < 0.05, \*\* *p* < 0.01, NS = *p* ≥ 0.05; a, b: Means within the same row with common superscript letters are not significantly different (*p* ≥ 0.05).

#### *Animals* **2019**, *9*, 362

However, during the grower period, the live bodyweight of the broiler fed on D1 and D2 was not different from that of C (1241.29 g/pen/period and 1165.50 g/pen/period vs. 1207.10 g/pen/period) and was higher than those of D3, D4, D5, D6, D7, and D8 (*p* < 0.01). D1 and D2 were associated with the lowest feed conversion ratio (*p* < 0.01) with mean values of 2.33 and 2.48, respectively. During the finisher phase, feed consumption was affected (*p* < 0.05) by dietary treatment. Feed intake of broilers fed on C, D3, D6, and D7 were statistically similar (*p* > 0.05) (1851.90, 1862.00, 1945.10, and 1872.80 g/pen/d, respectively) and were higher (*p* < 0.05) than 1564.40 g/pen/day (D5).

However, live body weights were similar between dietary treatments (*p* > 0.05) with mean values of 976.53 g/pen/period and 949.27 g/pen/period, respectively. The FCR of this period was not affected (*p* > 0.05) by dietary treatment.

#### *3.2. Carcass Characteristics*

The results obtained for carcass parameters (economical carcass segments, body organ segments, and gut organs) are shown in Table 4.

Our results indicate that, with the exception of thigh weights, economical carcass segments were not affected by dietary treatment (*p* > 0.05). The neck weights of the bird given D1 and D8 were the highest (*p* > 0.05) with mean values of 64.50 g and 64.63 g, respectively, compared to 79.57 for the control diet. D2 was associated with the highest back thoracic vertebrae weight (*p* > 0.05) at 103.99 g versus 45.11 g for birds given the diet D8. However, all other body segments (heart, liver, gizzard, and abdominal fat) weights were not affected by dietary treatment (*p* > 0.05). With the exception of proventriculus weight, gut organ weights were not affected (*p* < 0.05) by dietary treatment. Our data showed that the dietary supplementation of lysine and methionine at 30% with a level of L-carnitine equal to NRC and of 15% of lysine, methionine, and carnitine increased (*p* < 0.05) proventriculus weight from 10.77 g to 11.26 and 11.43 g, respectively.

NRC L-carnitine at 75% in excess of NRC; D8 = control diet

SEM = standard error of the mean; \* *p* < 0.05; NS = *p* ≥ 0.05; a, b: Means within the same row with common superscript letters are not significantly different (*p* ≥ 0.05).

recommendations,

 and L-carnitine at 75% in excess of NRC; D7 = control diet

supplemented

 with lysine at 30% in excess of NRC, methionine at 30% in excess of NRC, and L-carnitine at 75% in excess of NRC;

supplemented

 with lysine at 15% in excess of NRC, methionine at 15% in excess of NRC, and

#### *3.3. Intestine Segments*

The effects of dietary treatment on the length, weight, width, and diameter of small intestine segments (duodenum, jejunum, and ileum) in broilers are shown in Table 5.

The dietary supplementation of amino acid decreased (*p* < 0.05) duodenum weight and length. Birds given a supplemented diet with 30% of lysine and methionine in excess of NRC and 75% of L-carnitine in excess of NRC had the lowest weight, with a mean value of 12.99 g. The lowest duodenum length was recorded when birds were given 15% of lysine and methionine in excess of NRC plus 75% of L-carnitine in excess of NRC. A combination of L-carnitine (75%) and lysine-methionine (30%) increased the duodenum width significantly (*p* < 0.05) from 7.50 mm to 9.38 mm. Concerning the duodenum diameter, the dietary addition of lysine-methionine at a level equal to NRC plus 15% of L-carnitine in excess of NRC (D3), of lysine-methionine and L-carnitine at a rate of 15% in excess of NRC (D4), and of lysine-methionine and L-carnitine at a rate of 30% in excess of NRC plus 15% of L-carnitine in excess of NRC (D5) increased the diameter from 0.94 mm (C) to, respectively, 1.61 mm, 1.67 mm, and 1.68 mm.

Dietary treatments did not affect (*p* > 0.05) the jejunum weight and length. The dietary addition of lysine-methionine at a level equal to NRC plus 15% of L-carnitine in excess of NRC (D3) and of lysine-methionine and L-carnitine at a rate of 15% in excess of NRC (D4) increased the jejunum width (*p* < 0.05) from 9.26 mm (C) to 9.50 mm (D3) and 9.80 mm (D4). The dietary supplementation of amino acids significantly decreased (*p* < 0.05) the ileum weight from 9.10 g (C) to 2.67 g (D4) and 2.97 g (D7) and the length from 18.16 mm to 9.26 mm (D8) and 8.00 mm (D7). The ileum width of the broiler group fed with lysine-methionine at a level of 15% plus L-carnitine at 75% in excess of NRC was the highest (*p* < 0.05) compared to the control diet 7.33 mm versus 7.97 mm (D7). Dietary treatment did not affect (*p* > 0.05) ileum diameter.

Caecum weights were not different between treatments and ranged from 12.62 g (D5) to 17.13 g (C) (*p* > 0.05). The colon weight of broiler bird fed with lysine-methionine at a level equal to NRC plus 15% of L-carnitine in excess of NRC (D4) was the highest (*p* < 0.05), with a mean value of 2.36 g. Rectum weights were similar between all the treatments groups and ranged from 1.78 g (D6) to 2.32 g (D7) (*p* > 0.05).

(Control) = diet with lysine, methionine, and L-carnitine equal to NRC recommendations; D1 = control diet supplemented with lysine at 15% in excess of NRC, methionine at 15% in excess of NRC, and L-carnitine equal to NRC; D2 = control diet supplemented with lysine at 30% in excess of NRC, methionine at 30% in excess of NRC, and L-carnitine equal to NRC; D3 = control diet supplemented with lysine equal to NRC, methionine equal to NRC, and L-carnitine at 15% in excess of NRC; D4 = control diet supplemented control diet supplemented with lysine at 15% in excess of NRC, methionine at 15% in excess of NRC, and L-carnitine at 15% in excess of NRC; D5 = control diet supplemented lysine at 30% in excess NRC, methionine at 30% in excess of NRC, and L-carnitine at 15% in excess of NRC; D6 = control diet supplemented with lysine equal to NRC recommendations, methionine equal to NRC recommendations, and L-carnitine at 75% in excess of NRC; D7 = control diet supplemented with lysine at 15% in excess of NRC, methionine at 15% in excess of NRC, and L-carnitine at 75% in excess of NRC; D8 = control diet supplemented with lysine at 30% in excess of NRC, methionine at 30% in excess of NRC, and L-carnitine at 75% in excess of NRC, SEM= standard error of the mean; \*\* *p* < 0.01, \* *p* < 0.05; NS = *p* ≥ 0.05; a, b, c, d: Means within the same row with common superscript letters are not significantly different (*p* ≥ 0.05).

of #### *3.4. Humoral Immune Response*

#### 3.4.1. Humoral Immune Response against Sheep Red Blood Cell (SRBC)

The dietary effect of amino acids supplementation on primary and secondary antibody responses are shown in Table 6. IgT and IgG titers against SRBC for both primary and secondary responses were not affected by dietary treatments (*p* > 0.05). Birds receiving diets supplemented with excesses of amino acids (D1, D2, D3, D4, D5, D6, D7, and D8) had significantly lower titers of IgM than that of those receiving C for primary response (*p* < 0.05), with mean values of 2.33 log10 versus 2.00 log10 (D1), 1.66 log10 (D2), 1.33 log10 (D3), 1.33 log10 (D4), 1.00 log10 (D5), 1.33 log10 (D6), 1.33 log10 (D7), and 2.00 log10 (D8). Dietary treatment did not affect (*p* > 0.05) IgM titer after a secondary challenge.

C (Control) = diet with lysine, methionine, and L-carnitine equal to NRC recommendations; D1 = control diet supplemented with lysine at 15% in excess of NRC, methionine at 15% in excess of NRC, and L-carnitine equal to NRC; D2 = control diet supplemented with lysine at 30% in excess of NRC, methionine at 30% in excess of NRC, and L-carnitine equal to NRC; D3 = control diet supplemented with lysine equal to NRC, methionine equal to NRC, and L-carnitine at 15% in excess of NRC; D4 = control diet supplemented control diet supplemented with lysine at 15% in excess of NRC, methionine at 15% in excess of NRC, and L-carnitine at 15% in excess of NRC; D5 = control diet supplemented lysine at 30% in excess of NRC, methionine at 30% in excess of NRC, and L-carnitine at 15% in excess of NRC; D6 = control diet supplemented with lysine equal to NRC recommendations, methionine equal to NRC recommendations, and L-carnitine at 75% in excess of NRC; D7 = control diet supplemented with lysine at 15% in excess of NRC, methionine at 15% in excess of NRC, and L-carnitine at 75% in excess of NRC; D8 = control diet supplemented with lysine at 30% in excess of NRC, methionine at 30% in excess of NRC, and L-carnitine at 75% in excess of NRC, SEM = standard error of the mean; \* *p* < 0.05, NS = *p* ≥ 0.05; a, b: Means within the same column with common superscript letters are not significantly different (*p* ≥ 0.05).

#### 3.4.2. Humoral Immune Response against Bronchitis, Newcastle and Gumboro Diseases

The influence of different amino acids dietary supplementations on serum antibody titer in chickens vaccinated against IB, NCD, and Gumboro virus are shown in Table 7. Serum antibody titers in broilers vaccinated against bronchitis were not affected (*p* > 0.05) by dietary treatment on day 23. However, a significant increase (*p* < 0.05) in serum antibody titers in broilers vaccinated against NCD and Gumboro on the 27th and the 30th days was observed from, respectively, 3.66 log10 (C) to 6.00 log10 (D3) and from 3.38 log10 (C) to 3.59 log10 (D6). Chickens fed with 30% of lysine-methionine in excess of the NRC and of L-carnitine equal to the NRC supplemented diet had the heaviest thymus (*p* < 0.01) with a mean value of 24.60 g versus 15.59 for the control diet. However, chickens fed on L-carnitine (15%) and lysine-methionine (15%) in excess of the NRC had the lightest thymus with a mean value of 7.17 g. Bursa of fabricius and spleen weights were not affected (*p* > 0.05) by dietary L-carnitine and lysine-methionine supplementation.

C (Control diet) with lysine, methionine, and L-carnitine equal to NRC recommendations; D1 control diet supplemented with lysine at 15% in excess of NRC, methionine at 15% in excess of NRC, and L-carnitine equal to NRC; D2 = control diet supplemented with lysine at 30% in excess of NRC, methionine at 30% in excess of NRC, and L-carnitine equal to NRC; D3 = control diet supplemented with lysine equal to NRC, methionine equal to NRC, and L-carnitine at 15% in excess of NRC; D4 = control diet supplemented control diet supplemented with lysine at 15% in excess of NRC, methionine at 15% in excess of NRC, and L-carnitine at 15% in excess of NRC; D5 = control diet supplemented lysine at 30% in excess of NRC, methionine at 30% in excess of NRC, and L-carnitine at 15% in excess of NRC; D6 = control diet supplemented with lysine equal to NRC recommendations, methionine equal to NRC recommendations, and L-carnitine at 75% in excess of NRC; D7 = control diet supplemented with lysine at 15% in excess of NRC, methionine at 15% in excess of NRC, and L-carnitine at 75% in excess of NRC; D8 = control diet supplemented with lysine at 30% in excess of NRC, methionine at 30% in excess of NRC, and L-carnitine at 75% in excess of NRC; SEM = standard error of the mean; \*\* *p* < 0.01, \* *p* < 0.05, NS = *p* ≥ 0.05; a, b, c, d: Means within the same row with common superscript letters are not significantly different (*p* ≥ 0.05).

#### **4. Discussion**

It has been observed that L-carnitine supplementation can enhance broiler productivity [26]. However, the effect of administrating an excess of L-carnitine may depend on the magnitude of its endogenic biosynthesis from lysine and methionine [20,29–31]. In the current study, we are reporting the effect of dietary supplementation of different levels of L-carnitine with or without an excess of lysine-methionine on growth performance, carcass traits, and some humoral immunity markers of broiler chicken.

#### *4.1. Growth Performance*

The results relative to the growth performance show that a combination of lysine and methionine at 15% and 30% in excess of the NRC recommendations was without impact on the feed intake, live body weight gain, and FCR of broiler chicken during the whole experiment period.

Live body weights at the end of the experimental trial varied from 2600.70 g to 2886.20 g. These values are consistent with those reported by Bouyeh and Gevorgyan [39], who showed that dietary supplementations of lysine-methionine at 0, 1.1, 1.2, 1.3, or 1.4% higher than NRC recommendations [11] led to body weight gains from mean values of 2960 g to 2920 g (1.1%), 2850 g (1.2%), 2970 g (1.3%), and 2730 g (1.4%), respectively.