Insight View on the Role of in Ovo Feeding of Clenbuterol on Hatched Chicks: Hatchability, Growth Efficiency, Serum Metabolic Profile, Muscle, and Lipid-Related Markers

Simple Summary This study examined the effects of ovo injection of clenbuterol on fat deposition and growth performance in chickens, which is prejudicial to poultry consumers and muscle growth-related genes, egg hatchability, and fertility. The achieved result showed a definite effect of clenbuterol on body gain and hatchability. It decreased fat deposition and upregulation of muscle growth-related gene expressions accompanied by modulation of fatty and amino acid composition, reflecting a new insight into the intracellular pathways of clenbuterol supplementation on chicks. Abstract The present study aimed to assess the in ovo administration of clenbuterol on chick fertility, growth performance, muscle growth, myogenic gene expression, fatty acid, amino acid profile, intestinal morphology, and hepatic lipid-related gene expressions. In this study, 750 healthy fertile eggs from the local chicken breed Dokki-4 strain were analyzed. Fertile eggs were randomly divided into five experimental groups (150 eggs/3 replicates for each group). On day 14 of incubation, in addition to the control group, four other groups were established where 0.5 mL of worm saline (30 °C) was injected into the second group of eggs. In the third, fourth, and fifth groups, 0.5 mL of worm saline (30 °C), 0.9% of NaCl, and 10, 15, and 20 ppm of clenbuterol were injected into the eggs. Results suggested that clenbuterol increased growth efficiency up to 12 weeks of age, especially at 15 ppm, followed by 10 ppm, decreased abdominal body fat mass, and improved hatchability (p < 0.01). Clenbuterol also modulated saturated fatty acid levels in the breast muscles and improved essential amino acids when administered at 10 and 15 ppm. Additionally, clenbuterol at 15 ppm significantly decreased myostatin gene expression (p < 0.01) and considerably increased IGF1r and IGF-binding protein (IGFBP) expression. Clenbuterol administration led to a significant upregulation of hepatic PPARα, growth hormone receptor, and Lipoprotein lipase (LPL) mRNA expression with a marked decrease in fatty acid synthase (FAS) and sterol regulatory element-binding protein 1 (SREBP-1c) expression. In conclusion, the current study revealed that in ovo injection of clenbuterol showed positive effects on the growth of hatched chicks through reduced abdominal fat deposition, improved intestinal morphology, and modulation of hepatic gene expressions in myogenesis, lipogenesis, and lipolysis.

A total of 200 healthy hatched chicks (40 birds/group with four replicates for each: control group, normal saline, clenbuterol 10, 15, and 20 ppm/egg) were selected and used for the feeding trial. The chicks grew for 12 weeks, and standard feed was provided for local chicken strains. Chicks were maintained in brooder pens for two weeks after hatching and transferred into separate locations. The chicks (8 birds/m 2 ) were housed at the Kafrelsheikh University, Egypt, in an environmentally managed space. The house was kept at a temperature that was dependent on the bird's age. The temperature was regulated through an air conditioner. During the experiment, air humidity was maintained at almost 70% [35]. The basal diet formulation was confirmed with the following [36] ( Table 1). Body weight was measured at the nearest 0.1 g, and the ratio of feed conversion (FCR) was calculated. The birds were vaccinated against the common diseases in Egypt. Hitchner vaccine B1 (HB1) and Gumboro vaccine were administered through eye drops at the age of 7 and 10 days, respectively, and, at 13 days, intramuscular feedings of killed N.D.V., Reo, Gumboro, and infectious bronchitis vaccines were administered. Vaccines were intramuscularly injected with killed avian influenza viruses (AIV; H5N2) when they were 15 days of age, while Gumboro and LaSota were administered through eye drops at 20, 30 and 40 days of age. Subsequently, LaSota booster doses were administered at 51 days of age and then through an eye drop on a biweekly basis [37].

Growth Efficiency and Yield of Carcass
The experiment was finally completed at 12 weeks of age, 20 birds/group, five birds from each replicate per group were slayed for carcass yield estimation. The birds were weighed individually, and the FCR was calculated as the real consumption of feed (FI) divided by the body production. The slaughtering technique was performed following the Malaysian institute's method [38]. The carcasses were sprayed, dipped cool at 2 • C for 30 min, and allowed to drain effectively for 5 min, and the carcass yields were measured as live body weight percentages. Abdominal fat was eliminated and evaluated according to Baziz et al. [39].

Blood and Tissue Sampling
At 12 weeks of age, two blood samples were obtained by wing vein puncture from five birds from each replicate, randomly selected from each group, under gentle restraint. One sample was obtained for hematological analysis. Non-heparinized syringes were used in one sample for serum collection, separated at 3000× g/15 min by centrifuge of the clotted blood at 4 • C and stored at −20 • C for additional biochemical analysis.

Hematological Analysis
Blood samples were used to assess the hemoglobin content (g/dL) with Drabkin's technique utilizing the colorimetric form of cyanmethemoglobin after centrifugation [40]. Blood was smeared on a glass slide, left to dry, and then coated with Giemsa stain. Differential leukocyte counting was performed. One hundred leukocytes, including heterophils and lymphocytes, were counted on each blood film. H/L ratio was obtained by dividing the number of heterophils by the number of lymphocytes. Three slides were scored, and the means in each bird were calculated [41].

Blood Biochemical Analysis
The spectrophotometric analysis was conducted to evaluate global protein (g/dL), globulin, and albumin (g/dL) concentrations [42]. Biodiagnostic Company, Giza, Egypt, provided the commercial test kits. The lipoprotein fractions (VLDL, LDL, and HDL) were isolated using two sequential ultracentrifugation steps. the density was adjusted appropriately by adding NaCl (Sigma # S9888) and NaBr (Sigma # 310506), as detailed elsewhere [43] following [44]. In brief, plasma (5 mL) was transferred to quick seal tubes (Beckman Instruments, Palo Alto, CA, USA) and centrifuged for 18 h at 40,000 rpm, 4 • C in a 40.3 Ti fixed-angle rotor ultracentrifuge (Beckman, Brea, CA, USA). The 1.006 g/mL top fraction (VLDL) was brought back to a volume of 2.5 mL with saline (0.85%). The bottom fraction was adjusted to a relative of 1.063 with KBr and centrifuged for 18 h at 40,000× g to obtain the LDL (top) and the HDL (bottom). After centrifugation, each lipoprotein sample was dialyzed extensively against Tris-buffered saline (TBS; 10 mM Tris-HCl, 140 mM NaCl, and 5 mM EDTA (pH 8.0)) for 24 h to remove NaBr. For each of the lipoproteins that were purified individually, Total lipid (mg/dL), triglyceride, cholesterol (mg/dL), high-density lipoprotein, lower-density lipoprotein (mg/dL) measurements were obtained using commercially available kits. As per the manufacturer's instructions, maximum antioxidant efficiency was also calculated using commercial kits (Diamond Diagnostics). Serum aspartate aminotransferase (AST) and alanine aminotransferase (ALT) activities were determined [45].

Antioxidant Activity in Breast Muscles
Breast muscle samples in 100 mM cold potassium phosphate buffer, pH 7.2, were homogenized. Homogenate muscles were spun at 1500× g at 4 • C for 20 min, and the supernatant was added for further evaluation. The manufacturer's protocol was used to calculate malondialdehyde concentration with a biodiagnostic kit (Biodiagnostic # MD 2529, Egypt). The biodiagnostic package (Biodiagnostic, # GP 2524, Egypt) was tested following the protocol for GSH-Px. The biodiagnostic kit (Biodiagnostic # SD 2521, Egypt) protocol was used to test SOD. MDA, GSH-Px, and SOD contents were measured at 534 nm, 340 nm, and 560 nm, respectively, using a UV-VIS spectrophotometer (NanoDrop One C , Thermo Scientific, Wilmington, DE, USA) using the software Excel 2016 (Microsoft, Redmond, WA, USA).

Immunity Markers
Polymorphonuclear cell phagocytosis using Candida albicans was achieved according to [46]. The following aliquots were combined in the plastic tube: 100 µL fetal calf serum, 100 µL heat-killed C. Albicans (5 to 106/mL), and 100 µL blood. The tubes had been combined and incubated for 30 min at 37 • C, during which they were centrifuged for 5 min. The supernatant was removed, leaving a droplet in which the sediment was resuspended. Smears from the deposit were prepared, dried in the air, fixed with methyl alcohol, and stained with Giemsa stain. There were 200 heterophils examined, and the percentage of Candida-ingested heterophils was counted and expressed. A check for agarose gel cell lysis assessed serum lysosomal activity, as previously defined [47].

Amino Acid and Muscle Fatty Acid Profiles
Five birds from each replicate per group (20 birds/group) were selected to estimate muscle fatty acids and amino acid patterns. Extraction of fat was performed in the breast muscle using the chloroform-methanol (2:1) mixture to extract lipids, centrifuged for 10 min at 3000 rpm. The esterification procedure was achieved by adding the supernatant to 2 mL of methanol-sulfuric acid mix (95:5). The free fatty acids were provided by Sigma-Aldrich (Sigma, St. Louis, MO, USA). Fatty acid quantity was assessed using Agilent gas chromatography techniques (7890A GC). The flow rate conditions through the GC column and the splitless feeding mode were applied [48]. The amount of free amino acids (AA) in the breast muscle was evaluated [49]. Briefly, 2 g of muscle sample was homogenized with 20 mL of trichloroacetic acid (2%) for 2 min at 17.100 g. Subsequently, the homogenate was centrifuged at 3000× g/15 min, filtered through a 0.5 µm membrane, and dried. The derivatized samples and AA standards were inserted into the column for separation by high-performance liquid chromatography using a Nova-PakTM C18 column (4 µm, 3.9 × 4.6 mm).

Gene Expression Analysis
Total RNA was extracted from the tissue samples using the manufacturer's easy-RED Total RNA Extraction Kits (iNtRON Biotechnology, Inc., Seongnam-Si, Korea). The RNA integrity was tested by agarose gel electrophoresis, and NanoDrop's spectrophotometer was used to analyze the sample quantities. The first-strand cDNA was achieved by the HiSenScript cDNA package (iNtRON Biotechnology, Inc., Korea). The selected genes, with GAPDH as a standard gene, were amplified with specific primers and stable in the sample groups ( Table 2). The mRNA expression was performed using the Stratagene MX3005P real-time PCR (Agilent Technologies, CA, USA) and the TOPreal™ PreMIX SYBR Green qPCR master blend (Enzynomics, Daejeon, Korea) as indicated by the manufacturer. Tools were used for MxPro QPCR. A 2−a technique, described above, was used to test the relative concentrations of gene expression. The relative intensities of gene expression were assessed using the 2−∆∆ct method as outlined in [50]. Glyceraldehyde-3-phosphate dehydrogenase. IGF1R, insulin growth factor1 receptor, PPARα, peroxisome proliferator-activated receptors, SREBP-1c, Sterol regulatory element-binding protein 1, FAS, Fatty acid synthase, LPL, Lipoprotein lipase, MSTN, Myostatin gene, GHR, growth hormone receptor, IGFBP2, Insulin-like growth factor-binding protein 2.

Histomorphometric Examination
Tissue samples from both ascending and descending limbs of the duodenum and the bursa of Fabricius and spleen were collected from five chickens from each treated group. The samples were fixed in 10% formaldehyde solution and then dehydrated in graded ethanol. The dehydrated samples were cleared in xylene and then embedded in paraffin. Moreover, 5-µm-thick paraffin-tissue sections were stained with hematoxylin and eosin. The stained sections were examined under a light microscope (Leica). The obtained images were subjected to morphometric analysis, including intestinal villi length of duodenum, total cell count of splenic parenchyma, and lymphoid follicles of the bursa of Fabricius using image analysis software (NIH, Bethesda, MD, USA). A total of 5 images from each bird were selected and the average was calculated (Mean ± SE) [57].

Data Analysis
Data analysis was performed using SPSS version 23 (IBM Corp, Armonk, NY, USA) [58]. A one-way study of variance accompanied by the multiple ranges of Duncan determined the significant difference between treatments at a p-value < 0.05. Before conducting this test, Shapiro-Wilk and Levene's experiments assessed normality. Polynomial contrasts were applied to find linear and quadratic impacts of different clenbuterol levels on the different parameters [59].

Growth Performance Analysis and Carcass Traits
The effects of in ovo feeding on growth and carcass traits are shown in Table 3, in which the clenbuterol-injected group at 15 ppm showed higher (p = 0.023) weight gain and final body weight (p = 0.01) compared to other treated groups. Moreover, clenbuterol at 15 ppm improved the hatchability (p < 0.01) concerning normal saline and control groups and recovered the hatchability and fertility percentage of injected eggs. Additionally, there was a significant improvement in carcass yield (Table 3). Moreover, clenbuterol at 10 ppm also showed a markedly substantial difference in control and normal saline groups (p = 0.041). Furthermore, there was a significantly decreased abdominal fat weight percentage at 10 and 15 ppm dose of clenbuterol concerning other treated groups.

Blood Biochemical and Hematological Markers
There were no significant differences (p > 0.05) in RBC count, Hb level, and H/L ratio in different treated groups (Table 4). In ovo feeding of clenbuterol decreased the cholesterol, TG, total lipid, and LDL levels (p = 0.013, 0.016, 0.012, and 0.020 respectively) compared to other treated groups, with a noteworthy increase in HDL level (p = 0.011). Clenbuterol feeding resulted in considerably increased total protein and albumin levels compared to other treated groups. Moreover, clenbuterol had no considerable effect on AST and ALT levels. Additionally, clenbuterol groups showed a nonsignificant alteration in the total antioxidant capacity in other treated groups.

Immunity and Antioxidant Activity
MDA, GSH-PX, and SOD are presented in Table 5. The embryos' results in ovo feeding with clenbuterol revealed normal antioxidant activities (GSH-PX and SOD; Table 4). There was no statistically significant difference in MDA level between different treated groups (p > 0.05) ( Table 5). Lysosomal activity, phagocytic activity, and phagocytic index are shown in Table 5. The obtained results showed that in ovo feeding with clenbuterol 10 and 15 ppm in embryos led to a regular immune pattern in other treated groups.   Table 6 shows that the in ovo feeding of clenbuterol at 10 and 15 ppm resulted in significantly decreased (p < 0.05) low saturated fatty acids (myristic, palmitoleic, stearic, and palmitic) contents of the breast muscles compared to the other treated groups. Meanwhile, the current study revealed no significant variations of the in ovo feeding of clenbuterol to modulate the polyunsaturated FA (PUFA) contents in the breast muscles in other treated groups, including α-linolenic acid linoleic acid, docosahexaenoic acid, and eicosapentaenoic acid. As shown in Table 7, the in ovo feeding of clenbuterol at 10 and 15 ppm showed a marked increase in lysine, threonine, leucine, phenylalanine, methionine, valine, and isoleucine muscular contents compared with the control and normal saline groups. Moreover, in ovo feeding of clenbuterol significantly increased the nonessential AA contents in chicken breast muscles, such as serine, alanine, arginine, proline, and aspartic acid, especially at 15 ppm concentration in other treated groups.   Figure 1 shows that in ovo feeding of clenbuterol led to marked downregulation and myostatin gene expression in the breast muscle (p < 0.01) in the control and normal saline groups. Clenbuterol at 15 ppm showed significant upregulation of IGF1r, and clenbuterol significantly upregulated IGFBP expression at 15 ppm concentration in other treated groups; in the same context, clenbuterol feeding showed significant upregulation of hepatic PPARα in other treated groups (p < 0.05, p < 0.01, p < 0.05) for clenbuterol at 10 ppm, 15 ppm, and 20 ppm, respectively. Clenbuterol feeding showed significant upregulation in LPL mRNA expression with a marked decrease in FAS mRNA expression. Moreover, there was significant downregulation in SREBP-1c expression in clenbuterol groups in other treated groups with marked upregulation in GHr expression, as shown in Figure 2. 15 ppm, and 20 ppm, respectively. Clenbuterol feeding showed significant upregulation in LPL mRNA expression with a marked decrease in FAS mRNA expression. Moreover, there was significant downregulation in SREBP-1c expression in clenbuterol groups in other treated groups with marked upregulation in GHr expression, as shown in Figure 2.

Histomorphometry of the Duodenum, Spleen and Bursa of Fabricius
The duodenum of the control group was formed of mucosa, submucosa, muscularis, and serosa. The mucosa was thrown into the intestinal villi in the intestinal lumen and mucosal glands in the lamina propria. The intestinal villi became more numerous and branched, in addition to a significant increase in villi length (p < 0.001) in the clenbuterol 10 ppm group compared with the control and clenbuterol 15 ppm groups (Figure 3). The normal saline in ovo injected group showed no difference from the control group. The spleen of the chicken was formed of white and red pulp. The white pulp was composed of lymphatic follicles and periarterial lymphoid sheath. The red pulp was formed of blood sinusoid and blood cells. The lymphatic nodule contained blood vessels. The fourth group's spleen revealed a marked increase in the number of small lymphocytes in addition to the size of lymphatic nodules in the clenbuterol 10 ppm and clenbuterol 15 ppm groups compared with the other groups ( Figure 4). The histopathological examination of the bursa of Fabricius revealed that the mucosal layer was thrown into several folds lined by pseudostratified columnar epithelium. Each fold contained several lymphoid follicles. Each follicle was surrounded by loose connective tissue from the lamina propria-the follicle composed of a darkly stained cortex and lightly stained medulla due to dispersed lymphocytes. The two layers were separated by undifferentiated cells with acidophilic cytoplasm. The cortex and medulla of the clenbuterol 10 ppm and 15 ppm groups were darker than those of the control and clenbuterol 20 ppm groups due to an increase in the density of lymphoid cells ( Figure 5).

Histomorphometry of the Duodenum, Spleen and Bursa of Fabricius
The duodenum of the control group was formed of mucosa, submucosa, muscularis, and serosa. The mucosa was thrown into the intestinal villi in the intestinal lumen and mucosal glands in the lamina propria. The intestinal villi became more numerous and branched, in addition to a significant increase in villi length (p < 0.001) in the clenbuterol 10 ppm group compared with the control and clenbuterol 15 ppm groups (Figure 3). The normal saline in ovo injected group showed no difference from the control group. The spleen of the chicken was formed of white and red pulp. The white pulp was composed of lymphatic follicles and periarterial lymphoid sheath. The red pulp was formed of blood sinusoid and blood cells. The lymphatic nodule contained blood vessels. The fourth group's spleen revealed a marked increase in the number of small lymphocytes in addition to the size of lymphatic nodules in the clenbuterol 10 ppm and clenbuterol 15 ppm groups compared with the other groups ( Figure 4). The histopathological examination of the bursa of Fabricius revealed that the mucosal layer was thrown into several folds lined by pseudostratified columnar epithelium. Each fold contained several lymphoid follicles. Each follicle was surrounded by loose connective tissue from the lamina propria-the follicle composed of a darkly stained cortex and lightly stained medulla due to dispersed lymphocytes. The two layers were separated by undifferentiated cells with acidophilic cytoplasm. The cortex and medulla of the clenbuterol 10 ppm and 15 ppm groups were darker than those of the control and clenbuterol 20 ppm groups due to an increase in the density of lymphoid cells ( Figure 5).

Discussion
Clenbuterol has been shown to increase skeletal muscle mass in mammals [60]. Clenbuterol is a selective 2-adrenoceptor agonist with the ability to cross the blood-brain barrier that works by binding to 2-adrenoceptors and activating the enzyme adenylyl cyclase, which causes an increase in intracellular concentrations of cyclic adenosine monophosphate and, as a result, protein kinase A activation [61]. As a result of its numerous adverse effects on humans, such as cardiomyopathy and acute hepatitis, clenbuterol has been banned in several nation [62] Our study examined the effect of in ovo feeding of clenbuterol on fertility, hatchability, growth performance, and multiple molecular and physiological parameters concerning the pathway by which clenbuterol exerts its action. Table 3 shows the effects of in ovo feeding on growth and carcass traits, in which the clenbuterol-injected group at 15 ppm showed significantly higher weight gain and final body weight, and improved hatchability and fertility percentage. Additionally, there was a considerably enhanced carcass yield and significantly decreased abdominal weight percentage at 10 and 15 ppm doses of clenbuterol in other treated groups. Our obtained result was in agreement with those of previous studies [63,64]. They proved that b-adrenergic agonists could boost weight gain when added to feed, and the proportion of tissue fat is reduced [65]. These findings are attributed to increased nitrogen accrual and deterioration in the saturated fatty acid concentration [66].
Additionally, Spurlock, et al. [67] reported that clenbuterol administration stimulated anabolic activity. All previously mentioned studies support in ovo feeding of clenbuterol findings concerning weight gain and abdominal fat deposition. Our results are consistent with Hamano [68], who reported significant weight decreases in the abdominal fat in chicken fed 0.25 mg/kg of clenbuterol. In the same line, clenbuterol caused decreased abdominal fat [69]. Our histopathological results support clenbuterol's overall growth

Discussion
Clenbuterol has been shown to increase skeletal muscle mass in mammals [60]. Clenbuterol is a selective 2-adrenoceptor agonist with the ability to cross the blood-brain barrier that works by binding to 2-adrenoceptors and activating the enzyme adenylyl cyclase, which causes an increase in intracellular concentrations of cyclic adenosine monophosphate and, as a result, protein kinase A activation [61]. As a result of its numerous adverse effects on humans, such as cardiomyopathy and acute hepatitis, clenbuterol has been banned in several nation [62].
Our study examined the effect of in ovo feeding of clenbuterol on fertility, hatchability, growth performance, and multiple molecular and physiological parameters concerning the pathway by which clenbuterol exerts its action. Table 3 shows the effects of in ovo feeding on growth and carcass traits, in which the clenbuterol-injected group at 15 ppm showed significantly higher weight gain and final body weight, and improved hatchability and fertility percentage. Additionally, there was a considerably enhanced carcass yield and significantly decreased abdominal weight percentage at 10 and 15 ppm doses of clenbuterol in other treated groups. Our obtained result was in agreement with those of previous studies [63,64]. They proved that b-adrenergic agonists could boost weight gain when added to feed, and the proportion of tissue fat is reduced [65]. These findings are attributed to increased nitrogen accrual and deterioration in the saturated fatty acid concentration [66].
Additionally, Spurlock, et al. [67] reported that clenbuterol administration stimulated anabolic activity. All previously mentioned studies support in ovo feeding of clenbuterol findings concerning weight gain and abdominal fat deposition. Our results are consistent with Hamano [68], who reported significant weight decreases in the abdominal fat in chicken fed 0.25 mg/kg of clenbuterol. In the same line, clenbuterol caused decreased abdominal fat [69]. Our histopathological results support clenbuterol's overall growth success, which may be due to the increased height of the villus in all small intestine segments. Moreover, with in ovo feeding, clenbuterol has no significant effect on RBC count, Hb level, or H/L ratio, as shown in Table 4. There are no marked changes in normal liver activity enzyme. Similarly, Mohamed et al. [20] found that clenbuterol at five and ten ppm had no significant effect on liver function and white blood cells, reflecting the nonstressful condition of in ovo clenbuterol administration.
In contrast, a significant increase in total protein and albumin levels was observed in the ovo clenbuterol treated group at 10 and 15 ppm. Takahashi et al. [64] found that clenbuterol enhanced the carcass protein, in which the beta-agonist eased protein breakdown and increased the metabolic protein rate [70]. Moreover, Mohamed et al. [20] reported that clenbuterol significantly increased the total protein concentration because it increased protein synthesis and decreased degradation [22].
Lipid markers decreased with in ovo clenbuterol feeding relative to saline feeding and control, as shown in Table 4. Our finding was in harmony with [20] who reported that lipid profile was reduced with clenbuterol administration in fish ascribed to the role of clenbuterol in the impaired synthesis of cholesterol in the liver and body fat adipocytes that affect its release to muscle tissue. Ijiri et al. [22] found that cholesterol decreased in chicks injected with clenbuterol. In ovo feeding of clenbuterol showed no effect on the overall antioxidant activity. Our result was supported by [71], who reported that clenbuterol administration had no significant effect on the antioxidant activity in the control group with an ischemia-induced injury in an isolated rat heart. In ovo feeding of clenbuterol showed no significant difference in phagocytic activity and lysosomal activity in different treated groups. As shown in Table 6, in ovo feeding of clenbuterol at 10 and 15 ppm significantly decreased (p < 0.05, p < 0.01) lower saturated fatty acid (myristic, stearic, and palmitic) content of the breast muscles compared to other treated groups.
Meanwhile, the current study revealed no significant variations of in ovo feeding of clenbuterol to modulate the PUFA contents in the breast muscles concerning other treated groups: α-linolenic acid, linoleic acid, and docosahexaenoic acid. These data were consistent with [72], in which they reported that beta-adrenergic agonist therapy reduced the unsaturated fatty acids and increased the saturated fatty acids in M. longissimus Dorsi steers treated with beta-adrenergic agonist and showed that stearic acid might be negatively cholesterolemic, which decreased the cholesterol level. No previous studies have examined the effect of in ovo clenbuterol feeding on the amino acid contour of chicken muscle. In this context, the current study suggested that, as shown in Table 7, in ovo feeding of clenbuterol at 10 and 15 ppm showed a marked increase in lysine, threonine, leucine, phenylalanine, methionine, valine, and isoleucine muscular contents compared with the control and normal saline groups.
Moreover, in ovo feeding of clenbuterol significantly increased the nonessential amino acid contents in chicken breast muscles, such as serine, alanine, arginine, proline, and aspartic acid at 15 ppm in other treated groups. These data may be accredited to improving the protein content of muscles compared to the control group. Kheiri and Alibeyghi [73] revealed that the carcass yield and growth performance could be upgraded with the increase in lysine and threonine levels, which supports our growth markers.
Additionally, it is crucial to investigate the transcriptomic pathway of clenbuterol concerning muscle growth. It was found that in ovo feeding of clenbuterol led to marked downregulation and myostatin gene expression (p < 0.01) in the control and normal saline groups. Similarly, Ijiri et al. [22] found that clenbuterol feeding resulted in decreased muscle myostatin expression. Myostatin is an essential regulator for skeletal muscle growth and contributes to clenbuterol-induced muscle growth and mass [74]. Lalani et al. [75] showed that IGF-1 had a positive regulatory impact on muscle growth. These reports supported our finding in which clenbuterol at 15 ppm had significant upregulation of IGF1r. These findings were consistent with [74,76]. They found that clenbuterol leads to IGF-1 upregulation. Moreover, Abo et al. [2] showed that an insulin-like growth factor is well established as a fundamental part of embryonic muscle development and proliferation. However, clenbuterol at concentrations of 10 and 20 ppm did not affect the IGF-1 receptor level. These data were in harmony with [22], who reported that clenbuterol feeding on a one-day-old chick did not change the IGF-1 expression. These data were also supported by IGFBP expression in which clenbuterol significantly upregulates IGFBP expression at 15 ppm concentration in other treated groups. These data were in line with [77], who reported that muscle growth stimulated by clenbuterol is coupled with a local increase in muscle IGFBP content.
GHR is a growth hormone transmembrane receptor, an important hormone for normal growth [78]. Clenbuterol significantly upregulates hepatic GHr expression; this result was consistent with those of [79], who reported the importance of growth hormone in the postnatal growth of skeletal muscles, and IGF-1 and GHR both have good growth regulators, which leads to anabolic effects on proteins and carbohydrate metabolism, and mediates growth hormone activity [80]. In the same context, clenbuterol feeding led to significant upregulation of PPARα in other treated groups (p < 0.05), (p < 0.01), (p < 0.05) for clenbuterol at 10 ppm, 15 ppm, and 20 ppm, respectively. PPARα is highly expressed in the liver and plays a key role in lipid metabolism-boosting fatty acid oxidation [81]. This supports our finding that in ovo clenbuterol feeding possibly lessens fat deposition by the oxidation of long-chain fatty acids in the mitochondria and fatty acid oxidation in the liver [82]. Clenbuterol feeding showed significant upregulation in LPL mRNA expression with markedly decreased FAS mRNA expression, and this was consistent with [20], who reported that clenbuterol upregulated expression of the liver LPL gene. Clenbuterol modulates mRNA expression levels through attenuation of lipogenic activity (downregulated levels of the FAS in the liver) and fatty acid oxidation (increased levels of LPL in the liver gene expression), which facilitate lipid catabolism in agreement with our results. Kim et al. [82] reported that clenbuterol increased the rate of lipolysis and decreased the lipogenesis rate for adipose tissues.
Moreover, there was a significant downregulation in SREBP-1c expression in clenbuterol groups than in other treated groups. Sterol regulatory element-binding proteins-1 and -2 (SREBP-1 and -2) are key transcript components implicated in cholesterol and fatty acid biosynthesis. Our data were in harmony with [9]. They reported that clenbuterol reduced the SREBP-1c expression, which supports our earlier result.

Conclusions
In ovo feeding of clenbuterol improved hatchability, fertility, and growth efficiency, promoted lipolysis, modulated lipid markers, and decreased abdominal fat. Moreover, clenbuterol enhanced poultry body gain via upregulation of insulin growth factor 1 receptor and insulin-like growth factor-binding protein 2 expression, downregulated myostatin gene, and increased protein synthesis. In addition, clenbuterol increased the intestinal villi without significant alterations in the histopathology of the bursa of Fabricius and spleen. The in ovo clenbuterol feeding led to higher oxidation of fatty acids and increased growth weight.