MATERIALS AND METHODS

Experimental animals: Eleven growing male camels were a proximately 30 months of age and body weight average of 359.09±33.00 kg were used in this study. The camels were kept for 5 months finishing period, in which each camel was offered certain amounts of commercial concentrate mixture (12% crude protein), whereas berseem hay (Trifolium alexandrinum) was offered ad libitum. The amounts of concentrate mixture were adjusted every 2 weeks according to the live body weight change, so that the feed intake measured as DMI was adjusted to cover the nutritional requirements according to Farid et al.¹⁴.

Meat measurements: By the end of the finishing period, neck length, fore shank height and height at hump were recorded for each animal. Then, all camels were fasted for 24 h and then weighed and slaughtered according to the islamic rules. The ambient temperature on slaughter day was 21°C. After completion of bleeding, camels were skinned, eviscerated and dressed.

Weight of contents of the digestive tract was obtained and subtracted from the pre-slaughter weight to determine empty body weight of each camel¹⁵. Camel carcass was longitudinally split down at the middle line of the backbone into right and left sides. Right and left sides were divided into 4 and hind-quarters by cutting between the 11th and 12th ribs. Neck and hump were separated during the cutting process. Weights of the fore and hind-quarters, neck and hump were recorded. The carcasses were cooled at 4°C for 24 h and then weighted. Histological sections of camel Longissimus Dorsi (LD) were prepared and stained with hematoxylin and eosin stain according to the method described by Kiernan¹⁶. The morphometric analysis was performed using the Leica Qwin 500 Image Analyzer (LEICA Imaging Systems Ltd., Cambridge, England). The examined slides were placed on the stage of the microscope and focused at low power magnification (100X). The slides were screened to determine the boundaries of the tissue to be measured. Two bundles were assigned for each animal and the number of fibers in each bundle was obtained (Fig. 1).

Fig. 1:	Histological examination of the Longissimus Dorsi (LD) meat tissue, where A: Bundle and B: Fiber

Table 1:	PCR primers, annealing temperature (Ta) and amplicon size of the genes understudy

DNA extraction and purification: Before slaughtering the animals, blood samples were collected from jugular vein under aseptic conditions on vacationers containing EDTA as anti-coagulant and kept in the refrigerator until DNA extraction. The DNA was extracted from the blood using gene JET whole blood genomic DNA purification Mini Kit (Thermo Scientific K0721) following the manufacture instructions. The DNA concentration was measured on UV spectrophotometer (shimadzu Uv 2401 pc) at 260 nm and diluted to 50 ng μL^–1.

PCR amplification and DNA sequencing: The polymerase chain reactions were performed using specific primers for each gene part understudy. Full details of the primer sequences and their amplification conditions are presented in Table 1.

The PCR was performed in a reaction volume of 25 μL containing 100 ng of DNA, 0.2 mM of each primer, 1X PCR buffer, 2.5 mM MgCl₂, 0.2 mM of each dNTP and 0.5 U of green DreamTaq DNA polymerase (Thermo Scientific). The PCR cycle was: Initial denaturation at 94°C for 5 min followed by 35 cycles of denaturation 94°C for 30 sec, annealing (50-56°C) for 40 sec and extension at 72°C for 60 sec. The final extension was lasted for 10 min at 72°C. The PCR products were visualized on 2% agarose gel electrophoresis stained with ethidium bromide and the images were captured using bio-radgel documentation system. The PCR products were purified and sequenced at Macrogen Company (South Korea) using automated Genetic Analyzer 3730XL (Applied Biosystems Inc., USA).

Obtained sequences were compared with the available sequences in the international GenBank and analyzed by using the free online BLASTn software program (http://www. ncbi.nlm.nih.gov/) search engine of the National Center for Biotechnology Information (NCBI) and were aligned with the available dromedarius, bacterian and llama camels. All sequence alignments and distance calculations were made by Molecular Evolutionary Genetics Analysis software (MEGA version 6.0) developed by Tamura et al.¹⁹. In order to identify the effects of different SNPs, obtained sequences were converted to amino acids sequences using the software named: ExPASy translate tool-select²⁰.

Statistical analysis: Estimation of SNP frequencies and tests for deviation from Hardy Weinberg equilibrium were carried out using the Excel Microsatellite Toolkit²¹.

The relationship between normalized frequencies of (MYF5 and GH) and meat quality traits were calculated using Pearson correlation in PASW statistics 18.0 software (SPSS, Inc., Somers, NY, USA). Association analysis was carried out between frequencies SNPs and values of meat and carcass quality traits in animals using the least square means of General Linear Model (GLM ) procedure²².

RESULTS

Histological traits: The histological traits of camel meat were higher than the range of most meat animals²³. The present results of histological traits of meat (Fig. 1) indicated that camel meat had number of fibers 104.45±28.20. The histological traits of meat are the result of many factors including postmortem proteolysis, intramuscular fat, intramuscular connective tissue and the contractile state of the muscle are the most important characteristics that influence meat tenderness²⁴.

Myogenic factor 5 gene (MYF5) analysis: Successful PCR amplification for the gene (MYF5) produced a DNA fragment with different sizes in different animals and ranged from 400-422 bp (Fig. 2).

Sample of sequence analysis for the PCR products, their alignment with that in the GenBank is presented at Fig. 3.

Summary of the observed nucleotide changes for the animals understudy for the MYF5 gene are presented in Table 2. The number of nucleotide changes ranged from 7-25 with sequence identity ranged from 91-97%. Some nucleotide changes were found to be repeated in many animals in the nucleotide positions: 278, 305, 309, 317, 339, 377, 434, 515, 521, 522, 527, 528, 543, 549, 556, 557, 559, 570, 573 and 585. The SNPs identification showed one SNP at the nucleotide number 377, which was a change of T nucleotide to either A or C nucleotide with frequencies of 0.73 and 0.27, respectively.

The obtained sequences were submitted and accepted at the international GenBank and got the accession numbers: KR909026.1and KR909027.1.

Fig. 2:	PCR products for the myogenic factor 5 gene after running on agarose and staining with ethidium bromide showing a band size ranging from 400-422 bp. Lane 1: DNA marker size phi X 174/ HAIII, Lane 2: Negative control and Lane: 3-13 amplified samples

Fig. 3:	Example of sequence alignment for the MYF5 gene in dromedarius camel with the available sequence at the GenBank

Table 2:	Summary of the observed nucleotide changes in MYF5 gene for the dromedary camels understudy

Growth hormone gene: Since the primer set used in the amplification of the growth hormone gene region 5 and 3 UTR was taken from a previous study on llama camels, the PCR conditions were optimized to study on dromedarius DNA, the optimized cycle condition was: 94°C (60 sec), 56°C (45 sec), 72°C (60 sec) for 5 UTR and 94°C (60 sec), 50°C (45 sec), 72°C (60 sec) for 3 UTR.

Fig. 4:	PCR products for the growth hormone region 3 UTR after running on agarose and staining with ethidium bromide showing a band ranging from 221-244 bp. Lane 1: DNA size marker, Lane 2-12: Camel samples and Lane13: -ve control

Fig. 5:	Example of sequence alignment for the growth hormone gene 3 UTR region in dromedaius camel with the available at the GenBank

Figure 4 is showing the successful amplification of the growth hormone region 3 UTR, which produced a fragment of size length ranging from 221-244 bp.

Sample of sequence analysis for the PCR products, their alignment with that in the GenBank is presented in Fig. 5.

Summary of the observed nucleotide changes for the animals understudy for the UTR 3 of growth hormone gene are presented in Table 3. No SNPs were confirmed for this gene part in the animals studied.

Table 3:	Summary of the observed nucleotide changes for the dromedary camels understudy for the growth hormone 3 UTR region

Fig. 6:	PCR products for the growth hormone region 5 UTR after running on agarose and staining with ethidium bromide showing a band ranging from 600-630 bp, Lane 1: DNA size marker100 bp, Lane 2: -ve control and Lanes 3-13: Male of camels

Fig. 7:	Example of sequence alignment for the growth hormone 5 UTR region in dromedaius camel with the available at the GenBank

The obtained sequences submitted and accepted at the international GenBank and got the accession numbers: KR902742.1 and KR902743.1.

Regarding the amplification of the 5 UTR region of the growth hormone gene, the successful amplification produced a fragment of size length ranging from 600-630 bp (Fig. 6).

Sample of sequence analysis for the PCR products, their alignment with that in the GenBank is presented in Fig. 7.

Summary of the observed nucleotide changes for the animals understudy for the UTR 5 of growth hormone gene are presented in Table 4. The number of recorded SNPs ranging from 10-36 with sequence identity ranged from 92-99%.

The obtained sequences were submitted and accepted at the international GenBank and got the accession numbers: KR902744.1 and KR902745.1. Some SNPs were found to be repeated in many animals in the nucleotide positions: 91, 111, 116, 380, 419, 607, 621 and 669. The SNPs positions showed 2 hot spot at the nucleotides numbers 111(G>A, G>C) with frequencies of 0.73 and 0.27, respectively and 380 (G>A) with a frequency of 0.55.

DISCUSSION

It is well known that myogenic factor 5 is a protein that in humans is encoded by the MYF5 gene²⁵.

Table 4:	Summary of the observed nucleotide changes for the dromedary camels understudy for the growth hormone 5 UTR region

Table 5:	Correlations between different traits and the genomic parts understudy
*Significant at 5% level

Table 6:	Pearson correlation (r) between myogenic factor 5 gene (SNP 377) and phenotypic measurements in dromedary camel
*Single SNP correlation is significant at the 0.05 level (2-tailed), Allele frequencies of MYF5, 377 A>T are 0.67 and 0.33 and LD: Longissimus dorsi

It is a protein with a key role in regulating muscle differentiation or myogenesis. The MYF5 and MyoD, help the myogenic cells to progress normally during the determination stage of myogenesis. In this study, the myogenic factor 5 gene gave a band around 422 bp. This band size was similar to that reported by Shah et al.¹⁷ who studied the same exon part in the Pakistani dromedary camel. This study indicated that MYF5 gene is associated with carcass width at brisket and heart weight (Table 5). This gene has been reported to influence meat characters not only in camel but also in other mammalian species. The MYF5 gene is one of a family of 4 myogenic determination genes that control the skeletal muscle differentiation in many animals including: Pig²⁶, beef¹³ and sheep. In the present study, one SNP was found at the nucleotide number 377, this SNP was found to be associated with carcass width at brisket and fat thickness of longissimus dorsi in the dromedary camel (Table 6). The obtained sequences were converted into amino acid sequences using the ExPASy translate tool-select²⁰.

Table 7:	Pearson correlation (r) between growth hormone gene (SNP 380) and phenotypic measurements in dromedary camel
*Single SNP correlation is significant at the 0.05 level (2-tailed), Allele frequencies of GH, 380 G>A are 0.55 and 0.45

The conversion proved the change in the codon number 377 from A>T and the translation into the amino acids from methionine to lysine. This SNP may be used as a candidate gene for meat quality traits in dromedary camel. This SNP could be useful as a candidate gene for meat quality traits in dromedary camel. Regarding the growth hormone gene, it has been studied two parts: 3 UTR and 5 UTR regions. The PCR amplification of the 3 UTR regions produced a DNA fragment size of about 244 bp. This band size was similar to that reported by Daverio et al.¹⁸ after their study of the same region in llama (Lama glama). In this study, data cleared that the 3 UTR region is associated with neck length, fore shank height and height at hump (Table 5). In other hand, confirmed SNPs in the present study was not find. The genomic part 5 UTR of the camel growth hormone produced a DNA of band size in the range of 630 bp. This band size was similar to that reported by Daverio et al.¹⁸ for the same region in llama (Lama glama). It has been found that this genomic part is linked with dressing percentage with hump, dressing percentage with liver and number of fibers (Fig. 1). These results are similar to that reported by Afifi et al.²⁷ on dromedary camel. In the present study, it has been found two SNPs for this region at the nucleotides 111 (G>A or G>C) and 380 (G>A). The effect of these SNPs was found to be linked with many meat characters including: Dressing percentage with hump, dressing percentage with liver and number of fibers hump (Table 7 and 8).

Table 8:	Pearson correlation (r) between growth hormone gene (SNP 111) and phenotypic measurements in dromedary camel
**Single SNP correlation is significant at the 0.01 level (2-tailed), *Single SNP correlation is significant at the 0.05 level (2-tailed), Allele frequencies of GH, 111 G>A and 111 G>C are 0.73 and 0.27

Unfortunately there are no previous studies for the same region in camel to compare this results with. The camel Growth Hormone (GH) gene extends over about 1900 bp and like other mammalian GH genes, it splits into 5 exons and 4 introns¹¹. Growth hormone is a polypeptide hormone with diverse biological activities including somatogenic, lactogenic, insulin-like and diabetogenic effects. The biological effects of GH are coordinated through changes in tissue metabolism, including nutrient partitioning and thus can play a key role in increasing growth performance or milk yield²⁸.

CONCLUSION

Three SNPs were found in 2 genomic parts in the dromedarius camel. The first was at the 377 in the MYF5 gene, while the others were in the 5 UTR region of the growth hormone at nucleotides 111 and 380. These SNPs were found to associate with some meat characters and subsequently we recommend them to be taken as candidate genes for meat characters in camels.

ACKNOWLEDGMENT

This study has been achieved within the PROCAMED project funded by European Union within the program ENPI-CBC-MED, reference number I.B/1.1/493. The content of the present document is under the responsibility of the PROCAMED partners and could not be considered as the position of European Union.