Experimental Animals
The current study was conducted on Barki sheep flock, belongs to Maryout Research Station, 35 km south of Alexandria, Desert Research Center, Ministry of Agriculture and Land Reclamation, Egypt. Thirty-eight Barki lambs were kept and fed individually after weaning at 3 months of age (Initial body weight) till to 12 months of age (Final body weight). The lambs were kept in a cubical cement box of dimensions (120 width*150 length*135 height cm), and has access to water and ration. Lambs were fed as per standard schedule (NRC, 1985) to cover their nutritional requirements. Lambs were fed a certain amounts of commercial concentrate mixture (12% crude protein) plus alfalfa hay (Trifolium alexandrinum). However, the amounts of concentrate feed mixture (CFM) offered to lambs were adjusted bi-weekly according to the live body weight changes. Moreover, water was available all the time for experimental animals.
Experimental Design
Lambs were weighed weekly after weaning and divided into 3 groups according to their growth rates (Fast, intermediate and slow growing). In addition, three samples of main body tissues (eye muscle, liver and fat tail) from three animlas representing each group were taken for gene expression profiling at the end of the fattening period (12 months of age). The genes selected in real-time PCR expression are regulators of different molecular pathways such as protein biosynthesis (RPL7), fat deposition or lipogenesis (ADIPOQ), fatty acid oxidation or lipolysis (CPT1 and FABP4) and muscle development (CAPN3).
Slaughtering procedure and carcass trait
Twenty-three lambs were slaughtered at 12 months of in meat processing unit at Maryout Research Station to evaluate carcass traits following the stranded protocol (Frild et al., 1963) lambs were fasted for approximately 24 hours before slaughtering. After slaughtering and bleeding, carcass were skinned and eviscerated before weighing. Weights of all non-carcass components (trachea, lungs, heart, liver, testes, spleen, kidneys, and kidney fat) were immediately weighed after removal from the body. The rumen and reticulum were cleaned and washed under cold running water, and then they were weighed. Empty body weights were recorded and all carcasses were held in a chiller at 4 °C for 24 h to evaluate cold carcass weight (Frild et al., 1963).
Blood and tissue sampling
Nine blood samples (3 from each group) were collected during slaughtering in tubes that contain EDTA as anticoagulant substance. Tissue samples (liver, tail fat, and muscle) were taken immediately from lambs after slaughter and kept in RNA later then kept at -80 ºC till RNA extraction was performed in Cairo University Research Park, Faculty of Agriculture, Cairo University, Egypt.
Analysis of blood T3 and T4 hormones profile
Blood samples were collected in tubes containing EDTA as anticoagulant substance. Samples were centrifuged at 3000 rpm for 20 minutes. Plasma was stored at -20 ºC until estimation of T3 and T4 hormones using an enzyme immunoassay test kit (Chemux Bioscience Inc, USA, CA). The sensitivity value reported to be according to manufacturer information. The intra and inter-assay variation coefficients were 5.0 and 13.0%, respectively.
Measurement of blood total protein level
The profile total protein (g/dl) was done using colorimetric methods according to instructions provided by the manufacturer company (Bio diagnostic, Giza, Egypt). The plasma samples (0.025 ml) were mixed well with biuret reagent (1.0 ml) and incubated at 37°C for 10 min the absorbance for standard and samples were measured using the spectrophotometer at 550 nm wavelengths.
Measurement of blood glucose concentration
The blood glucose profile (mg/dl) was measured using colorimetric methods according to instructions provided by the manufacturer company (Bio diagnostic, Giza, Egypt).
Measurement of blood total lipids level
The blood total lipids (mg/dl) was measured using colorimetric methods according to instructions provided by the manufacturer company (Bio diagnostic, Giza, Egypt).
Measurement of blood calcium level
The blood calcium level (mg/dl) was measured using colorimetric methods according to instructions provided by the manufacturer company (Bio diagnostic, Giza, Egypt).
Gene expression Profile
Total RNA extraction
The procedure of RNA isolation was performed using GeneJet RNA purification kit (Thermofisher Scientific, Vilnius, Lithuania) according to manufacture instructions. Approximately 20 mg of tissue was weighed and grind with a pestle in a mortar using liquid nitrogen till powder was formed. The powder was transferred to a 1.5 ml micro-centrifuge tube with 300μl of lysis buffer and 20μl of β-mercaptoethanol and vortexed for 20 seconds. A volume of 600μl of diluted Proteinase K (10μl of included Proteinase K diluted in 590μl of Tris-EDTA (TE) buffer) was added and vortexed for 20 minutes then incubated for 10 minutes at room temperature and finally centrifuged for 10 minutes at 12000 xg. A volume of 450μl of ethanol was added and mixed by pipetting. 700μl of lysate was transferred to the GeneJet RNA purification column and was inserted into a collection tube and then it was centrifuged for 1 minute at 12000 xg.
The flow through was discarded and placed the purification column in the tube. The previous step was repeated until all the lysate was transferred and centrifuged. The collection tube was discarded, which contained the flow though solution. The GeneJet RNA purification column was placed into a new 2ml collection tube. A volume of 700μl of wash buffer 1 was added to the GeneJet RNA purification column and centrifuged for 1 minute at 12000 xg. The flow through was discarded and the purification column was placed back into the collection tube, then 250μl of wash buffer 2 was added to the GeneJet RNA purification column and centrifuged at 12000 xg for 2 minutes.
The collection tube containing the flow through solution was discarded and the GeneJet RNA purification column was transferred to a sterile 1.5 ml RNase free micro-centrifuge tube. Nuclease free water (100μl) was added to the GeneJet RNA purification columns and centrifuged at 12000 xg for 1 minute. The purification column was discarded. For DNA digestion, 1 ml of DNAse (Thermo Scientific, California, USA) was added to 9 μl of RNA sample and the mixture (10 μl) was incubated in a thermal cycler for 30 minutes at 37°C. After that, 1 ml of EDTA was added to the mixture. The mixture (RNA, DNAse, and EDTA) was incubated in a thermal cycler (Thermofisher Scientific, CA, USA) for 10 minutes at 65°C. Finally, the purity and concentration of extracted RNA were measured at A260/280 nm ratio (1.9-2.1) using NanoDrop 2000C (Thermofisher Scientific, Wilmington, DE, USA). The samples were stored at -80°C freezer until cDNA synthesis.
cDNA synthesis
After adjusting the RNA concentration of all isolated RNA samples, synthesis of cDNA was done using revertAid First Strand cDNA Synthesis Kit (Thermofisher, USA). The reaction mix consisted of 1 μl of oligo (dT) 18 primer to 11 μl of the adjusted RNA, 4 μl of 5x reaction buffer, and 2 μl of 10 mM dNTP for each RNA sample. Finally, 1 μl of RiboLock RNase inhibitor and 1 μl of revertaid reverse transcriptase was added to reach a final volume of 20 μl and samples were incubated in a thermal cycler (Thermofisher Scientific, CA, USA) for 60 minutes at 42°C followed by 70°C for 5 minutes. The cDNA samples were stored in -20°C freezer till used for real-time PCR runs.
Quantitative Real-time PCR
A pair of primers (forward and reverse) was designed for each specific gene (GAPDH, ADIPOQ, CPT1, FABP4, RPL7and CAPN3) using Primer3 software (http://primer3.wi.mit.edu//) as shown in Table 1. The design of primers was based on gene sequences described in the GenBank database (www.ncbi.nlm.nih.gov). Real-time PCR was performed using glyceraldehyde 3-phosphate dehydrogenase (GAPDH) as housekeeping gene The real-time PCR was done in the 96 well plate (Thermofisher Scientific, Wilmington, DE, USA). The real-time PCR reaction mix is composed of 2μl of cDNA sample, 12μl of Maxima SYBR Green/ROX qPCR Master Mix (Thermofisher Scientific, CA, USA), 0.2μl of specific reverse primer, 0.2μl of specific forward primer and 7.6μl of nuclease free water. Reaction was incubated in StepOnePlus™ Real-Time PCR (Applied Biosystems, CA, USA). The reaction mix was incubated at 50°C for 2 minutes; initial denaturation was done for 10 minutes at 95°C and 40 cycles at 95°C (Denaturation) for 15 minute and final step at 60°C for 1 minute (annealing), at 95°C for 15 seconds and then 60°C for 1 minute. The results were expressed as Ct values and relative gene expression profile was estimated delta delta Ct analysis (2-ΔΔCT method) as done in our recent study (Ghanem et al., 2021).
Statistical analysis
The growth performance data and gene expression profile of the three different lambs of each experimental group (Fast, intermediate and slow growing animals) were analyzed using the procedure of General Linear Model (SAS, 2011). The statistical analysis was performed to test the effect of animal growth rate, carcass traits and biochemical measurements on gene expression profile of selected candidate transcripts by applying the following formula:
Yij = µ + Gi + eij.
Y ijk= individual observation; µ= Overall mean; Gi= Effect of growth rate; eij = Experimental error; i = 1,2,3 (fast, intermediate and slow).
The mean values were compared for statistical significance using Duncan’s range test (1955). Differences were considered statistically significant at P ≤ 0.05.