I. rats will be carried out as follows: 60

I.                   Study design:

This is an experimental study that will be conducted in research laboratories, Department of Medical Laboratories, College of Applied Medical Sciences, Qassim University.

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II.                Study subjects :

 The experiments on rats will be carried out as follows: 60 rats will be included in this study. They will be housed under hygienic conditions, in the animal house of the Applied Medical Science, Qassim University at 21°C–24°C in a 12 hr/12 hr light/dark cycle and will be freely fed on the laboratory food pellets, food and water during the two weeks of accommodation before starting the experiments.

Primarily 10 mL blood will be withdrawn from the all rat tails on EDTA as anticoagulant and separated equally. 5ml blood will be used for DNA extraction and determination the MTHFR gene. 

The remaining 5 ml of blood will be centrifuged at 4,000 rpm for 10 minutes to collect the plasma samples for estimating the FSH, LH, and testosterone levels. 

The animals will be divided according to MTHFR gene genotypes as follows: The genomic DNA will be extracted from the peripheral leucocytes. Polymerase Chain reaction –restriction fragment length polymorphism (PCR-RFLP) and will be used to detect the MTHFR C677T genotypes.  

According to the MTHFR C677T  genotypes the rats will be grouped according the genotypes to the: 

 MTHFR CC carriers (Group 1); MTHFR CT carriers (Group2) and MTHFR TT carriers (Group3).  

-All rat groups will be undergone to seminal fluid analysis in the beginning of the study.

-Sex hormones estimation (FSH , LH, testosterone ) will be measured to all rat different group genotypes using ELISA technique.

– Folic acid supplementation will be given to the C677T CT and TT genotype carriers who are supposed to have infertility for 4 weeks and re-examine the seminal fluid and sex hormones to measure the therapeutic effectiveness on male rats fertility who carry the MTHFR C677T SNP.   

II- Blood and Seminal Sample collection :

  Ten mL blood will be withdrawn from the rat tails of each rat on EDTA as anticoagulant and separated to 5ml each, the genomic DNA will be extracted from the peripheral leucocytes. Polymerase Chain reaction –restriction fragment length polymorphism (PCR-RFLP) and will be used to detect the MTHFR C677T genotypes.  

-The remaining 5 ml of blood will be centrifuged at 4,000 rpm for 10 minutes to collect the plasma samples for estimating the FSH, LH, and testosterone levels.

-Then at the end of the study the whole rats with different genotypes will be sacrificed for the measurements of plasma testosterone, FSH, and LH, along with sperm count, sperm motility, and sperm malformation rate, and any pathological disorders in the testicular tissue will be recorded seminal analysis       

– One testicle from each rat will be dissected and instantly fixed with 10% formalin solution for 24 hrs, then dehydrated with ethanol, embedded in paraffin, sectioned into slices, and hematoxylin-eosin (HE) will be used for staining for visualizing seminiferous tubules under the microscope.

III.             Measurement of sex hormones  (14):

Plasma samples for estimating the FSH, LH, and testosterone levels using ELISA kit. Results for the samples can be calculated directly using a standard curve (14).  

 

IV.             Estimation of plasma Homocysteine (Hcy) levels by ELISA kits(15 )

 Plasma samples for determination of Hcy levels using ELISA kit. Results for the samples can be calculated directly using a standard curve (15).  

V.                MTHFR C677T gene SNP Determination (16)(17) :

Ten mL blood will be withdrawn from the rat tails of each rat on EDTA as anticoagulant and separated to 5ml, the genomic DNA will be extracted from the peripheral leucocytes using Biospin Blood Genomic DNA Mini-prep Kit (BioFlux, Iran) (Bubbon, 1985) ( 16) . Polymerase Chain reaction –restriction fragment length polymorphism (PCR-RFLP) and will be used to detect the MTHFR C677T genotypes. (17)(Angeline, 2007)    

PCR amplification and PCR restricted segments detection: A 198 base pairs (bp) of the MTHFR gene will be carried on following this procedure: incubating the 120 g of the genomic DNA in 50 ul of both forward and backward primers of the MTHFR C677T SNP as follows: 5′-TGAAGGAGAAGGTGTCTGCGGGA-3′ and 5′-AGGACGGTGCGGTGAGAGTG-3′, respectively.

 Restriction enzyme HinfI at 37°C for 3-4 hours in the buffer will be used for restriction the 198 bp product of the MTHFR DNA segment , yielding to CC , CT and TT genotype fragment length as ( 198, 198, 175,23, and 175, 23 ) , respectively. 

The resulted PCR fragments will be separated applying the agarose gel (3%) including 5 mg/ml ethidium bromide and measured by 100 Base-Pair Ladder (Bioron).

 

VI.             Clinical Examination & Laboratory check-up

  -All participated examined rats will be fed freely with regular foods and water and will be weighed by the end of each week for one month. The MTHFR C677T CT and TT genotypes rat carriers will be fed with folic acid to test the effect of folic acid therapy on the improvement of the fertility status by estimation their sex hormones and seminal analysis.