ABSTRACT
Fetal DNA in maternal plasma and serum has been shown to be a useful material for prenatal fetal sex determination during early gestational ages. Non-invasive prenatal diagnosis is now possible at 8[th] week of pregnancy, by maternal blood sample testing. The purpose of this study was to evaluate two DNA extraction methods from mother plasma and its routine clinical application in bovine fetus gender determination with non-invasive method. Maternal blood samples were taken from 40 pregnant cows during the 8[th]-38[th] weeks of gestation. DNA was extracted from 350 micro l of maternal plasma with two salting-out and phenol-chloroform methods. The absorption in A[260] and purity [A[260]/A[280]] of extracted DNA were detected by ultraviolet spectrophotometer. Three micro l of the extracted DNA with phenol-chloroform method was used as a template. The PCR reaction was carried out to amplify the fragments of X and Y chromosomes of amelogenin, TSPY and BC1.2 genes. The difference between the mean absorption of DNA extracted by phenol-chloroform method and salting-out method was not significant in A260 [p>0.05, p=0.3549], but the difference between mean purity [A260/A280] of DNA extracted by phenol-chloroform method and salting-out method was significant [p<0.001]. X chromosome fragment was detected in all 40 samples and Y chromosome fragments were detected in 25 plasma samples which were delivered a male calf. The sensitivity and specificity of test was 100% with no false negative and false positive results. The results showed that phenol-chloroform method is a simple and sensitive method for isolation of fetal DNA in maternal plasma
ABSTRACT
In order to establish a reliable non-invasive method for sex determination in a bovine fetus in a routine setting, the possibility of identifying specific sequence in the fetal X and Y-chromosomes has been evaluated in maternal plasma using conventional multiplex polymerase chain reaction [PCR] analysis. The aim of this study was to provide a rapid and reliable method for sexing bovine fetuses. In this experimental study, peripheral blood samples were taken from 38 pregnant heifers with 8 to 38 weeks of gestation. DNA template was extracted by phenol-chloroform method from 350 micro l maternal plasma. Two primer pairs for bovine amelogenin gene [bAML] and BC1.2 were used to amplify fragments from X and Y chromosomes. A multiplex PCR reaction has been optimized for amplification of 467 bp and 341 bp fragments from X and Y bAML gene and a 190 bp fragment from BC1.2 related to Y chromosome. The 467 bp fragment was observed in all 38 samples. Both 341 and 190 bp fragments were detected only in 24 plasma samples from male calves. The sensitivity and specificity of test were 100% with no false negative or false positive results. The results showed that phenol-chloroform method is a simple and suitable method for isolation of fetal DNA in maternal plasma. The multiplex PCR method is an available non-invasive approach which is cost efficient and reliable for sexing bovine fetuses
Subject(s)
Animals , Animals, Laboratory , Multiplex Polymerase Chain Reaction , Prenatal Diagnosis , Pregnancy , DNA , Cattle , Pregnancy, AnimalABSTRACT
Pentoxifylline [PX] is a methyxanthin derivative that influences the sperm motion characteristics. In general, PX has been reportedly effective in preserving sperm motility in vitro, also when administered orally to the asthenozoospermic patients. The main objective of this prospective clinical trial study was to rule out the effect of oral administration of PX on sperm progressive motility of asthenozoospermic ejaculates obtained from men with or without mild testicular varicoceles. In addition, the role of patient's age on sperm motility following PX administration was investigated. A total of 68 infertile men with asthenozoospermia were allocated to this study. Following physical examination, 20 cases were found with mild varicocele of testis. A dosage of 400 mg PX/ twice daily for duration of 3 months was administered to each patient. Two semen samples [one before and one after the PX therapy] were evaluated under blind condition. Semen parameters of sperm concentration, total and fast progressive motility [%] and morphology [%] were analyzed for each sample. Also, the sperm motion characteristics of asthenozoospermic patients with testicular varicocele were compared with cases lacking varicocele. The subjects were divided into two age groups of <30 and >/= 30 years old. PX was significantly effective on the fast progressive motility of sperm [p<0.01]. Also, total progressive motility was enhanced from 26.82 +/- 16.8 to 29.60 +/- 22.2 with PX therapy. However, PX did not have any negative effect on other semen parameters. Oral therapy of PX was also effective in improving the fast progressive motility of sperm of samples from cases with or without mild testicular varicocele [p<0.01]. Fast progressive motility was also significantly enhanced in ejaculates of men from both age groups. Our results demonstrate that low dose of oral therapy of PX is significantly useful in enhancing fast progressive motility of sperms from infertile men with asthenozoospermia. Also, testicular varicocele did not interfere with enhancing effect of PX on sperm motility