Fresh semen from fifty bulls was evaluated for ejaculate volume, individual motility, concentration, lives sperm and abnormalities as well as acrosome integrity. Bulls were classified according to semen motility into two groups; good and poor. DNA was extracted from semen of both groups, then the PCR followed by single-strand conformation polymorphism (SSCP) techniques were performed for mutation detection in gonadotropin releasing hormone receptor (GnRHR) gene through multiple sequence alignment. The results showed that the percentages of sperm motility, sperm concentration and live sperm, were significantly higher in good than poor semen quality bulls. However, semen volume, percentage of acrosome integrity and abnormalities did not differ between good and poor. The PCR amplification of 240-bp fragment and the results of SSCP appeared a genetic polymorphism with two patterns. Pattern I was seen in all good bulls, with incidence of 100%. Pattern II appeared only in poor semen quality bulls with a frequency of 31.25%. The sequence analysis of the PCR product for the two patterns showed two single nucleotide polymorphisms (SNPs) as a transversion base substitution mutation at positions 20 (T/A) and 193 (A/T). The GnRHR gene could be used as a genetic marker related to semen quality in buffalo due to the good semen bulls had a unique pattern.
HIS STUDY aimed to evaluate the sperm characteristics in buffalo characteristics in buffalo bulls and to screen the genetic genetic polymorphisms in PIT-1 gene as bases for selection of bulls with good breeding value. The study was performed on 60 buffalo bulls aged 2-8 years. The animals were divided into three groups according to the age. The first group were between 2 to <3 years (n= 35). The second group were between 3 to <5 years (n=14). The third group were between 5 to <8 years (n=11).Three semen collections were obtained from each animal at 15-day intervals and evaluated for volume, individual motility, live sperm and chromatin integrity %. The semen samples were stored at 20 °C until DNA extraction then polymerase chain reaction (PCR) and DNA amplification were carried out. Restriction fragment length polymorphism (RFLP) was used for genotyping of pituitary-specific transcription factor gene using HinfI-RFLP. The results showed that, ejaculate volume, individual motility and live sperm % were significantly lower in old than adult and young buffalo bulls. While chromatin damage percentage had no significant difference among groups. All buffalo bulls were genotyped as BB with the predominance of B allele where PCR 451 bp fragment was digested into two fragments 244 and 207 bp. It may be concluded that age had adverse effect on semen quality. M onomorphic pattern of the amplicon 451 bp in PIT-1HinfI locus in exon 6 was fixed in Egyptian buffalo with the predominance of B allele and BB genotype in a high frequency (100%).
Background Extracellular vesicles (EVs) are a promising biomarker and play a vital role in cell–cell communication. This study aimed (I) to identify and characterize EVs from low volume uterine lavage (LVL) and serum in mares with endometritis, compared to healthy controls and (II) to measure serum levels of interleukin 6 (IL-6), and prostaglandins (PGF2α and PGE2). Mares were divided into 30 sub-fertile (endometritis) and 20 fertile (controls). Serum and LVL was collected for EV isolation, and determination of serum levels of inflammatory mediators. Characterization and visualization of EVs were done by electron microscopy, dynamic light scattering and flow cytometry. Results Serial ultracentrifugation of LVL and use of a commercial kit for serum were strategies for EVs isolation. Mares with endometritis released higher amounts of larger size EVs. The EVs from mares with endometritis differentially expressed CD9 and CD63, compared to controls. Mares suffering from endometritis evoked higher levels of inflammatory mediators. Conclusions Thus, EVs could be used for a better understanding the regulatory mechanisms associated with developing endometritis in mares.
T HIS study investigated the genetic polymorphism of osteopontin (OPN) gene in Egyptian buffalo bulls in a trial for association with the quality of fresh semen. A total of 228 fresh semen ejaculates were collected from 57 buffalo bulls. Checking the fertility potential, the ejaculates were evaluated for volume, individual motility, live sperm, sperm abnormalities and concentration. The bulls' semen was grouped according to the individual motility into high (>60%, n=47) and low (<60%, n=10) quality. A fragment of 250 bp from intron 3 of OPN gene was amplified by PCR then genotyped by restriction fragment length polymorphism, single strand conformation polymorphism and DNA sequencing. The high-quality semen was significantly increased in individual motility % (p<0.0001), live sperm % (p<0.0001) and sperm concentration (p<0.05) than the low quality. Also, the monomorphic pattern of OPN gene in both groups of bulls was noticed. However, five mutations were discovered including three nucleotide insertions (T 44, A49 and A62) and two nucleotide substitutions (G11>C and A108>G) when comparing the sequence with that of buffalo in the GenBank. In conclusion, OPN gene might have no genetic variation and so it is not associated with semen quality in Egyptian buffalo bulls. Future studies with a larger number of populations on different regions of OPN gene are recommended.
The current study aimed to determine the genetic polymorphism of sex determining factor or sex determining region, Y-chromosome gene (SRY) and the association of semen parameters with field fertility of Egyptian buffalo bulls. Fresh semen ejaculates (n=264) from 66 bulls were collected and evaluated. The bulls (n= 56), with ejaculates ≥ 60% motility, were categorized according to pregnancy rate (PR) into high (PR≥50%, n=47) and low (PR<50%, n=7) fertile. SRY gene polymorphism was identified by the restriction fragment length polymorphism, the single strand conformation polymorphism, and the nucleotide sequencing. The results revealed a significant difference between high and low fertile bulls in PR (P< 0.001). A significant negative correlation (r=-.369, P< 0.01) was shown between sperm abnormalities and PR and a significant positive correlation (r=.273, P< 0.05) was obvious between live sperm and PR. The SRY gene showed no genetic variation among bulls. The Basic Local Alignment Search Tool (BLAST) of the sequence showed 100% identity with Pakistani buffaloes. In conclusion, sperm abnormalities and live sperm can be used as good predictors for fertility in buffalo. PR is more accurate than semen analysis for evaluating fertility. The SRY gene could be highly conserved in Egyptian buffalo. Further research is recommended by expanding SRY gene fragment and increasing number of samples.
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