The objective of this study was to establish the optimal conditions for hypoosmotic swelling (HOS) test to assess the functional integrity of the membranes of boar fresh or frozen/thawed spermatozoa. When pooled semen sample was incubated for 30 min at $37^{\circ}C$ with different test solution of varied osmolarity, the highest percentage of HOS positive spermatozoa was observed in a 150 mOsmol fructose/Na-citrate solution (33.6%). Incubation time did not affect significantly the score of HOS positive spermatozoa observed in a 150 mOsmol fructose/Na-citrate solution at $37^{\circ}C$, but the osmolarity affected the score of HOS positive spermatozoa under the same condition above. Fresh semen was significantly better than frozen/thawed semen in semen parameters evaluated such as motility, viability, membrane integrity and lipid peroxidation (p<005). In the relationships of sperm parameters, motility vs viability, motility vs membrane integrity and viability vs membrane integrity were positively correlated ($0.82{\sim}0.94$) but lipid peroxidation vs other estimated factors was negatively correlated ($- 0.90{\sim}- 0.98$). Among the evaluation methods, motility vs Viability, motility vs membrane integrity and lipid peroxidation vs other estimated factors were significantly correlated (p<0.05). These results of this. study indicate that the optimal condition of HOST in boar spermatozoa is a 150 mOsmol fructose/Na-citrate solution for 30 min incubation at $37^{\circ}C$ and HOST can substitute the examination of motility, viability and lipid peroxidation.
Objective: Several publications have established a relationship between sperm DNA damage and male factor infertility, based on data from America, Europe, and Asia. This study aimed to compare the extent of sperm DNA damage in sperm samples from Nigerian men with unexplained infertility and in sperm samples from a fertile group composed of sperm donors who had successfully impregnated a female partner naturally or through assisted conception. Methods: A total of 404 men underwent male fertility evaluation at Androcare Laboratories and Cryobank participated in this study. Semen analysis and a sperm chromatin structure assay (SCSA) were performed on all subjects. Results: The men in the unexplained infertility group were slightly older than the men in the fertile sperm group ($36{\pm}10$ years vs. $32{\pm}6$ years, p=0.051). No significant difference was observed between the two groups in semen analysis parameters ($p{\geq}0.05$). Men in the unexplained infertility group with normal semen parameters had a significantly higher DNA fragmentation index (DFI) than men in the fertile sperm group ($27.5%{\pm}7.0%$ vs. $14.1%{\pm}5.3%$, p<0.05). In the unexplained infertility group, 63% of the men had a DFI greater than 20%, compared to 4% in the fertile sperm group. In the unexplained infertility group, 15.2% of the subjects had a DFI greater than 30%, compared to 1% in the fertile sperm group. Conclusion: Our study showed that the SCSA may be a more reliable predictor of fertility potential than traditional semen analysis in cases of unexplained infertility.
Baek, Sun Young;Chung, Hak Jae;Hong, Joon Ki;Cho, Eun Seok;Choi, Inchul
Korean Journal of Agricultural Science
/
v.47
no.3
/
pp.657-665
/
2020
The objective of this study was to investigate whether supplementation of pentoxifylline (PTX; phosphodiesterase inhibitor) to thawed boar semen improves the post-thaw motility of sperm and affects the efficiency of artificial insemination (AI) and further development. To determine the concentration of PTX for AI, frozen-thawed semen was incubated with 0, 5, 10, and 20 mM PTX in an extender freezing medium, respectively, after thawing. Kinematic properties of sperm were examined with a computer-assisted semen analysis (CASA) system. In addition, viability and mitochondrial activity were also tested by LIVE/DEAD and a MitoTracker kit. There were no significant differences in the kinetic parameters of thawed sperm between control and treatment groups, but overall assessment parameters such as motility and rapid progressive were higher in the 10 mM PTX group. In the viability and mitochondrial assay, there were no significant differences observed in the PTX treatment, compared to the control. For further analysis, artificial inseminations were performed using frozen semen and 10 mM PTX treated cryopreserved semen, respectively. There were no differences in pregnancy rates and fetus weights among the groups until 30 and 40 days, but litter size was reduced and relatively low-birth weight was observed in the PTX group. In summary, our findings suggest that enhancement of in vitro sperm quality or non-toxicity supplemented by PTX may have detrimental effects on fetus development.
Nabi, Ali;Khalili, Mohammad Bagher;Eslami, Gilda;Vakili, Mahmood;Anbari, Fatemeh;Torki, Alireza
Clinical and Experimental Reproductive Medicine
/
v.49
no.1
/
pp.33-39
/
2022
Objective: Male genital tract infections have been associated with infertility, and Escherichia coli has drawn increasing attention as an important bacterium in this context. This investigation aimed to characterize and compare the distributions of O-antigen serogroups of E. coli in the semen samples of fertile and infertile men. Methods: In this case-control study, semen samples were collected from 618 fertile and 1,535 infertile men. The E. coli-positive samples were evaluated in terms of concentration, morphology, viability, and motility parameters according to the World Health Organization 2010 guidelines. Finally, different serogroups of E. coli were identified by multiplex polymerase chain reaction targeting the O-antigen variations of the bacterium. Results: The prevalence of E. coli among fertile men was significantly higher than among infertile men (p<0.001). The sperm morphology, viability, and motility in the E. coli-positive fertile group were significantly higher than in the E. coli-positive infertile group (p<0.001). E. coli O6 was the most prevalent serogroup found in both groups. However, there was no significant difference in the frequency of different serogroups of E. coil between the two groups (p=0.55). Conclusion: Despite the higher prevalence of E. coli among fertile men, E. coli had more detrimental effects on semen parameters in infertile men. There was no significant difference in E. coli serogroups between the fertile and infertile groups.
Objective: This study aims to identify heat shock protein70-2 (HSP70-2) and protamine-1 (PRM1) mRNA and protein in Madura bull sperm and demonstrate their relation as bull fertility biomarkers. Methods: The Madura bull fertility rates were grouped based on the percentage of first service conception rate (%FSCR) as high fertility (HF) (79.04%; n = 4), and low fertility (LF) (65.84%; n = 4). mRNA of HSP70-2 and PRM1 with peptidylprolyl isomerase A (PPIA) as a housekeeping gene were determined by quantitative real-time polymerase chain reaction, while enzyme-linked immunoassay was used to measure protein abundance. In the post-thawed semen samples, sperm motility, viability, acrosome integrity, and sperm DNA fragmentation index were analyzed. Data analysis was performed on the measured parameters of semen quality, relative mRNA expression, and protein abundance of HSP70-2 and PRM1, among the bulls with various fertility levels (HF and LF) in a one-way analysis of variance analysis. The Pearson correlation was used to analyze the relationship between semen quality, mRNA, proteins, and fertility rate. Results: Relative mRNA expression and protein abundance of HSP70-2 and PRM1 were detected and were found to be highly expressed in bulls with HF (p<0.05) and were associated with several parameters of semen quality. Conclusion: HSP70-2 and PRM1 mRNA and protein molecules have great potential to serve as molecular markers for determining bull fertility.
The aim of this experiments were to assess the time-interval change of motional characteristics in frozen-thawed semen of Korean native cattle (KNC) by using computer aided semen analysis (CASA) technology. Twenty-six KNC frozen semen straws were obtained from Korean KNC improvement department, livestock improvement main division, national livestock cooperatives federation in Korea. Specimens were allowed to thaw at $37^{\circ}C$ for 30 sec in water bath. Semen analysis was performed on semen image analysis system (SIAS, Medical supply, Korea) adjusted to the gate settings and used the semen droplet ($5{\mu}l$) placed on Makler counting chamber (Sefi medical instrument, Israel) prewarmed at $37^{\circ}C$. The same person used the same micropipette to fill the Makler counting chamber. A total of 150 or more of sperms were analysed in each specimen by a single trained person by scanning at least 5 to 10 fields. The measurement parameters in SIAS were as follows ; frame rate = 30 frames per sec, image capture = 1 sec, minimum motile speed = $10{\mu}m/s$, maximum countable sperm number = 400. Statistical analysis was done by Student t-test with use of the Sigma plot program on a IBM personal computer. The dancemean(DNM) and hyperactivated sperm(HYP) of frozen-thawed KNC semen kinematics were significantly decreased(p < 0.05) after 10 min of incubation at $37^{\circ}C$ water bath. But, wobble(WOB) of same sample semen was significantly increased(p < 0.05) after 10 min of incubation and significantly decrease(p < 0.05) after 60 min of same incubation. And, after 30 mim of incubation, significantly differences were found most of motion kinematics, motifity(MOT), curvilinear velocity(VCL), straight line velocity(VSL), average path velocity(VAP), amplitude of lateral head displacement(ALH), beat cross frequency(BCF), mean angular displacement(MAD), dance(DNC), on same sample semen. The DNM of KNC semen sample was variable kinematics after 30 min of incubation. Also, the linearity(LIN) and straightness(STR) was significantly decreased(p < 0.05) from 60 min of incubation. In conclusion, the AI within 30 min after thawing of frozen semen can be an effective method for obtaining high fertility rate in KNC reproductive program.
Present study analyzed the changes in peripheral blood testosterone concentrations and testicular cytogram in relation to age and semen quality in crossbred males. Three different age groups of crossbred males viz. bull calves (6 months, n = 5), young bulls (15 months, n = 5) and adult bulls (4 to 6 years, n = 8) were utilized for the study. Testicular fine needle aspiration cytology technique was used to quantify testicular cytology and their indices. Peripheral blood testosterone concentrations were measured using enzyme-linked immunosorbent assay method. Semen samples collected from adult bulls were microscopically evaluated for quality parameters. Mean peripheral blood testosterone concentrations in bull calves, young bulls and adult bulls were $2.28{\pm}0.09ng/mL$, $1.42{\pm}0.22ng/mL$ and $5.66{\pm}1.08ng/mL$ respectively, and that in adult bulls were significantly different (p<0.01) from young bulls and bull calves. There was no significant difference between the proportion of different testicular cells in bull calves and young bulls. Between young and adult bulls, significant differences (p<0.01) were observed in the proportion of spermatocytes, spermatozoa, and sperm: Sertoli cell ratio. The proportions of Sertoli cells showed a significant difference (p<0.01) between the three age groups. The number of primary spermatocytes had a positive correlation with peripheral blood testosterone concentrations in bull calves (r = 0.719, p<0.01). Number of Sertoli cells per 100 germ cells was negatively correlated with blood testosterone concentration in young bulls (r = -0.713, p<0.01). Among different semen parameters in adult bulls, ejaculate volume (r = 0.790, p<0.05) had positive relationship, and sperm motility had significant negative correlation (r = -0.711, p<0.05) with testosterone concentrations. The number of Sertoli cells and Sertoli cell index had a positive correlation with various semen quality parameters (p<0.001). Results of the present study conclude that number of Sertoli cells and Sertoli cell index are good indicators of semen quality, but peripheral blood testosterone concentrations may not have a direct relationship with various seminal attributes in crossbred bulls.
In this study the effect of extenders and temperatures on sperm viability and fertilizing capacity of boar sperm during long-term storage was investigated. Acrosomal integrity, membrane integrity, motility and hypo-osmotic resistance were evaluated by fluorescence and light microscopy. An in vitro fertilization test was performed to assess the fertilizing capacity of stored spermatozoa. The five diluents tested were ranked according to their ability to maintain sperm functional parameters and Zorlesco (ZO) extender with BSA or with PVA instead of BSA produced the best results. Zorlesco extender substituted with PVA (ZO+PVA) was found to maintain motility both at 15 and 20$^{\circ}C$. within 5 days of storage, but the quality of semen stored at 15$^{\circ}C$ decreased thereafter as compared to semen stored at 20$^{\circ}C$ Semen stored at 5$^{\circ}C$ demonstrated rapid loss of motility already within 24 h. Both fertilization and cleavage of semen stored at 20$^{\circ}C$ in ZO substituted with PVA instead of BSA did not change significantly until day 8 of storage. It is therefore concluded that PVA can be used to substitute for BSA and 20$^{\circ}C$ was more suitable than 15$^{\circ}C$ for boar semen storage, and in vitro fertilizing capacity of spermatozoa was maintained for at least 8 days in ZO+PVA at 20$^{\circ}C$.
Objective: This study investigated the effect of adding seminal plasma to frozen-thawed semen on the quality of sperm and pregnancy following insemination in dromedary camels. Methods: In experiment 1, the frozen-thawed semen from 9 collections (3 bulls) was further diluted with either the base extender or homologous seminal plasma (HSP). In the second experiment, a pooled sample of frozen-thawed semen was diluted with either seminal plasma from another three bulls. Live percentage, total and progressive motility, functional and acrosome integrity, and sperm kinematics were evaluated at 15, 60, and 120 minutes post-thawing and compared to the non-treated control. In experiment 3, frozen semen was used to inseminate camels in the following experimental groups: 1-Single insemination with double dose undiluted frozen semen (n = 9); 2-Re-insemination in 6 hours with undiluted semen (n = 13); 3-Single insemination with HSP treated sperm (n = 14). Results: Frozen-thawed sperm diluted in HSP or the non-homologous seminal plasma from Bull C indicated an improvement in all parameters after 1 hour post-thawing incubation (p<0.05). The proportion of total and progressively motile sperm did not drop significantly at 60 minutes post-thawing when diluted with the seminal plasma of Bull C (p>0.05). Double insemination with nontreated sperm and single insemination with HSP-treated sperm resulted in similar pregnancy rates (15.3% vs 21.4%, p>0.05). None of the camels conceived with double-dose single insemination of nontreated sperm. Conclusion: Seminal plasma improves sperm longevity and motility after thawing in dromedary camel with a significant between-bull variation in effect. Low post-thaw sperm longevity might be the cause behind the low pregnancy rates in frozen semen insemination of dromedary camels.
This study was designed to evaluate the changes in sperm motility, viability, HOST(hypo-osmotic swelling test), IVP(in vitro penetration), SCSA(sperm chromatin structure assay) during storage of liquid semen collected from boars with different farrowing rates using AI, and to find the relationship between boar fertility through AI and sperm diagnostic parameters during semen storage. The results of HOST were significantly decreased according to the increasing of in semen storage days and the results of IVP were significantly decreased at 3 days of semen storage (P<0.05). The %Red was significantly different among the >80%, 7080% and <70% farrowing rate group at semen storage day 6(P<0.05). The correlation coefficients between the %Red and farrowing rate were increased according to the semen storage. In conclusion, these results suggest that the sperm parameters evaluated in these studies may be useful indicators to predict the fertility of AI and evaluate the semen quality in boars.
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