• Title/Summary/Keyword: sperm quality

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Development of Sperm MTT Assay for Its Application in Boar Semen

  • Jang, Hyun-Yong;Lee, Hee-Young;Cheong, Hee-Tae;Kim, Jong-Taek;Park, In-Chul;Park, Choon-Keun;Yang, Boo-Keun
    • Journal of Embryo Transfer
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    • v.25 no.4
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    • pp.229-235
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    • 2010
  • The MTT assay is one of superior evaluation methods widely used to analyze the viability of metabolically active cell. It can be used to determine the percentage of viable sperm through measurement of the reduction of MTT granules at mitochondria in sperm tail. The purpose of this study is to determine the optimal condition of a simple and easy MTT assay to validate boar sperm viability and compare the accuracy of this test with microscopic examination. The MTT reduction rate for sperm viability were analyzed in microtiter plates (96 well) from 1 hr to 5 hr incubation periods at $37^{\circ}C$ using spectrophotometer (microplate reader) at 550 nm wavelength. The remainder of semen sample was simultaneously examined to compare the correlation of accuracy between MTT assay and other sperm parameters. Those sperm parameters were included the motility, survival rates, membrane integrity, mitochondria activity and acrosome integrity. The OD values of MTT assay (MTT reduction rates) did not greatly change at 1 hr to 5 hr incubation periods in different proportion of live and freeze-killed sperms (dead sperm). The MTT reduction rates or survival rates were decreased according to the different concentration of live and dead sperm. The linear regression at 1 hr and 4 hr incubation periods in sperm MTT assay was y=291.55x-72.176 and y= 180.64x-44.569, respectively. There are high correlation between 1 hr and 4 hr incubation periods (p<0.001). The results of MTT assay and other sperm parameters has a positive correlation (p<0.01 or 0.05). The correlation coefficients for MTT assay was 0.88115 for motility, 0.89868 for survival rates, 0.91722 for membrane integrity and 0.77372 for acrosome integrity, respectively. In conclusion, the MTT assay can be used as a reliable and efficient evaluation method for boar sperm viability. It can be use practical means to evaluate the quality of boar sperm by a fast, inexpensive and easy method.

Mouse Embryo Culture as Quality Control for Human In Vitro Fertilization (생쥐 체외수정 정도관리의 유용성에 관한 실험적 연구)

  • Lim, Young-Kyung;Park, Hyun-Jeong;Lee, Yu-Il
    • Clinical and Experimental Reproductive Medicine
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    • v.18 no.1
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    • pp.49-53
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    • 1991
  • The development of 2-cell mouse embryos to the blastocyst stage in vitro has been used as a quality control for the media empolyed for human in vitro fertilization. There was a comparison between the quality control data of the culture medium as ascertained by 2-cell mouse embryos development and sperm motility and the data from fertilization and cleavage of human oocytes. However, there was no obvious association between fertilization and cleavage of human oocytes and the quality of the medium ascertained by mouse embryo development and sperm motility.

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Effect of Glycosaminoglycans on In vitro Fertilizing Ability and In vitro Developmental Potential of Bovine Embryos

  • Kim, Eun Young;Noh, Eun Hyung;Noh, Eun Ji;Park, Min Jee;Park, Hyo Young;Lee, Dong Sun;Riu, Key Zung;Park, Se Pill
    • Asian-Australasian Journal of Animal Sciences
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    • v.26 no.2
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    • pp.178-188
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    • 2013
  • The glycosaminoglycans (GAGs) present in the female reproductive tract promote sperm capacitation. When bovine sperm were exposed to 10 ${\mu}g/ml$ of one of four GAGs (Chondroitin sulfate, CS; Dermatan sulfate, DS; Hyaluronic acid, HA; Heparin, HP) for 5 h, the total motility (TM), straight-line velocity (VSL), and curvilinear velocity (VCL) were higher in the HP- or HA-treated sperm, relative to control and CS- or DS-treated sperm. HP and HA treatments increased the levels of capacitated and acrosome-reacted sperm over time, compared to other treatment groups (p<0.05). In addition, sperm exposed to HP or HA for 1 h before IVF exhibited significantly improved fertilizing ability, as assessed by 2 pronucleus (PN) formation and cleavage rates at d 2. Exposure to these GAGs also enhanced in vitro embryo development rates and embryo quality, and increased the ICM and total blastocyst cell numbers at d 8 after IVF (p<0.05). A real-time PCR analysis showed that the expression levels of pluripotency (Oct 4), cell growth (Glut 5), and anti-apoptosis (Bax inhibitor) genes were significantly higher in embryos derived from HA- or HP-treated sperm than in control or other treatment groups, while pro-apoptotic gene expression (caspase-3) was significantly lower in all GAG treatment groups (p<0.05). These results demonstrated that exposure of bovine sperm to HP or HA positively correlates with in vitro fertilizing ability, in vitro embryo developmental potential, and embryonic gene expression.

Effects of Storage Time on Bacteria Concentration and Sperm Parameters in Boar Semen (돼지 액상정액 보존 일수에 따른 정액내 세균과 정자 기능의 변화)

  • Chung, Ki-Hwa;Kim, In-Cheol
    • Reproductive and Developmental Biology
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    • v.36 no.3
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    • pp.163-166
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    • 2012
  • This study was conducted to determine the relationship between elapsed time after semen preservation on the changes of bacteria and semen quality. Semen was diluted with BTS(Beltsville Thawing Solution) extender without antibiotic for 7 days and sperm parameter and fertility were measured. Sperm motility was measured by CASA and total bacteria number was counted after 22~24 hr incubation from counting agar plate in which sperm dilute to $10{\sim}10^6$ in 0.9% saline solution and inoculate to agar. Acrosomal integrity was measured by Chlortetracycline (CTC) staining. CTC patterns were uniform fluorescence over the whole head (pattern F), characteristic of incapacitated acrosome-intact spermatozoa; fluorescence-free band in the post-acrosomal region (pattern B), characteristic of capacitated acrosome-intact spermatozoa; and almost no fluorescence over the whole head except for a thin band in the equatorial segment (pattern AR), characteristic of acrosome reacted spermatozoa. Total number of bacteria was significantly increased (p<0.0001) 3 days after preservation. Sperm motility, viability, and morphological abnormality on elapsed time after preservation were lower from 5 ($77.24{\pm}6.47$, p<0.001) and 7 days ($77.24{\pm}6.47$, p<0.001) after preservation compared to 1 ($15.71{\pm}7.18$) and 3 days($18.39{\pm}7.22$) after preservation, respectively. Sperm viability was significantly lower ($53.25{\pm}35.03$, p<0.0001) at 7 days after preservation. Morphological abnormality of sperm was lower (p<0.001) at 1 ($15.71{\pm}7.18$) and 3 ($18.39{\pm}7.22$) days compared to 5 ($21.84{\pm}7.91$) and 7 ($22.59{\pm}9.93$) days after preservation. Acrosomal integrity and capacitation rate (pattern F) were significantly lower (p<0.001) from 5 days after preservation. Based on the data we obtained from this study suggested that semen preserved more than 5 days without antibiotic would not recommend use for artificial insemination.

Sleep deprivation induces structural changes in the adult rat testis: The protective effects of olive oil

  • Fatemeh Karimi;Ali Noorafshan;Saied Karbalay-Doust;Maryam Naseh
    • Clinical and Experimental Reproductive Medicine
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    • v.50 no.1
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    • pp.19-25
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    • 2023
  • Objective: Sleep deprivation (SD) is a common problem in today's stressful lifestyle and have physiological consequences, including reproductive dysfunction and infertility. As an antioxidant, olive oil may be effective in reducing testicular and spermatological damage by decreasing the production of free radicals. Methods: This study investigated the effects of olive oil on sperm quality and testicular structure using stereological methods to assess rats with SD. Results: When comparing SD group to grid floor+distilled water (GR) group, we found that the sperm count and motility, as well as the percentage of slow progressive sperm was significantly lower in SD group (p<0.05), but the percentage of immotile sperm was higher (p<0.01). However, no improvement was observed in sperm count or motility after concomitant treatment of SD group with olive oil. Stereological examinations revealed no significant change in the total volumes of the seminiferous tubules, interstitial tissue, and germinal epithelium in the study groups. Conversely, the total number of testicular cell types was significantly lower in SD group than in GR group. Although the total number of Sertoli and Leydig cells was significantly higher in the S +olive oil group than in the untreated SD group, no significant difference in the total number of other testicular cell types was observed between the two groups. Conclusion: SD potentially induced structural changes in testis that affected sperm count and motility. However, olive oil only improved the total number of Sertoli and Leydig cells in the animals with SD and did not improve sperm count and motility.

Degenerative changes in testis, epididymis, and sperm quality in ICR mice treated with methoxychlor and bisphenol A

  • Juyeon Hong;Donghun Kang;Daeyoung Kim
    • Journal of Animal Reproduction and Biotechnology
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    • v.37 no.4
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    • pp.276-284
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    • 2022
  • Endocrine-disrupting chemicals found in many commercial products may interfere with the normal functioning of the endocrine system and are unsafe because of their cumulative effect on the human body. However, little is known about the effects of combinations of endocrine-disrupting chemicals in humans. Methoxychlor and bisphenol A are toxic to male reproductive organs. Therefore, we studied the effects of methoxychlor and bisphenol A on male reproductive function. Male mice were divided into four treatment groups: control, 400 mg methoxychlor, 1 mg bisphenol A, and 400 mg methoxychlor + 1 mg bisphenol A/kg/day. Methoxychlor and bisphenol A were dissolved in sesame oil and acetone and administered orally for 4 weeks. After administration, the weight and histological changes in the testicles and epididymis, sperm count and health were observed biochemical tests and whole blood counts were performed. The results showed that the mice in the bisphenol A and methoxychlor + bisphenol A groups gained more weight than those in the control and methoxychlor group. The weights of the testes and epididymis were higher in the experimental groups than in the control. Sperm motility and progression were significantly reduced in the bisphenol A and methoxychlor + bisphenol A groups. Histological observation showed a reduced number of sperm, smaller seminiferous tubules, and destroyed lumen in the methoxychlor + bisphenol A group compared to the other groups. In conclusion, our study showed that methoxychlor and bisphenol A destroy male reproductive tissues and decrease sperm quality.

Melatonin and selenium supplementation in extenders improves the post-thaw quality parameters of rat sperm

  • Shahandeh, Erfan;Ghorbani, Mahboubeh;Mokhlesabadifarahani, Tahereh;Bardestani, Fateme
    • Clinical and Experimental Reproductive Medicine
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    • v.49 no.2
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    • pp.87-92
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    • 2022
  • Objective: The aim of this study was to determine the effects of melatonin and selenium in freezing extenders on frozen-thawed rat sperm. Methods: Semen samples were collected from 20 adult male Wistar albino rats. Following dilution, the samples were divided into six groups: four cryopreserved groups with 1 mM and 0.5 mM melatonin and selenium supplements, and two fresh and cryopreserved control groups. The rapid freezing technique was used to freeze the samples. Flow cytometry was used to assess plasma membrane integrity, mitochondrial membrane potential, and DNA damage, while computer-assisted sperm analysis was used to assess motility. Results: Total motility was higher in the 1 mM melatonin supplementation group than in the cryopreserved control group (mean±standard error of the mean, 69.89±3.05 vs. 59.21±1.31; p≤0.05). The group with 1 mM selenium had the highest plasma membrane integrity (42.35%±1.01%). The cryopreserved group with 0.5 mM selenium had the highest mitochondrial membrane potential, whereas the cryopreserved control group had the lowest (45.92%±4.53% and 39.45%±3.52%, respectively). Conclusion: Cryopreservation of rat semen supplemented with 1 mM melatonin increased sperm motility after freeze-thawing, while supplementation with 0.5 mM selenium increased mitochondrial activity.

Long-Term Exposure of Sildenafil Citrate on Sperm Parameters in Rat

  • Suresh, Sekar;Prithiviraj, Elumali;Venkatalakshmi, Nagella;Ganesh, Mohanraj Karthik;Ganesh, Lakshmanan;Lee, Hyun-Jeong;Prakash, Seppan
    • Reproductive and Developmental Biology
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    • v.35 no.4
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    • pp.435-439
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    • 2011
  • Sildenafil citrate (SIL) a phosphodiesterase 5 inhibitor (PDE5I) has been used for long time as a first line oral drug for erectile dysfunction. Though it has beneficial effects on erectile organ it also has some adverse effects in other cells and/or tissues related to reproductive system when exposed to longer duration. The objective of the present study is to evaluate the long term effect of SIL on sperm parameters in Wistar albino rat. The animals are divided into two groups, for group I - rats were treated with saline (vehicle alone) and group - II oral administration of 5 mg/kg b.w. of SIL was administrated orally once in a day for 120 days. At the end of the trial period animals were sacrificed and epididymal sperm were subjected to various analysis. Results showed significant reduction in sperm count, motility, viability and morphologically intact sperm in long term PDE5I exposed animals when compared to control. Acrosomal status and fertility test also showed significant reduction in long term PDE5I exposed animals. The present study clearly indicated that long term SIL has shown to induce alteration in sperm quality and quantity, leading to decline in fertility rate. Indicate that SIL impinge on spermatogenesis as well as epididymal function. Understanding the molecular down-stream events involved in long-term exposure to PDE5 inhibitor can be valuable to supervise on related infertility issues and to suggest corrective measures.

Evaluation of DNA Fragments on Boar Sperm by Ligation-mediated Quantitative Real Time PCR

  • Lee, Eun-Soo;Choi, Sun-Gyu;Yang, Jae-Hun;Bae, Mun-Sook;Park, Jin-Young;Park, Hong-Min;Han, Tae-Kyu;Hwang, You-Jin;Kim, Dae-Young
    • Journal of Embryo Transfer
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    • v.25 no.2
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    • pp.111-116
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    • 2010
  • Sperm chromatin integrity is essential for successful fertilization and development of an embryo. Reported here is a quantification of DNA fragments which is intimately associated with reproductive potential to provide one of criteria for sperm chromatin integrity. Three sperm populations were considered: CONTROL (no treatment), UV irradiation (48mW/$cm^2$, 1h) and $H_2O_2$ (oxidative stress induced by hydrogen peroxide, 10 mM, 50 mM and 100 mM). DNA fragments in boar sperm were evaluated by using ligation-mediated quantitative real-time polymerase chain reaction (LM-qPCR) assay, which relies on real-time qPCR to provide a measure of blunt 5' phosphorylated double strand breaks in genomic DNA. The results in agarose gel electrophoresis showed no significant DNA fragmentation and no dose-dependent response to $H_2O_2$. However, the remarkable difference in shape and position was observed in melting curve of LM-qPCR. This result supported that the melting curve analysis of LM-qPCR presented here, could be more sensitive and accurate than previous DNA fragmentation assay method.

Pentoxifylline treatment of frozen pig sperm affects sperm motility and fetal numbers

  • Baek, Sun Young;Chung, Hak Jae;Hong, Joon Ki;Cho, Eun Seok;Choi, Inchul
    • Korean Journal of Agricultural Science
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    • v.47 no.3
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    • pp.657-665
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    • 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.