• Journal of Embryo Transfer
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• v.19 no.1
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• pp.11-18
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• 2004

The aim of this study was to evaluate the improvement of testicular sperm motility following different culture conditions such as human follicular fluid (hFF) and temperature. Testicular tissues obtained from azoospermia (n=21) were minced into small pieces by blade and recovered sperm suspension were cultured in Ham's F10 with or without 40% hFF at different temperatures (Group I: 37$^{\circ}C$/with hFF, Group II: 32$^{\circ}C$/withGroup III: 37$^{\circ}C$/without, Group IV:32$^{\circ}C$ /without The motility and viability of sperm were monitored during culture for 48 hours. Initial motility of testicular sperm was 10.9$\pm$1.9%. After 24 hours culture, sperm motility was 23.5$\pm$2.1% (Group I), 8.1$\pm$1.1% (Group II), 10.4$\pm$ 1.4% (Group III) and 4.0$\pm$0.8% (Group IV), respectively. After 48 hours, the motility had been changed as 32$\pm$2.3% (Group I), 14.3$\pm$1.7% (Group II), 5.3 $\pm$1.4% (Group III) and 4.3$\pm$0.9% (Group IV). In hFF group (I and II), sperm motility of group I cultured at 37$^{\circ}C$ was higher than those of group II at 32$^{\circ}C$. But, sperm viability of group I cultured at 37$^{\circ}C$ was lower than those of group II at 32$^{\circ}C$ (54.4$\pm$4.1% vs. 59.4$\pm$3.7%) after cultured for 48 hours. We acquired the best motility of testicular sperm when performed in vitro culture for 48 hours in hFF supplemented medium at 37$^{\circ}C$. Increase of sperm motility by in vitro culture could be useful tool fur human TESE-ICSI program.

• Journal of Animal Reproduction and Biotechnology
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• v.37 no.2
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• pp.130-135
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• 2022

Epididymal sperm cryopreservation provides a potential method for preserving genetic material from males of endangered species. This pilot study was conducted to develop a freezing method for tiger epididymal sperm. We evaluated post-thaw sperm condition using testes with intact epididymides obtained from a Siberian tiger (Panthera tigris altaica) after castration. The epididymis was chopped in Tyrode's albumin-lactate-pyruvate 1x and incubated at 5% CO₂, 95% air for 10 min. The Percoll separation density gradient method was used for selective recovery of motile spermatozoa after sperm collection using a cell strainer. The spermatozoa were diluted with modified Norwegian extender supplemented with 20 mM trehalose (extender 1) and subsequent extender 2 (extender 1 with 10% glycerol) and frozen using LN₂ vapor. After thawing at 37℃ for 25 s, Isolate^® solution was used for more effective recovery of live sperm. Sperm motility (computerized assisted sperm analysis, CASA), viability (SYBR-14 and Propidium Iodide) and acrosome integrity (Pisum sativum agglutinin with FITC) were evaluated. The motility of tiger epididymal spermatozoa was 40.1 ± 2.0%, and progressively motile sperm comprised 32.7 ± 2.3%. Viability was 56.3 ± 1.6% and acrosome integrity was 62.3 ± 4.4%. Cryopreservation of tiger epididymal sperm using a modified Norwegian extender and density gradient method could be effective to obtain functional spermatozoa for future assisted reproductive practices in endangered species.

• Journal of Life Science
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• v.26 no.8
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• pp.943-947
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• 2016

The objective of this study was to see if fairly simple magnetic nano-particle treatment enhances boar semen qualities. Boar semen samples were prepared from the swine AI center and samples were divided by 4 different motility groups (1, >90%; 2. 80~90%; 3. 70~80%; 4. <70%) using computer assisted sperm analysis (CASA) evaluation. Boar semen was extended using BTS extender and same number of magnetic nano-particles as total number of spermatozoa in each sample was treated for 20 min and collected for 5 min at room temperature. Sperm qualities such as motility and viability were evaluated by the CASA before and after treatment. Sperm abnormality and degree of agglutination were also evaluated under the microscopic examination before and after treatment. There were significant changes (p<0.05) on sperm motility from all 4 different groups in the average of 7.11% after treatment. The enhancement of sperm motility changes was more clear in the groups of lower sperm motile groups (<70% and 70~80%; 19.12±1.08% and 5.67±0.71%, p<0.05). The sperm motility character in terms of curvilinear velocity (VCL), straight line velocity (VSL), average path velocity (VAP) and linearity (LIN, %) showed also similar pattern but motility enhancement wear more clear in below 70% motile group. Average sperm viability was increased to 4% by magnetic nano-particles (p<0.05). The percentage of sperm abnormality was also reduced significantly (p<0.05) to the range of 3.7~4.5% before after treatment. The degree of sperm agglutination was also reduced in lower motility groups by the magnetic nano-particle purification.

• Journal of Embryo Transfer
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• v.33 no.4
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• pp.321-326
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• 2018

In this study, two epididymal spermatozoa recovery methods in relation to sperm number, motility, viability and acrosome reaction were examined. Seven bulls were castrated and 7 testicles with epididymides were transferred to the laboratoy. Epididymis in each bull was randomly used for flushing and mincing methods with semen extender (Optixcell, IMV, France). The recovered spermatozoa with adjusted sperm concentration to $40{\times}10^6cells/mL$ was diluted with optixcell and cryopreserved. In experiment 1, the difference in the total number of spermatozoa using flushing and mincing methods was insignificant (2570.0 and $2505.2{\times}10^6cells/mL$, respectively). For experiment 2, the percentage of motile spermatozoa and motility parameters between flushing and mincing methods were studied through the use of sperm class analyzer after frozen-thawing. The percentage of total motile sperm between flushing and mincing methods was almost the same with $89.5{\pm}12.8$ and $91.4{\pm}7.9%$, respectively. The same is the case with experiment 3 wherein the viability and acrosomal integrity of frozen-thawed epididymal spermatozoa by flushing and mincing was insignificantly different. The results from the study showed that both flushing and mincing methods can be used for epididymal spermatozoa recovery in bull.

• Korean Journal of Agricultural Science
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• v.49 no.2
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• pp.259-267
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• 2022

Although pesticides are recognized as necessary substances to improve agricultural production, exposure to pesticides is known to have a direct or indirect adverse effect on the reproductive function of mammals. The present study examines the effects of mancozeb, a well-known fungicide, on the fertility capacity of spermatozoa. Boar spermatozoa exposed to varying concentrations of mancozeb (0.01 - 0.5 µM) were evaluated for motility, motion kinetic parameters, viability, acrosome integrity and the generation of intracellular reactive oxygen species (ROS) after 30 min or 2 hrs of incubation. A significant reduction in the motility of spermatozoa was observed upon exposure to mancozeb. Similarly, there was a significant reduction of the motion kinematics of sperm treated with mancozeb as compared to untreated controls (p < 0.05). The sperm viability percentage and acrosome integrity also showed dose-dependent decreases upon exposure to mancozeb. High concentrations of mancozeb (0.2 - 0.5 µM) induced higher levels of intracellular ROS production, which resulted in the loss of the sperm membrane and decreased sperm motility due to oxidative stress. Taken together, the results here indicate that direct exposure to mancozeb affects the sperm fertility capacity. Hence, careful research that examines the interaction between reproduction and environmental toxins is crucial to prevent fertility disorders in animals.

• Journal of Veterinary Clinics
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• v.10 no.1
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• pp.11-18
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• 1993

Four extenders such as tris-fructose-citrate, Tris-glucose-citrate, glycine-glucose-citrate and lactose that the more frequently utilized types of semen extenders used for freezing dog semen evaluated with sperm motility, viability and acrosomal score in the processing procedures to prior freezing and after frozen-thawing respectively. Each extender contained 4% glycerol and 20% egg yolk were treated by same methods in dilution, freezing, storage and thawing. The results were obtained as follows; 1. The sperm motility and viability in procedure from dilution to frozen-thawing appeared superiorly with recovery rate of 53.2%, 54.8% in tris-fructose-citrate but appeared inferiorly with recover rate of 8.4%, 8.3% in lactose to others. 2. In the processing procedure course to prior-freezing, glycine-glucose-citrate appeared superiorly with decrease rate of 5.4% in motility, and lactose with decrease rate of 4.6% in viability, but tris-glucose-citrate appeared inferiorly with decrease rate of 12.2%, 11.4% in the sperm motility and viability. 3. During frozen-thawing, tris-fructose-citrate appeared superiorly with decrease rate of 35.2% in motility and 30.7% in viability but lactose appeared inferiorly with decrease rate of 76.7% in motility and 75.7% in viability. 4. The variation of acrosome morphology in the total processing procedures appeared that glycine-glucose citrate were superior with acrosome score of 0.1191$\pm$0.029, that tris-fructose-citrate were inferior with acrosome score of 0.1941$\pm$0.045 to others.

• Reproductive and Developmental Biology
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• v.33 no.1
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• pp.35-40
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• 2009

The objectives of this study was to evaluate the efficiency of the bacteria eliminated sperm by percoll gradient method on sperm quality and embryo cleavage in vitro in pig. The semen of miniature pig collected by gloved-hand method pre-warmed ($37^{\circ}C$) in thermos bottle, and separated by 65% percoll. Analysis of sperm ability was estimated by examining viability, capacitation and acrosome reaction using chlortetracycline (CTC) and the abnormality. Also, fertility of sperm was monitored with cleavage rate of embryo after IVF using separated and un-separated sperm by percoll. The result, viability of separated sperm was significantly(p<0.05) higher($83.6{\pm}$2.0 vs $59.0{\pm}4.4%$) than un-separated sperm. The results of CTC analysis showed the percentage of F- and B-patterned separated sperm was higher in separated that than un-separated sperm. On the contrary, the percentage of AR-patterned form unseparaed sperm was significantly(p<0.05) higher($13.6{\pm}0.8$ vs $8.1{\pm}0.6%$) than separated sperm. Also, abnormality of un-separated sperm was significantly(p<0.05) higher($2.2{\pm}0.4$ vs $16.8{\pm}2.8%$) than separated sperm. However, the cleavage rates of embryo using separated sperm by percoll and un-separated sperm had not significantly difference on 2 cell stage(9.25 vs 11.88%), 4 cell stage(26.76 vs 24.51%) and >4 cell stage(63.99 vs 63.61%) at 48h of IVF. Therefore, the sperm separated by percoll method showed improvement in sperm quality than un-separated sperm in miniature pig.

• Journal of Animal Science and Technology
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• v.49 no.5
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• pp.585-592
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• 2007

This research was carried out in order to establish the production technique for Poong-san dog’s frozen semen, by examining the semen characteristic and the volume of glycerol added to the dilution solution, thawing temperature and sperm motility and viability as well as the motility using CASA according to time variation. Average semen volume was 5.9ml, sperm concentration 116.3×106 sperm/ml, total sperm number 789.3×106 sperm, motility 88.7±1.7% and viability 87.6±7.8%. When it was cryopreservation and thawed at different glycerol concentrated extender, it showed 52.7% motility and 57.7±10.3% viability at 7% glycerol, compared to other treatments. For semen cryogeny, at conditions of 5, 7cm and a height of 10cm for pre-cryogeny and maintaining the semen at 7cm from the surface of liquid nitrogen resulted in profitable motility and viability.

• Journal of Embryo Transfer
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• v.28 no.3
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• pp.193-198
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• 2013

This study was designed to evaluate the effect of L-cysteine on sperm characteristics and oocyte cleavage in vitro in Korean native cattle. For this study, the freezing of diluted semen were added with Triladyl containing 20% egg-yolk and/or 0, 5, 10 and 20 mM L-cysteine before cryopreservation. The viability in frozen-thawed sperm were estimated by SYBR14/PI double stain, acrosome damage with FITC-PNA, mitochondria intact with Rhodamin123 and hydrogen peroxide($H_2O_2$) level with carboxy-DCFDA by flow-cytometry. The developmental capacity was also assessed with cleavage rates in oocytes fertilized in vitro by frozen-thawed sperm. In results, the sperm viability was significantly increased in 10 mM and 20 mM concentrations of L-cysteine than other groups (p<0.05). In addition, acrosome damage was significantly decreased in 10 mM and 20 mM concentrations of L-cysteine than other groups (p<0.05). The mitochondria intact was also significantly increased in 10 mM and 20 mM concentrations of L-cysteine than other groups (p<0.05). On the other hand, the cleavage rates were significantly increased in 0 mM, 5 mM and 10 mM groups than 20 mM concentration of L-cysteine (p<0.05). The oocyte degeneration of oocytes were significantly decreased in 0 mM, 5 mM and 10 mM groups than in 20 mM L-cysteine group (P<0.05). However, there are no significantly differences among the L-cysteine treatment groups. We suggest that concentration of 10 mM L-cysteine have beneficial impact for sperm cryopreserved in Korean native cattle. This result also could be recommended for artificial insemination program if supported by an improvement in the fertility results and required further study.

• Reproductive and Developmental Biology
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• v.33 no.4
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• pp.273-281
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• 2009

Antioxidants partially ameliorated the detrimental effects of reactive oxygen species (ROS) on sperm characteristics during in vitro storage. The objective of the present study was to investigate the single or synergetic antioxidative effect of curcumin and Vit. E on the characteristics of fresh boar sperm during in vitro storage. The sperm viability in curcumin, Vit. E supplementation and curcumin+Vit. $E+H_2O_2$ groups remained over 85.0% in 3 hr incubation period, but in 6 hr incubation period, curcumin+Vit. $E+H_2O_2$ groups was sharply dropped than those of curcumin and Vit. E group. The membrane intergrity in all evaluated groups except for $H_2O_2$ group did not significantly difference in 3 hr incubation period. The viability in curcumin or Vit. E supplementation were significantly increased than in curcumin+$H_2O_2$ and Vit. $E+H_2O_2$ group in 6 hr incubation period. The percentage of mitochondrial activity and acrosome intergrity obtained similar trends within same incubation periods irrespective of treatment. The lipid peroxidation of spermatozoal plasma membrane ranged from $11.6{\sim}17.5\;nM/l{\times}10^6$ and $14.0{\sim}19.0\;nM/l{\times}10^6$ in 3 hr and 6 hr incubation periods. In conclusion, curcumin or Vit. E surpplementation alone or cooperatively improved sperm viability index (motility, membrane intergrity, viability and survival rates) and fertility index (mitochondria activity, acrosome intergrity and lipid peroxidation) of fresh boar sperm, indicating that curcumin and Vit. E have a antioxidative properties through its scavenging activity against hydrogen peroxide.