• Clinical and Experimental Reproductive Medicine
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• v.21 no.1
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• pp.1-11
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• 1994

The occurrence and time course of capacitation, acrosomal loss, and hyperactivated motility require quantitative definition in order to characterize fertile human sperm. Recently the method has been developed to estimate the quality of spermatozoa by using kinematic parameters such as curvilinear velocity(VCL), average path velocity(VAP), linearity(LIN), straightness(STR), amplitude of lateral head displacement(ALH), and beat cross frequence(BCF) from Computer Assisted Sperm Analysis (CASA). In this study, using the Hamilton Thorn motility analyzer HTM 2030(Hamilton Thorn Research, Beverly, MA), we attempted to identify the spermatozoa with hyperactivated motility (HA) objectively and to monitor hyperactivation of human spermatozoa during incubation in capacitating media and after treatment of calcium ionophore as compared with acrosome status. And we examined whether HA are related to the result of SPA. Semen samples obtained from 16 healthy men were prepared by swim up technique and preincubated in a capacitating media(modified BWW medium) for 5 hours and treated with calcium ionophore solution. The acrosome reaction was detected with PSA-FITC labelling of the acrosome and in vitro sperm ferilizing capacity was assessed by the zona free hamster ovum penetration assay (SPA). The incidence of hyperactivated sperm was 2.6% in fresh semen, 14.3% of the swim up population, 13.7% after 5h of incubation. Significant increase of percentage of hyperactivated sperm was observed after the incubation (p<0.05) but after treatment, no significant changes of percentage of hyperactivated sperm(l1.8%) in contrast to significant rise in the percentage of acrosome reacted cells. Correlation analysis failed to show any significant relationship between the percentage of sperm with HA and SPA score. In conclusion, although no direct correlations were found between the results of SPA and HA, hyperactivation of sperm is associated with capacitation and monitoring hyperactivated sperm will be expected as a method of evaluating the functional quality of sperm such as SPA.

• Clinical and Experimental Reproductive Medicine
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• v.29 no.2
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• pp.105-115
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• 2002

Objective : To evaluate the effects of the reactive oxygen species (ROS) generated with a xanthine (X) and xanthine oxidase (XO) system on sperm function, the change of sperm characteristics, lipid peroxidation, and DNA fragmentation in bovine spermatozoa. Materials and Methods: ROS were produced using a combination of 1000 uM X and 50 mU/ml XO. The ROS scavengers: superoxide dismu tase (SOD) (200 U/ml) and catalase (500 U/ml) were also tested. Spermatozoa were incubated for 2 hours in BWW medium with a combination of X-XO supplemented with or without ROS scavengers at $37^{circ}C$ under 5% $CO_2$ incubator. Sperm movement characteristics by CASA (computer-aided sperm analysis), HOST (hypoosmotic swelling test), Caionophore induced acrosome reaction, malondialdehyde formation for the analysis of lipid peroxidation, the percentage of DNA fragmentation using the method of TdT-mediated nick end labelling (TUNEL) by flow cytometry were determined after 2 hours incubation. Results: The action of ROS on bovine spermatozoa resulted in a decreased in capacity for sperm motility, Ca-ionophore induced acrosome reaction and membrane integrity, an increased in malondialdehyde formation and the percentage of sperm with DNA fragmentation. In the effects of antioxidant, catalase completely alleviated the toxic effects induced by the ROS in terms of sperm function and characteristics, however SOD exhibited no capacity to reduce the toxic effects. Conclusion: The ROS can induce significant damages to sperm functions and characteristics. The useful ROS scavengers can minimized the defects of sperm function and various damages of spermatozoa.

• Journal of Embryo Transfer
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• v.28 no.3
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• pp.297-302
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• 2013

Cryopreservation of epididymal spermatozoa offers a potential tool for rescuing genetic material from males of genetically elite populations. Castration, catastrophic injury, sudden death or any other event that makes semen collection or mating impossible may prematurely terminate a stallion reproduction. Stallion epididymal spermatozoa vary widely in the loss of progressive motility, acrosomal integrity, and viability during freezing and thawing. The objective of this work was to investigate the effect of (1) freezing package types on cryopreservation efficiency, (2) thawing temperatures (37, 56 or $70^{\circ}C$) on Computer Assisted Sperm Analysis (CASA) parameters and (3) post-thawing incubation time (0, 1, 2 or 4h) on castrated stallion epididymis. Post-thawed sperm motility ranged between 59.69% and 64.28% ($56^{\circ}C$ and $37^{\circ}C$) in various thawing temperatures. When stallion epididymis sperm was frozen, straw was better than freezing tube on VCL (Velocity of Curvilinear Line) and VAP (Velocity of Average Path) parameter. Higher percentage of motility was observed at $37^{\circ}C$ thawing temperature even though no significant difference was observed among various temperatures. The motility, VCL, ALH (Amplitude of Lateral Head displacement), VAP, BCF (Beat-Cross Frequency) and STR (Straightness index) parameter of post-thawed sperm were significantly decreased by increasing the incubation time for all thawing temperatures. The present study showed that type of freezing package (Straw vs. Freezing tube) was not significantly different on cryopreservation efficiency. Furthermore, stallion epididymal spermatozoa frozen-thawed at $37^{\circ}C$ for 1 min resulted the highest proportion of motility and velocity movement. In addition, motility and viability of frozen-thawed stallion epididymal spermatozoa were also decreased by incubation.

• Asian-Australasian Journal of Animal Sciences
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• v.14 no.11
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• pp.1527-1533
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• 2001

Garole is a prolific, rare, less known and small size Indian sheep breed found in low and humid Sunderban region of West Bengal. Although information on stored Garole ram liquid semen upto 24 h is available, but there is a need to further investigate the short-term and long-term preservability of Garole ram semen for extensive utilization of this valuable germplasm by artificial insemination. The aim of the present study was to apply computer-assisted sperm analysis technique for assessing the motion characteristics of Garole ram semen stored (i) in liquid state at refrigeration temperature for short-term preservation upto 48 h and (ii) in frozen state at $-196^{\circ}C$ for long-term preservation after packaging in mini straws. Short-term preservation had a significant effect on motility (p<0.01) as the motility progressively decreased from 90.1% at 0 h to 85.5% and 73.2% after 24 and 48 h of storage, respectively. Although the decline in rapid moving sperms was also significant (p<0.01) on storage but the decrease was more pronounced at 48 h as compared to 24 h of storage period. Storage of chilled semen had also a significant effect on % linearity (p<0.05), % straightness (p<0.01), sperm velocities (p<0.01), amplitude of lateral head displacement (p<0.01) and beat frequency (pO.Ol) of spermatozoa. The replication had a significant effect for all the variables except average path and straight line velocity. However, the interactions of short-term storage and replication were non-significant for most of the variables except % of medium moving sperms, sperm velocities and beat frequency. On long-term preservation of Garole ram spermatozoa under controlled conditions the mean post-thaw recovery of 70.4 and 71.4% motile spermatozoa was achieved having 48.8 and 48.9% of rapidly motile spermatozoa, respectively in both the replicates. The effect of replication on cryopreservation was significant (p<0.05) on amplitude of lateral head displacement and beat frequency, but there was no significant effect on motility, rapidly motile spermatozoa, linearity, straightness and sperm velocities of frozen-thawed spermatozoa. It can be concluded from these results that an average 70% motility can be achieved on storage of Garole ram semen in chilled liquid state upto 48 h or in liquid nitrogen after freezing under controlled conditions in straws. However, further studies are required to evaluate the fertility of short-term and long-term preserved Garole ram semen for extensive use of this prolific sheep breed.

• Journal of Animal Reproduction and Biotechnology
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• v.34 no.4
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• pp.272-279
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• 2019

The study was conducted to evaluate the seminal attributes and cryobanking of Achhami (Bos indicus) bull semen. Of two Achhami bulls, 8 ejaculates from each bull were evaluated for seminal attributes. For semen freezing and cryo-banking, 4 ejaculates (having ≥2 mL semen volume, ≥75% of sperm motility and ≥1,000 × 10⁶ cells/mL of sperm concentration) from each bull were used. Semen samples were diluted in egg-yolk-tris-citrate extender using a two-step dilution protocol, and were frozen in liquid nitrogen (LN₂) vapour in a styrofoam box. The mean semen volume, colour, sperm mass activity, motility, viability, concentration, abnormal acrosome, midpiece and tail and, abnormal head of two Achhami bulls were 4.4 ± 0.5 mL vs. 2.5 ± 0.2 mL, 2.5 ± 0.1 vs. 2.4 ± 0.1, 3.5 ± 0.1 vs. 3.5 ± 0.1, 77.0 ± 1.1% vs. 78.3 ± 1.3%, 94.4 ± 0.5% vs. 91.0 ± 0.6%, 1137.7 ± 73.7 × 10⁶ cells/mL vs. 1060.0 ± 44.3 × 10⁶ cells/mL, 10.2 ± 0.5% vs. 10.3 ± 0.5% and 6.7 ± 0.5% vs. 8.2 ± 0.3%, respectively. The post-thawed sperm motility and viability were 53.0 ± 2.0% vs. 50.0 ± 0.0% and 80.2 ± 0.4% vs. 73.2 ± 0.7%, while evaluating by computer-assisted sperm analysis (CASA) system, the percentage of the progressive motility, fast motility, slow motility, local motility and immotile sperm were 75%, 68%, 7.4%, 16.6% and 8.6%, respectively. A total number of 620 doses semen straw were cryo-banked. Due to the acceptable post-thawed sperm motility and viability recorded, cryopreservation of Achhami semen is hereby recommended so as to preserve the Achhami breed. For further validation, the fertility will be observed from the produced frozen semen.

• Journal of Embryo Transfer
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• v.25 no.4
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• pp.263-266
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• 2010

Prediction of semen's fertilizing ability used in artificial insemination (AI) is one of very important factors on pig reproductive performance. In vitro fertilization (IVF) has been used for indirect evaluation of sperm's fertilizing ability and it has been showed as highly correlated index. In swine industry, increasing interest in preservation of boar semen raises questions on the sperm motility from semen used in commercial AI centers. Mitochondria in sperm mid-piece generate the energy to support motility and could be an explanation of impaired fertility. Objective of this study was to suggest usable sperm motility to farms in measuring the effect of sperm motility and sperm abnormality on in vitro production of embryo in which sperm's fertilizing ability can be determined indirectly. Semen samples were provided from local AI center and used within 3 days after collection. Semen samples were divided by 4 different motile groups (>70%; 61~70%; 51~60%; <50%) using CASA (computer-assisted sperm analysis) on the days of IVF. Developmental rate to the blastocyst stage from over 61% motile sperm group showed significantly higher rate than below 60% motile sperm group ($16.5{\pm}0.7{\sim}18.4{\pm}0.8%$ vs $6.3{\pm}0.8{\sim}11.5{\pm}0.7%$, p<0.05). In experiment to determine the relationship between sperm motility and viability and abnormality, over 61% motile sperm groups showed significantly higher viability rate compared to below 60% motile sperm groups ($84.8{\pm}4.0{\sim}88.1{\pm}4.0%$ vs $69.1{\pm}4.0{\sim}74.2{\pm}4.0%$, p<0.05). On the other hand, morphological sperm abnormality showed significantly higher in over 70% motile sperm group ($10.2{\pm}2.2$ vs $16.0{\pm}2.2{\sim}21.0{\pm}2.2%$, p<0.05). In experiment to find the correlation between sperm motility of 4 different motile groups and amount of mitochondria, lower motility group also showed lower level of mitochondria (p<0.05). The mitochondria parameter used in this study showed another possibility to differentiate the sperm motility. Taken together, because below 60% motile semen used in AI reduce the fertility, AI centers should provide the over 60% motile sperm to the farms at the time of AI.

• Journal of Embryo Transfer
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• v.29 no.4
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• pp.397-401
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• 2014

The boar sperm has more lipid droplets and specialty of seminal plasma compared with other species, causing difficulties of freezing sperm and decreases for the utilization of frozen semen into the artificial insemination. However, several studies reported significant results for the recovery of sperm motility and reproductive by addition of cryoprotectants and seminal plasma after thawing. This study was designed to investigate the effects of supplementation of trehalose or glycerol in the LEY (lactose and egg yolk in BTS) solution for the conventional freezing and vitrification process. Two boars aged 16 months were used to collect semen for 2 times in a week. The samples were allotted to 3 freezing solutions (LEY + glycerol 10.5% + OEP 1.5%, LEY + trehalose 1M + OEP 1.5%, and sucrose 1.5M + trehalose 1 M + OEP 1.5%) after centrifugation at 800 g for 10 minutes. Semen was equilibrated in freezing solutions for 10 minutes and injected into plastic straws with 2~3 air bubbles to minimize freezing damages. Vitrification was performed to locate sperm in 5 cm above $LN_2$ for 5 minutes, and the conventional freezing was conducted with an automatic freezer. Motility and survival rates were measured by CASA (Computer assisted sperm an alyzing system) and FITC (Fluorescein isothiocyanate), respectively after thawing semen at $50^{\circ}C$ for 12 seconds. The results were analyzed by ANOVA with STATVIEW statistical program. The vitrificatioin solution (LEY + 10.5% glycerol + 1.5% OEP) presented higher motility (20.9%) than other solutions while the solution (LEY + 1M trehalose + 1.5% OEP) showed the lowest (motility : 5.2%). However, survival rates of vitrified sperms detected by FITC showed 1~4% live sperms in almost of dead sperms at all vitrification solutions' groups, but survival rate of freezing solution of LEY + 1M trehalose + 1.5% OEP LEY and LEY + 10.5% glycerol + 1.5% OEP were showed 49%, and 79%, respectively. There were differences (P<0.05) survival rate of conventional freezing in LEY + 10.5% glycerol + 1.5% OEP and LEY + 1M trehalose + 1.5% OEP and the remaining showed no differences. The results suggested that vitrified boar semen was not enough to be utilized for the artificial insemination, but it showed possibility to utilize for ICSI and conventional freezing with glycerol would be useful method for artificial insemination in pig while we choose the outstanding semen against tolerance to freezing damages.

• Journal of Animal Reproduction and Biotechnology
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• v.34 no.1
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• pp.57-63
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• 2019

This study was conducted to find out the effect that ${\kappa}-Carrageenan$ has on the properties of dog sperm when it was added to the cryoprotectant. Extender basically was contained 1.21 g Trizma base, 0.67 g citric acid, 0.4 g glucose, 0.03 g penicillin G, 0.05 g streptomycin sulfate. Extender1 was added with 0.1%, 0.2%, 0.3%, and 0.5% carrageenan, while extender2 was supplemented with glycerol. After freezing-thawing, the motility, viability, acrosome integrity, apoptosis, and ROS (reactive oxygen specifications) of sperm were measured to analyze the effects of the supplementation of carrageenan. Total Motile (TM), Rapid Progressive Motile (RPM), Medium Progressive Motile (MPM), and Immotile were measured through the CASA system after thawing in 37 degree water. Extender with 0.2% ${\kappa}-carrageenan$ ($64.26{\pm}0.49$) was significantly higher than control ($40.24{\pm}8.27$) (p < 0.05). RPMs of extender with 0.1%, 0.2% ${\kappa}-carrageenan$ ($57.64{\pm}6.34$, $56.47{\pm}1.35$) were significantly higher than the other groups (p < 0.05). Acrosome integrity was measured by dyeing to PSA-FITC with an epifluorescence microscope. Normal acrosome ratio of extender with 0.5% ${\kappa}-carrageenan$ ($61{\pm}8.03$) was higher than the other groups (p < 0.05). Apoptosis was measured with a FACSCalibur flow cytometer using FITC (FITC Annexin V Apoptosis Detection Kit). Treated groups of ${\kappa}-carrageenan$ of 0.1% ($0.81{\pm}0.05$), 0.2% ($0.85{\pm}0.05$) were significantly higer (p < 0.05) than control. Modified SYBR/PI staining was used for determination of viability and DCF staining was used for evaluation of ROS. Viability and ROS were not significantly different from other groups. In conclusion, adding a certain concentration of carrageenan to the extender of cryopreservation, carrageenan contributes to the improvement of the sperm motility, acrosome integrity and prevention of apoptosis.

• Journal of Embryo Transfer
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• v.32 no.1
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• pp.1-8
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• 2017

Cryopreservation of germ cells from genetically proven animals could be a source of restoration tools from the risk of extinction or disappearance of wanted characteristics. Using frozen semen, the genetic gains of Korean native cattle have been increased greatly for 70 years. The preservation of genetic resources as a form of frozen semen straw has limited availability due to the numbers. To circumvent this weakness of frozen semen, we tested two re-freezing methods with different initial thawing temperatures using frozen Korean proven semen and rare breed semen from albino, black and chikso breeders. It has been known that human sperm could resist to cryo-damages by repeated freeze-thaw cycles, but not for Korean proven bulls number (KPN) or for rare breeds. Total 7 frozen semem from brindled(2), black(1), Korean Albino(2) and KPN(1) bulls were used for our research. After thawing straws under $5^{\circ}C/2min$ or $37^{\circ}C/40sec$ with low temperature water bath and thermo jug, spermatozoa were re-diluted with triladyl diluents after first thawing and re-frozen. Sperm motilities were compared between animals and treated groups after re-thawing. Mean values of motility and viability of refrozen/thawed sperm for expansion of the number of straws were significantly higher in $5^{\circ}C$ than in $37^{\circ}C$ (P < 0.05). However, the activity of viable sperm thawed at $5^{\circ}C$ was significantly decreased before refreezing. It is estimated that re-freezing of frozen semen from rare Korean native cattle is possible with resistant properties of survived spermatozoa.