• Reproductive and Developmental Biology
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• v.35 no.3
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• pp.329-334
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• 2011

This study was conducted to establish a freezing method of miniature pig spermatozoa. The semen 더aculated from PWG M-type miniature pig was collected by gloved-hand method. The semen was diluted with same volume extender (m-Modena B). The frozen solution used frozen solution of four different (LEY, TCG, BF-5 and m-Modena+egg yolk) for find optimal frozen solution in miniature pig sperm. The diluted semen for frozen rate assay was added to LEY solution (solution I: 11% lactose+egg yolk; solution II: solution I+glycerol+OEP), and frozen depending on freezing rate by the three different freezing methods (A: until $5^{\circ}C$ for 1 hrs, holding at $-102^{\circ}C$ for 10 min; B: until $5^{\circ}C$ for 2 hrs, holding at $-102^{\circ}C$ for 10 min; C: until $5^{\circ}C$ for 3 hrs, holding at -80 and $-102^{\circ}C$ for 10 min). Semen cooled until $5^{\circ}C$ was added with glycerol 1, 3 and 5%, and take a equilibrium time for 0, 10 and 30min. Frozen-thawed sperm were evaluated for viability, acrosomal status and morphological abnormality. The results of frozen-thawed sperm ability by frozen solution, viability was higher in LEY solution compared to other three different frozen solution. AR pattern of LEY solution were lower than other three different frozen solution. The results of freezing rate, viability was higher in B method compared to other methods (p<0.05). Acrosomal statute was intacted in A and B methods than C method. The experiment for glycerol condition was showed that sperm viability was higher in extender with 1% and 3% glycerol and equilibrium time of 0 min. The acrosome damage was lower in extender with 1% glycerol and equilibrium time of 10 min than other conditions. In conclusion, the optimal conditions for cryopreservation of miniature pig spermatozoa obtained in LEY frozen solution, cooling rate of 1~2 hrs, 1~3% glycerol concentrations and glycerol equilibrium time of 0~10 min.

• Korean Journal of Animal Reproduction
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• v.10 no.1
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• pp.19-26
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• 1986

This experiment was carried out to investigate the effect of bovine serum ablumin (BSA), sugars, glycerol equilibration time, straw size and thawing method on the survival index and the morphology of frozen boar spermatozoa. The results obtained were summarized as follow: 1. When the semen frozen in BF5 dilutor as pellet form was thawed in BTS at 37$^{\circ}$and 50$^{\circ}C$, BF5 dilutor with fructose showed higher sperm survival index than that with dextrose, however, when the semen was thawed on dry test tube at 37$^{\circ}C$, BF5 dilutor with sucrose showed higher sperm survival index than with other sugars. 2 When the semen forzen in BF5 dilutor with straw and thawed at 37$^{\circ}C$, BF5 dilutor with dextrose showed higher sperm survival index than those with other sugars, and there was no difference in sperm survival index between 0.5 and 1.0 ml straws. 3. The sperm survival index of frozen sperm was significantly (P<0.05) improved due to addition of BSA (0.05%) to BF5 dilutor. 4. When the extended semen with BF5 dilutor contatining 0.01 to 0.05% of BSA was frozen in the straw, the semen without glycerol equilibration showed significantly (P<0.05) higher sperm survival index than those with 2, 4 and 6 hrs glycerol equilibration time. 5. The sperm frozen in BF5 dilutor with dextrose or fructose, sucrose and raffinose showed 77 to 88% in normal acrosome rate and no difference among sugars. 6. The frozen semen showed lower normal acrosome rate than the first and second diluted semen, whereas the frozen semen showed higher swollen, damaged and missing acrosome rate than the first and second diluted semen. 7. Damaged and missing acrosome rate of sperm head due to freezing was somewhat inhibited by addition of BSA (0.01 to 0.05) to the BF5 dilutor.

• Molecules and Cells
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• v.26 no.6
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• pp.558-565
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• 2008

Although the fertilizing ability of spermatozoa is greatly reduced after freezing, complete understanding of alterations induced by cryopreservation has not been elucidated. The present study evaluates the effects of cryopreservation on the $Ca^{2+}$ handling of boar spermatozoa using several sperm activators. Intracellular $Ca^{2+}$ signals from single spermatozoa were measured using confocal $Ca^{2+}$ imaging of unfrozen samples and of other spermatozoa after having been frozen. Elevation of the external $K^{2+}$ concentration elicited a three times larger $Ca^{2+}$ increase in fresh spermatozoa than in cryopreserved spermatozoa. Caffeine elicited $Ca^{2+}$ transients with some oscillations in the fresh spermatozoa, but not in the thawed spermatozoa. Depletion of the $Ca^{2+}$ store with thapsigargin induced a rapid rise in $Ca^{2+}$ in the control but generated a smaller increase of $Ca^{2+}$ after thawing. Exposure to progesterone induced a biphasic rise of the $Ca^{2+}$ level in the fresh spermatozoa only. Sperm viability was reduced by cryopreservation. Resting $Ca^{2+}$ levels in fresh and cryopreserved spermatozoa were similar. Longer incubation (2.5 h) of thawed spermatozoa partly recovered the $Ca^{2+}$ response to the interventions. These results suggest that cryopreservation reduces the responsiveness of spermatozoa to depolarization, modulators of the internal $Ca^{2+}$ store and progesterone in terms of the $Ca^{2+}$ signal, thus providing a possible mechanism for reduced fertility observed in cryopreserved boar spermatozoa.

• Journal of Embryo Transfer
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• v.14 no.2
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• pp.131-137
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• 1999

This experiment was carried out to investigate the effects of osmolarity of thawing diluents, seminal plasma added in thawing diluents on the sperm viability and the effects of thawing temperature, the temparature of the thawing diluents on the sperm viability and acrosomal morphology of boar spermatozoa by the straw method. The result obtained were summarized as follows: 1. The sperm viablilty after thawing of the frozen semen was shown greater in the high osmolarity(392~492mOsm) than low osmolarity(300mOsm) in thawing diluent. The added levels of seminal plasma in thawing diluent did not affect the viability of frozen-thawed boar semen. 2. In terms of thawing temperature, the sperm viability was shown higher in the frozen semen thawed at 5$0^{\circ}C$ for one min. (p<0.01) than those thawed at 2$0^{\circ}C$ or 37$^{\circ}C$ for one min. The sperm viability was not significant at the diluent temparature of 2$0^{\circ}C$or 37$^{\circ}C$ after thawing: but the sperm viability was higher in thawing diluent at 2$0^{\circ}C$ than in that at 37$^{\circ}C$. However, the effects of thawing temperature and diluent solution on normal acrosomal rate were not significant. 3. Cleavage rates of oocytes fertilized with frozen semen were 46.4% and 43.3%, respectively, which were thawed at 5$0^{\circ}C$ for one min. and then diluted in mBTS medium at 2$0^{\circ}C$or 37$^{\circ}C$. To sum up, the sperm viability was shown greater at the high of thawing diluents of frozen boar semen. In terms of thawing conditions, the sperm viability was shown greater, when semen was thawed at a high temperature for a short time and then diluted at the same temperature as that in the straw.

• Reproductive and Developmental Biology
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• v.29 no.3
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• pp.149-154
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• 2005

The purpose of this study was the analysis of sperm ability in Specific Pathogen Free (SPE) miniature pig for production of bio-organ. The collected semen was diluted with extender and stored at $17^{\circ}C$t for up to 7 days. The semen samples were evaluated at 0, 1, 3, 5, and 7 days of storage for analysis of sperm ability. Sperm ability was evaluated by examining viability, progressive motility, sperm abnormality and intensity of the sperm membrane. Also, the semen was processed according to the convenient freezing method, and frozen-thawed sperm was evaluated by examining viability, capacitation and acrosome reaction using chlortetracycline (CTC) staining. Motility of spermatozoa of SPF miniature pig was significantly (P<0.05) lower on 3 days or later compared to the Duroc, Yorkshire and Landrace in domestic boar. The percentage of abnormal spermatozoa of Landrace were significantly (P<0.05) higher than in SPF miniature pig, Duroc and Yorkshire that had a similar percentage on 5 or 7 days of sperm storage. The percentage of spermatozoa with coiled tail decreased during the storage period but there were no significant difference. On the other hand, viability of frozen-thawed spermatozoa had a significantly (P<0.05) lower in SPF miniature pig than in other domestic boars. CTC patterns had no significant difference, but SPF miniature pig had higher percentage of capacitated spermatozoa and lower percentage of acrosome-reacted it than domestic boars. Therefore, this study suggest that it is necessary to develop the suitable extender and freezing methods methods for the high viable rate and fertilizing ability in vitro.

• Clinical and Experimental Reproductive Medicine
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• v.27 no.2
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• pp.201-209
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• 2000

Objective: This study has been carried out to evaluation the effect of fertilization promoting peptide (FPP) on the kinematic parameters, capacitation and acrosome reaction of the frozen-thawed human spermatozoa. Methods: After FPP treatment, we examined kinematic parameters, capacitation and acrosome reaction, using the methods of computer-aided sperm analysis (CASA) and chlortetracycline (CTC) fluorescence analysis. Results: We have obtained the evidence that FPP can promote the capacitation and inhibit the spontaneous acrosome reaction of frozen-thawed human spermatozoa in vitro. FPP ($25{\sim}100$ nM) induced a significant increase in the proportion of B-pattem capacitated spermatozoa, and a significant decrease in the proportion of F-pattem uncapacitated ones without significant stimulation of acrosomal exocytosis. In the kinematic parameters treatment, FPP treated groups maint3ined higher LIN, BCF and STR than those of control. The VAP, VSL, VCL and ALH were not different. Therefore it is suggested that FPP in human seminal plasma may play a positive role in promoting human sperm function.

• Journal of Animal Science and Technology
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• v.54 no.4
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• pp.283-290
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• 2012

Cryopreservation induces sublethal damage to the spermatozoa, which leads to their reduced fertile life. The objective of this study was to investigate the effect of taurine, hypotaurine and trehalose as antioxidants on the function of the freezing-thawed sperm in Korean Jeju Black Bull. The semen was cryopreserved with tris egg yolk extendercontaining 7% glycerol and treated with 20mM taurine, hypotaurine and trehalose. Frozen-thawed sperms were evaluated for sperm motility, viability, membrane integrity, acrosome integrity and sperm penetration ability. The results were compared to semen cryopreserved in tris egg yolk extender containing 7% glycerol only as control. Frozen-thawed semen evaluation clearlyindicated that the addition of taurine or hypotaurine significantly improved (p<0.05) the motility and viability compared to control spermatozoa. Moreover, in membrane integrity, swollen sperm ratio was significantly increased (p<0.05) in taurine, hypotaurine or trehalose compared to control. In sperm acrosome integrity, F pattern ratio was increased (p<0.05) in hypotaurine among treatments, and AR pattern was significantly lowered (p<0.05) in taurine, hypotaurine and trehalose. In assessed sperm fertilizing ability, taurine, hypotaurine or trehalose significantly improved (p<0.05) the ratio of pronucleus formation and SFI. Finally, compared with the control, addition of taurine, hypotaurine or trehalose as an antioxidant to the freezing extender showed more positive effects on the frozen-thawed spermatozoa. It is concluded that the addition of taurine, hypotaurine, or trehalose to the freezing extender could reduce cryodamage of the Korean Jeju Black Bull spermatozoa.

• Korean Journal of Veterinary Research
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• v.47 no.1
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• pp.135-138
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• 2007

A 3 year-old female Afghan hound came to the Veterinary Referral Hospital of the College of Veterinary Medicine, Seoul National University for artificial insemination (AI) with frozen semen. In order to inseminate, semen was frozen in USA 3 years ago. Frozen semen was sent by air from Santiago to Seoul for AI. The stud died 2 years ago, so we could only use a limited amount of frozen semen in that estrus cycle. The number of total motile spermatozoa was $59.4{\times}10^6$and the total volume was 1.2 ml. The frozen spermatozoa were thawed in $70^{\circ}C$ water for 8 sec, which were then deposited at the bilateral uterine horns by a surgical method. The number of corpus luteum was 6. Sixty days after artificial insemination resulted in the birth of 4 puppies, all of which are alive and healthy.

• Journal of Animal Reproduction and Biotechnology
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• v.36 no.2
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• pp.82-90
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• 2021

In the present study, we examined if deep uterine artificial insemination (DUAI) can improve the pregnancy rate of artificial insemination (AI) using epididymal spermatozoa (ES) in Hanwoo cattle. The estrus cycles of 88 Hanwoo cows were synchronized, and 17 cows were artificially inseminated using the DUAI method with ES, 20 cows were artificially inseminated via the uterine body (BUAI) method with ES, and as a control, 51 cows were inseminated by using the BUAI method with ejaculated spermatozoa from 1 proven bull after frozen thawing. The pregnancy rate of the DUAI method (58.8%) was higher than that of the BUAI method (25.0%, p = 0.0498). The motility of ES was examined immediately after thawing and after 3 and 6 h of incubation. The rapid progressive sperm motility of the control group was significantly higher than that of the ES group immediately after thawing and after 3 and 6 h of incubation (p < 0.05). The straight line velocity and average path velocity of the ES group after 6 h of incubation were significantly lower than those in the control group (p < 0.05). The linearity and amplitude of lateral head of ES were lower than those at 6 h (p < 0.05). The flagellar beat cross frequency and hyperactivation of ES were lower than the control spermatozoa immediately after thawing and at 3 h (p < 0.05). These motility parameters suggested that ES had a low motility and fertilization ability compared to the control spermatozoa. After frozen-thawing and 3 h of incubation, the percentage of live spermatozoa with intact acrosomes in the ES was significantly lower than that in ejaculated spermatozoa (p < 0.05). Our findings suggested that the DUAI method can overcome the low pregnancy rate of ES, despite the low motility, viability, and fertilization ability of ES.

• 대한생식의학회:학술대회논문집
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• 2001.11a
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• pp.106-106
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• 2001