• 한국수정란이식학회지
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• 제33권4호
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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.

• 한국동물생명공학회지
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• 제36권2호
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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.

• 대한수의학회지
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• 제15권2호
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• pp.207-213
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• 1975

Skimmed goat milk heated at $92^{\circ}C$ for 10 minutes was used as a basal extender for bull semen. The extenders for liquid semen were prepared by adding simultaneously at various ratio of 5% dextrose solution and egg yolk to skimmed goat milk. After bull seven was diluted with the extenders at the rate of 20 million spermatozoa per ml of the extenders. The extenders were stored at $5^{\circ}C$ and the survival rates of spermatozoa were examined at 4 and 24 hours, and 3, 5 and 7 days after dilution. The extenders for frozen semen were prepared by adding various ratlo of glycerol to skimmed goat milk containing 20 parts of 5% dextrose solution and 3 parts of egg folk to 77 parts of skimmed goat milk. After bull semen was diluted with the extenders at the rate of 40 million spermatozoa per ml of the extenders, the extenders were frozen in liquid nitrogen tank. The frozen extenders were thawed at $40^{\circ}C$ for 2 minutes, and the revival rates of the spermatozoa in the extenders were examined. These thawed extenders were stored at $5^{\circ}C$ and the survival rates of the spermatozoa were examined at 10 minutes and 24 hours and 3 and 5 days after thawing. The results obtained were as follows: 1. Among the extenders stored at $5^{\circ}C$, the survival rate of the sperm was the highest in the extender including 20 parts of 5% dextrose solution and 3 parts of egg yolk to 77 parts of skimmed goat milk, and the survival rate was significantly higher that of the spermatozoa in egg folk-2.9% sodium citrate (1 : 4) extender. (P<0.05) 2.Among the extenders frozen in liquid nitrogen tank, the revival rate of the spermatozoa was the highest in the extender containing 7ml of glycerol per 100ml of the extender with consisted of 77 parts of skimmed goat milk, 20hparts of 5% dextrose solution and 3 parts of egg yolk, and the revival rate was significantly higher than that of the spermatozoa in egg yolk-2.9% sodium citrate (1 : 4) extender containing 8ml of glycerol per 100ml of the extender (p<0.01). 3. Among the extenders stored at $5^{\circ}C$ after thawing, the survival rate of the spermatozoa was the highest in the extender containing 7ml of glycerol per 100ml of extender which consisted of 77 parts of skimmed goat milk, 20 parts of 5% dextrose solution and 3 parts of egg yolk, and the survival rate was significantly higher than that of the spermatozoa in egg yolk -2.9% sodium citrate (1 : 4) extender containing 8ml of glycerol per 100ml of the extender (p<0.01).

• 한국동물생명공학회지
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• 제35권4호
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• pp.307-314
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• 2020

In the present study, we examined the effect of straw size on spermatozoa motility, viability, acrosome integrity, mitochondrial membrane potential, and plasma membrane integrity after freezing-thawing. Hanwoo semen was collected from three bulls and diluted with an animal protein-free extender, divided into two groups, namely, 10 million spermatozoa in 0.25 mL and 20 million spermatozoa in 0.5 mL straw, and cryopreserved. In Experiment 1, the motility and motility parameters of the frozen-thawed spermatozoa were evaluated. After freezing-thawing, the spermatozoa motility parameters fast progressive, straight line velocity, and average path velocity were compared between the 0.25 mL straw and 0.5 mL straw groups. They were 35.2 ± 1.0 and 32.3 ± 0.7%, 34.6 ± 0.7 and 31.8 ± 0.5 μm/s, 51.4 ± 1.3 and 47.1 ± 1.1 μm/s, 0.25 mL straw and 0.5 mL straw groups, respectively. In Experiment 2, the viability, acrosome membrane integrity, and mitochondrial membrane potential of the frozen-thawed spermatozoa were assessed. After freezing-thawing, the percentages of spermatozoa with live, intact acrosomes and high mitochondrial membrane potential were compared between the in 0.25 mL straw and 0.5 mL straw groups. They were 48.0 ± 2.6% and 35.6 ± 2.8% between the 0.25 mL straw and 0.5 mL straw groups. In Experiment 3, the plasma membrane integrity of frozen-thawed spermatozoa was compared. After freezing-thawing, the plasma membrane integrity was higher for the in 0.25 mL straw group than the 0.5 mL straw group. They were 62.0 ± 2.2 and 54.1 ± 1.3% between the 0.25 mL straw and 0.5 mL straw groups. In conclusion, our results suggest that freezing semen in 0.25 mL straw improves the relative motility, viability, and acrosomal, mitochondrial membrane potential, and plasma membrane integrity of Hanwoo bull spermatozoa.

• 한국가축번식학회지
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• 제13권1호
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• pp.18-25
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• 1989

This study was carried out to investigate the general semen characteristics of the Korean native goat and the effect of temperature, incubation time, dilution rate, freezing rate and glycerol concentration on motility and NAR (normal apical ridge) acrosome of fresh and frozen Korean native goat spermatozoa. 1. Average semen volume per ejaculate, motility, concentration and pH of fresh Korean native goat spermatozoa were 0.19${\pm}$0.09 ml, 94.5${\pm}$0.47%, 26.17${\times}$108${\pm}$1.68/ml and 6.63${\pm}$0.18, respectively. 2. Motility and NAR acrosome of fresh spermatozoa during incubation were higher at 22$^{\circ}C$ than at 5$^{\circ}C$ or 37$^{\circ}C$(P<.01). 3. Motility and NAR acrosome of spermatozoa diluted 1:4 during incubation were higher at 22$^{\circ}C$ than at 5$^{\circ}C$ or 37$^{\circ}C$(P<.01). 4. Motility and NAR acrosome of spermatozoa during incubation were higher for samples diluted 1:1, 1:2, or 1:4 than for samples diluted 1:6(P<.01). 5. Motility and NAR acrosome of post-thaw spermatozoa were higher at freezing rate of 12$^{\circ}C$/min than at freezing rate of 1$^{\circ}C$/min or 24$^{\circ}C$/min when glycerol concentration was 9%(P<.01).

• Reproductive and Developmental Biology
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• 제31권2호
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• pp.121-126
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• 2007

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.

• 한국임상수의학회지
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• 제21권4호
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• pp.363-368
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• 2004

To investigate the effects of seeding during freezing procedure on post-thaw viability, motility, and acrosome integrity of boar spermatozoa, semen from 5 Yorkshire boars were collected for this experiment. Raw semen were diluted with Merck I, subsequently added with cooling diluent containing lactose and egg yolk and with freezing diluent containing glycerol. The diluted semen were frozen on the rack in the styrofoam box filled with liquid nitrogen at the distance of 5 cm or I cm above LN2 level. Seeding was performed to only a group of straws frozen at 5 cm away on the surface of LN2. The frozen semen were thawed in $50^{\circ}C$C water and the viability and local motility were analyzed by sperm analysis imaging system. A part of thawed semen was taken for the examination of morphology of apical ridge of the acrosome to compare with the effect of seeding between the seeding-treated and non treated groups. I. Post-thaw viability was considerably higher in seeding-treated sperm than non-seeding group (p<0.01), however, no difference of local motility was obtained among the groups. 2. At three hours after thawing, viability was also higher in seeding-treated group than non-treated group (p<0.05), along with no difference of motility among the groups. 3. Higher normal acrosome integrity was obtained in the seeding-treated sperm than non-treated groups (p<0.01). 4. Between non-seeded groups, higher normal acrosome integrity was obtained in the sperm group frozen at 5cm upper on the surface of LN2 than that frozen at 1cm away (p<0.01). These results indicated that seeding treatment during freezing boar spermatozoa was beneficial to post-thaw viability and normal acrosome integrity.

• Reproductive and Developmental Biology
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• 제35권3호
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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.

• 대한수의학회지
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• 제47권1호
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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.

• 한국수정란이식학회지
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• 제25권4호
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• pp.237-245
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• 2010

Only an optimum number of viable spermatozoa in a frozen-thawed insemination dose can ensure conception at artificial insemination (AI). We report here the percentages of normal, abnormal and viable spermatozoa present in the frozen-thawed semen of 20 Black Bengal bucks used for commercial AI. Bucks in this experiment were of 19.3~46.1 months old and 25~42 kg body weight. Four semen straws (0.25 ml) from each buck were collected for evaluation of their kinetic parameters. Scrotal circumference was measured by using a scrotal tape, sperm motility was estimated on eye estimation and sperm concentration was determined by using a haemocytometer. Sperm morphology was studied in paraformaldehyde fixed spermatozoa under differential interference contrast (DIC) microscope. To determine the proportion of live (plasma membrane intact) spermatozoa, semen was stained with SYBR-14 and propidium iodide and examined under fluorescent microscope. Scrotal circumference, post-thaw sperm motility, sperm concentration per insemination dose and proportion of normal spermatozoa were $21.5{\pm}0.7\;cm$, $43.5 {\pm}5.4%$, $83.5{\pm}6.7$ million and $88.3{\pm}4.1%$, respectively. The percentages of spermatozoa with head shape and acrosome abnormalities were lower ($2.7{\pm}1.1$ and $1.4{\pm}1.3$, respectively), whereas higher percentages of abnormalities ($7.0{\pm}1.8$) were observed in mid piece and tail portion. The proportion of live spermatozoa was $28.5{\pm}5.4$. It is concluded that although a good number of morphologically normal spermatozoa are present in the insemination dose, the proportion of live spermatozoa is low, which warrants further improvements of buck semen freezing procedures to ensure good quality at AI.