• Reproductive and Developmental Biology
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• 제38권4호
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• pp.171-177
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• 2014

L-Carnitine is an antioxidant for the transport of fatty acids in mitochondria and breakdown of lipids for metabolic energy. Some studies have suggested that carnitine improves sperm motility in mammals. The objective of this study was to investigate the effect of L-carnitine on the characteristics in fresh semen of miniature pigs. The collected fresh semen was stored in modena B medium with L-carnitine (0, 1.0, 2.0, and 4.0 mg/ml) for 10 days at $18^{\circ}C$. The semen quality of viability, acrosome reaction and mitochondria integrity was analyzed on 0, 3, 7, and 10 day of semen storage. The percentages of live and dying sperm were not different among treatment groups with different concentrations of L-carnitine during the storage period. In acrosome reaction analysis, when the sperm stored for 7 day, the percentages of live sperm with acrosome reaction were significantly (p<0.05) lower in 1 ($9.0{\pm}0.9%$), 2 ($7.6{\pm}0.2%$) or 4 mg/ml ($7.9{\pm}0.8%$) L-carnitine-treated groups than the control group (0 mg/ml L-carnitine) ($11.12{\pm}0.2%$). However, there were no difference in percentages of live sperm with acrosome reaction for 3 and 10 days of storage with each concentrations of L-carnitine. When sperm was stored for 3 and 10 days, the percentages of live sperm with mitochondria integrity were significantly higher in 2 mg/ml of L-carnitine-treated group than control group (p<0.05). In conclusion, the L-carnitine has a positive effect on acrosome reaction and mitochondria integrity in liquid state of fresh semen in miniature pigs.

• 한국수정란이식학회지
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• 제33권3호
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• pp.185-194
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• 2018

The aim of this study was performed to evaluate the effects of ice-binding protein from the arctic yeast Leucosporidium (LeIBP) supplementation on cryopreservation of boar sperm. The collected semen was diluted ($1.5{\times}10^8/ml$) in lactose egg yolk (LEY) and cooled at $5^{\circ}C$ for 3 h. The cooled semen was then diluted ($1{\times}10^8/ml$) in LeIBP containing LEY with 9% glycerol and maintained at $5^{\circ}C$ for 30 min. The semen was divided into six experimental groups (control, 0.001, 0.005, 0.01, 0.05 and 0.1 mg/ml of LeIBP). The straws were kept on above the liquid nitrogen ($LN_2$) vapors for 20 minutes and then plunged into $LN_2$. After thawing, computer-assisted sperm analysis was used for sperm motility and flow cytometry was performed to assess the viability, acrosome integrity (FITC-PSA/PI), ROS (DCF/PI), lipid peroxidation (BODIPY C11/PI) and apoptosis (Annexin V/PI), respectively. No significant responses were observed for sperm motility. However, sperm viability was significantly increased on 0.05 and 0.1 mg/ml of LeIBP groups compared to control (P < 0.05). In addition, acrosome integrity was significantly increases LeIBP groups (P < 0.05) and both ROS and lipid peroxidation level were lower in all LeIBP groups than those of control (P < 0.05). On the other hand, a significant higher apoptosis rate was observed in 0.05 and 0.1 mg/ml of LeIBP groups compared to control (P < 0.05). It can be assumed that a supplementation of LeIBP in boar sperm freezing extender is an effective method to increase the sperm qualities after cryopreservation.

• 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.

• Reproductive and Developmental Biology
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• 제30권1호
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• pp.59-64
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• 2006

본 연구는 돼지정액의 보다 간편하고 손쉽게 동결시킬 수 있은 방법을 확립하기 위하여 수행하였다. 돼지정액은 3시간에 걸쳐 $5^{\circ}C$까지 냉각 후 Straw에 봉입하고 다양한 방법 및 step에 의해 스티로폼 용기 내에 들어있는 $LN_2$ 중에서 동결하였다. 정자의 생존성은 $LN_2$ 표면으로부터 10 cm 위에서 10분간 정치 후 침적할 경우 가장 높게 나타났다(54.0%). Straw를 $-102^{\circ}C$에서 10분간 정치시킨 처리구가 여타구보다 높은 생존성이 얻어졌다(74.0%, P<0.05). 응해 방법에 따른 동결정액의 생존성 실험에서는 $37^{\circ}C$의 융해구가 $52^{\circ}C$ 융해구보다 유의적으로 높은 결과를 나타냈다(P<0.05). 1단계 동결 방법과 3단계 동결방법으로 돼지 정액을 동결시킨 결과 정액의 일반적 특성 및 첨체의 이상 유무를 평가하는 CTC 검사에서는 커다란 차이가 인정되지 않았다. 동결정액을 이용한 체외수정 결과에서는 상실배기 이상 발육율에서 1-step이 3-step보다 높은 발육율을 나타내었다(27.5 vs 14.7%, P<0.05). 본 실험의 결과 돼지정액 동결 시 $-102^{\circ}C$에서 10분간 정치시키는 1단계 동결방법이 간편하면서 유리한 동결 방법으로 활용될 수 있음을 보여준다.

• 한국동물생명공학회지
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• 제34권1호
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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.

• Reproductive and Developmental Biology
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• 제35권3호
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• pp.363-367
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• 2011

The purpose of this study was to improve of frozen-thawed sperm using magnetized water in Korean native cattle. Before cryopreservation, without egg-yolk Triladyl$^{(R)}$ solution was flowed though magnet [0, 2000, 4000 and 6000 Gauss(G)] for S min. The freezing of dilluted semen added with Triladyl containing 20% egg-yolk. Analysis of frozen-thawed sperm was estimated viability with SYBR14/PI double stain, membrane intact with hypoosmotic swelling test (HOST), acrosome reaction with FITC-PNA, mitochondria membrane function with Rhodamin 123 by flow- cytometry. Sperm viability was significantly higher in 4000G group than other groups (p<0.05). However, the Hypoosmotic Swelling Test(HOST) was significantly higher in fresh, 4000 and 6000G than 0 and 2000G (p<0.05). In addition, mitochondria membrane damage and acrosome damage were significantly lower in 6000G group than other groups (p<0.05). in conclusion we suggest that magnetized water could be improve ability of sperm on cryopreservation in Korean native cattle.

• Asian-Australasian Journal of Animal Sciences
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• 제17권11호
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• pp.1501-1508
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• 2004

In this study the effect of extenders and temperatures on sperm viability and fertilizing capacity of boar sperm during long-term storage was investigated. Acrosomal integrity, membrane integrity, motility and hypo-osmotic resistance were evaluated by fluorescence and light microscopy. An in vitro fertilization test was performed to assess the fertilizing capacity of stored spermatozoa. The five diluents tested were ranked according to their ability to maintain sperm functional parameters and Zorlesco (ZO) extender with BSA or with PVA instead of BSA produced the best results. Zorlesco extender substituted with PVA (ZO+PVA) was found to maintain motility both at 15 and 20$^{\circ}C$. within 5 days of storage, but the quality of semen stored at 15$^{\circ}C$ decreased thereafter as compared to semen stored at 20$^{\circ}C$ Semen stored at 5$^{\circ}C$ demonstrated rapid loss of motility already within 24 h. Both fertilization and cleavage of semen stored at 20$^{\circ}C$ in ZO substituted with PVA instead of BSA did not change significantly until day 8 of storage. It is therefore concluded that PVA can be used to substitute for BSA and 20$^{\circ}C$ was more suitable than 15$^{\circ}C$ for boar semen storage, and in vitro fertilizing capacity of spermatozoa was maintained for at least 8 days in ZO+PVA at 20$^{\circ}C$.

• 한국수정란이식학회지
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• 제23권2호
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• pp.115-118
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• 2008

Glycerol is the cryoprotectant most frequently used to freeze semen in several of species. The objective of the present study was to compare the effect of three different glycerol concentrations (4, 6 or 8%, v/v) on frozen-thawed dog sperm survival rate. Ejaculates from 9 dogs collected by digital manipulation were pooled and assessed by macroscopic and microscopic criteria. Semen was divided into 3 aliquots, which were centrifuged and the sperm pellets rediluted with first Tris-glucose-citric acid extender. After 1 h cooling at $4^{\circ}C$, second extender containing 4, 6 or 8% glycerol was added, respectively. The semen was loaded into 0.25 ml straws and frozen and stored in liquid nitrogen and thawed. Sperm vigor, live:dead spermatozoa ratio using HOS test, and sperm morphology using $Spermac^{(R)}$ stain were evaluated. After thawing, there were no significant differences among groups in vigor, viability and morphology. In conclusion, the three glycerol concentrations (4, 6 or 8%) can be used successfully in cryopreservation of canine semen. Therefore the use of 4% glycerol in the extender has less toxic effect and reduces of freezing injuries.

• Animal Bioscience
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• 제36권10호
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• pp.1530-1535
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• 2023

Objective: Semen cryopreservation result in decreased sperm parameters and fertilization ability. Sericin exhibits antioxidant activity by reducing lipid peroxidation resulting from free radicals, which can potentially improve cryopreservation outcomes. The present study aimed to examine the efficacy of various sericin concentrations supplemented with a rooster semen-freezing extender on post-thaw semen quality and fertilizing ability of sperm after cryopreservation. Methods: Semen samples were collected from 40 roosters (5 reps), then were pooled, and divided into four groups by the levels of sericin supplementation (0%, 0.25%, 0.50%, and 0.75%) in a freezing extender. Semen suspensions were loaded in medium straw (0.5 mL) and cryopreserved with the traditional liquid nitrogen vapor method. Post-thawed semen was evaluated for sperm motility, sperm viability, and lipid peroxidation. Also, the fertility test was determined. Results: The results showed that supplementation of the freezing extender with 0.50% to 0.75% sericin resulted in greater total motility and progressive motility and lower malondialdehyde levels than the other groups after cryopreservation (p<0.05). However, the viability of 0.75% decreased compared with the value of 0.50% sericin supplementation (p<0.05). Moreover, the fertility and hatchability of total eggs were significantly higher in the 0.50% sericin group than in the other groups (p<0.05). Conclusion: In conclusion, 0.50% sericin is recommended as an alternative component of the freezing extender to improve cryopreserved rooster semen.

• Reproductive and Developmental Biology
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• 제30권1호
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• pp.27-34
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• 2006

This study was conducted to compare the reproduction ability of the wild type boar and recombinant human erythropoietin (hEPO) transgenic boar semen. Ejaculated boar semen was analyzed by flow cytometry, Elisa and IVF methods. In experiment 1, flow cytometric analysis showed that the live sperm ratio of transgenic boar sperm significantly lower (P<0.05) than that of wild type boar after incubation at 20, 22, 24 and 26 hr. In experiment 2, the presence and levels of various cytokines (IL-6, IL-10 and $TNF-{\alpha}$) to related animal reproduction in the seminal and blood plasma were examined using specific enzyme immunoassay. There was no significant difference between both groups. In experiment 3, the fertilizing capacity and developmental ability of both boar sperm were compared. The transgenic boar sperm had a significantly low capacity of penetration, sperm-zona binding, embryo development, and blastocyst formation compared to wild type sperm (P<0.05). These results suggest that transgenic boar sperm harboring hEPO gene has low sperm viability than wild type boar, and it is a reason to decrease of fertility and litter size.