The objective of the present study was to evaluate the effect of disaccharides supplementation in glycerol-free tris (GFT) on dog sperm cryopreservation with respect to pH adjustment of extender and post-thaw incubation. The spermatozoa collected from five dogs were resuspended (5×107 cell/ml) with GFT containing 100 mM of lactose (L), trehalose (T) or sucrose (S) or pH adjusted (6.85) 100 mM of lactose (LP), trehalose (TP) or sucrose (SP). The sperm (500 μl) were loaded in straws, cooled for 50 min at 4℃, frozen using liquid nitrogen (LN2) vapor for 20 min and plunged in LN2. After thawing at 37℃ for 25 s in a water bath, the spermatozoa were incubated at 24℃ for 30 min. The progressive motility, viability, mitochondrial membrane potential (MMP) and mRNA expression of SMCP gene were then assessed. The MMP was evaluated by combined JC-1 plus PI staining. The relative abundance of SMCP was assessed using quantitative real-time polymerase chain reaction (RT-PCR). Adjustment of pH in GFT extender supplemented with disaccharides did not improve sperrm motility and viability. In general, post-thaw incubation increased the progressive motility of spermatozoa. The sperm motility in the group S was significantly (P<0.05) higher than other groups regardless of post-thaw incubation time. Similarly, the sperm viability in the group S was significantly (P<0.05) higher following post-thaw incubation. The higher sperm motility in the group S was also supported with the significantly (P<0.05) higher live sperm having high MMP. There was no significant difference in mRNA expression of SMCP gene among the experimental groups. These results indicate that cryopreservation of dog sperm in GFT supplemented with S and 30 min post-thaw incubation at 24℃ could provide better freezability of dog spermatozoa with improved motility and higher MMP.

본 연구의 목적은 능성어와 자바리의 정자동결을 위한 간편한 실험법개발이다. 희석제와 동해 방지제가 정자동결에 미치는 효과를 파악하고자 운동성성과 생존율을 조사하였다. 동결실험에 서 300 mM glucose와 15% dimethylsulfoxide (DMSO)를 희석제와 동해방지제로 각각 사용하였 다. 동결실험결과, 능성어 정자는 동결 5개월 후 60% 이상의 운동성을 보였고, 자바리 정자는 동결 5개월후 90% 이상의 운동성을 보였다.

The cryopreservation of sperm has become the subject of research for successful artificial insemination technologies. Antifreeze proteins (AFPs), one of the factors necessary for effective cryopreservation, are derived from certain Antarctic organisms. These proteins decrease the freezing point of water within these organisms to below the temperature of the surrounding seawater to protect the organism from cold shock. Accordingly, a recent study found that AFPs can increase the motility and viability of spermatozoa during cryopreservation.To evaluate this relationship, we performed cryopreservation of boar sperm with AFPs produced in the Arctic yeast Leucosporidium sp. AFP expression system at four concentrations (0, 0.01, 0.1, and 1 μg/ml) and evaluated motility using computer assisted sperm analysis. DNA damage to boar spermatozoa was measured by the comet assay, and sperm membrane integrity and acrosome integrity were evaluated by flow cytometry. The results showed that motility was positively affected by the addition of AFP at each concentration except 1 μg/ml (p<0.001).Although cryopreservation with AFP decreased the viability of the boar sperm using, the tail DNA analyses showed that there was no significant difference between the control and the addition of 0.1 or 0.01 μg/ml AFP. In addition, the percentage of live sperm with intact acrosomes showed the least significant difference between the control and 0.1 μg/ml AFP (p<0.05), but increased with 1 μg/ml AFP (p<0.001). Our results indicate that the addition of AFP during boar sperm cryopreservation can improve viability and acrosome integrity after thawing.

This study was conducted to evaluate effect of α-linolenic acid (ALA) and bovine serum albumin (BSA) on viability, acrosome reaction and mitochondrial intact in frozen-thawed boar sperm. The boar semen was collected by gloved-hand method and cryopreserved using freezing extender containing 3 ng/mL ALA and/or 20 μg/mL BSA. Cryopreserved boar sperms were thawed in 37°C water-bath for 45 sec to analysis. Viability, acrosome reaction, and mitochondrial intact were analyzed using flow cytometry. In results, viability of frozen-thawed boar sperm was significantly higher in only ALA+BSA supplement group than control group (p<0.05), whereas there was no difference either in ALA or BSA supplement. However, acrosome reacted sperm in both of live and all sperm population were significantly decreased in all treatment groups than control (p<0.05). Interestingly, mitochondrial intact of boar sperm was enhanced in ALA and ALA+BSA groups compared with control (p<0.05). In this study, we showed that supplementation of ALA and BSA in freezing extender enhanced the sperm viability, mitochondrial intact and decrease acrosomal membrane damage. In conclusion, our findings suggest that quality of frozen-thawed sperm in mammalians could improve by using of ALA and BSA.

Spermatogonial stem cells (SCCs) is foundation for spermatogenesis throughout male adult life because they have ability of self-renewal and differentiation into spermatozoa. Storage of such SSCs is very important to study on male reproduction, which would contribute human male infertility to be treated. However, during cryopreservation, the most cells are damaged by cryoinjury such as apoptosis, necrosis, osmotic stress, oxidative stress and so on. For the reason, in cryopreservation technique, targeting purpose is what cells are stored stably without cryoinjury. The purpose of this study was to develop the cryoprotectant for decrease in cryoinjury of SSCs by using melatonin and necrostatin-1 as additive cryoprotectant. The SSCs with melatonin or necrostatin-1 was frozen for 1 month, and then thawed to evaluate survival, recovery and proliferation rate. The result showed that necrostatin-1 50 mM was significantly greater than DMSO control. Furthermore, we conducted the characterization of cryo-thawed SSCs with necrostatin-1 50 mM to confirm whether the SSCs could maintain the undifferentiated state. As a result, the normal expression of each marker, which is PLZF, GFRa1 and VASA, was observed except for C-kit, meaning that the cells could maintain the undifferentiated state regardless of cryopreservation. Therefore, the result indicates that the cryo-thawed SSCs have ability of proliferation and self-renewal. In conclusion, our finding verifies that cryopreservation of SSC with necrostatin-1 50 mM could be helpful to preserve the SSCs stably, contributing to various studies on male reproduction and infertility treatment

Cryopreservation of miniature pig sperm is essential because of high demand of organ transplant in mass production. However, miniature pig sperm are vulnerable to oxidative stress more than other mammals. Erythritol is a naturally occurring sugar alcohol with powerful antioxidant property. Thus, the aim of our study is to verify if erythritol could reduce lipid peroxide and enhance viability of frozen thawed miniature pig sperm. Ejaculated semen samples were frozen with cryoprotectant subjected to erythritol treatment (0, 10, 100, 500 mM). After frozen thawed, spematozoa viability were examined using the computer assisted sperm analysis (CASA) system. The product of lipid peroxidation, malondialdehyde (MDA) were quantified using spectrophotometer with DPPH and ABTS assays as ROS scavenger markers. Our result showed that erythritol enhanced sperm viability (p<0.05), reduced lipid peroxides significantly (p<0.05), proving the concentration of 100 mM erythritol to be an effective for lowing oxidative damage. Data from our study suggest that erythritol exhibits significant lipid peroxidation scavenging characteristics which may prevent oxidative damage, enhance viability of frozen thawed sperm and thus could be a effective additive as cryoprotectant.

Processes of cryopreservation consists of three steps: dilution with the extender/cooling (Step 1), addition of cryoprotectant (Step 2), and freezing/thawing (Step 3) and spermatozoa are exposed different kind of environment and stress in each step. We categorized sperm samples as good freezablitiy (GF), damaged by cryoprotectant (DCP), and damaged by freezing (DF) and identified characteristics of each group in different step of cryopreservation. In Step 2, DCP was significantly decreased in motility, rapid speed and increased in slow speed. DF was significantly decreased in only motility whereas there were no significant difference between GF and DF and significantly higher than DCP in Step 2. Motility, rapid, medium speed of all group were significantly decreased in Step 3 and GF was significantly higer than other groups. AR pattern of all groups were significantly increased in Step 3 whereas GF was significantly lower than other groups. Additionally AR pattern of DF was significantly increased in Step 2. F pattern of DF and DCP were significantly decreased in Step 3. There no difference of B pattern in whole process. Mitochondrial activity of DCP was significantly decreased in Step 2 and mtichondrial activity of all groups were significantly decreased in Step 3. However mtichondrial activity of GF was higher than other groups. Viability result shows same significant difference with mitochondrial pattern. The present study compared with various sperm parameters in different groups which has different freezability. We defined different two types of group that damaged from different step of cryopreservation. DF and DCP is mainly damaged in Step 3 and Step 2 respectively. The results of current study suggest that various sperm parameters can be used as physical markers in freezability.

The study aims to assess the embryo development and survivability of bovine embryos cultured in vitro by addition of cysteine. The rates of metaphase II formation are not significantly different among the three groups(73.8% for TCM199, 76.9% for TCM199 with 0.3mM cysteine and 83.8% for TCM199 with 0.5mM cysteine). No differences on cleavage rate(70.6~74.6%) was observed among three culture medium(70.6% for TCM199, 71.3% for CR1aa, and 74.6% for SOF) with 0.5mM cysteine. However, significantly(P<0.05) higher development rate was obtained in the blastocyst stage by adding 0.5mM cysteine in SOF medium(35.6%) than in TCM199(27.6%) or CR1aa(26.6%). No significant differences in the cleavage rates were observed among the three culture. After freezing the blastocysts cultured with 0.5M cysteine, the re-expansion rates ranged from 61.3% to 86.4% among groups, and hatching rates are from 26.3% to 46.9% among groups. The rates of re-expansion and hatching are significantly(P<0.05) higher in SOF medium(86.4% and 46.9%, respectively) than those in TCM199(61.3% and 26.3%) and CR1aa medium(87.1 and 44.4%). After thawing, the blastocyst re-expansion rate become significantly(P<0.05) higher in in vivo (87.1%) and in vitro (70.3%) embryos. In conclusion, our results demonstrate that supplementation of IVM and IVC media with 0.5mM cysteine improved the quality of in vitro production of embryo and post-thawed embryo. Future studies comparing these culture systems in well-designed trials should be performed.

Microalgae are of significant importance for future biotechnological applications. Many microalgae banks or laboratories attempt to maintain various microalgae for further research purposes. Cryopreservation has been preferred to reduce a labor-intensive and costly routine sub-culturing. Cryopreservation can also diminish the genetic drift risk. However, cryopreservation as a long term storage of microalgae method are still in developing progress because it cannot be generalized for all microalgae. Microalgae types, cryoprotectant agents (CPAs) types, freezing and thawing methods are the most important factors that should be considered for cryopreservation. In this short review the basic principles and the current advanced of microalgae cryopreservation methods are discussed with a suggested starting parameters for microalgae cryopreservation.

Cryopreservation has been applied successfully in many mammalian species. Nevertheless, pig embryos, because of their greater susceptibility to cryoinjuries, have shown a reduced developmental competence. The aim of this study was to evaluate the survival status of vitrified-warmed porcine embryos. Forced blastocoele collapse (FBC) and non-FBC blastocysts are vitrified and concomitantly cultured in culture media which were supplemented with/without fetal bovine serum (FBS). Porcine vitrified-warmed embryos were examined in four different methods: group A, non- FBC without FBS; group B, non-FBC with FBS; group C, FBC without FBS; group D, FBC with FBS. After culture, differences in survival rates of blastocysts derived from vitrified-warmed porcine embryos were found in group A∼D (39.5 (A) vs 52.5 (B) and 54.8 (C) vs 66.7% (D), respectively, p<0.05). Reactive oxygen species (ROS) level of survived blastocysts was lower in group D than that of another groups (p<0.05). Moreover, total cell number of survived blastocysts was higher in group D than that of other groups (p<0.05). Otherwise, group D showed significantly lower number of apoptotic cells than other groups (2.0±1.5 vs 3.2±2.1, 2.8±1.9, and 2.7±1.6, respectively, p<0.05). Taken together, these results showed that FBS/FBC improves the developmental competence of vitrified porcine embryos by modulating intracellular levels of ROS and the apoptotic index during the vitrification/warming procedure. Therefore, we suggest that FBS and FBC are effective treatment techniques during the vitrification/warming procedures of porcine blastocysts.

본 연구에서 생쥐 초기 배아를 이용하여 동결 보존된 배아의 생존율 또는 발달율에 영향을 미치는 요인들의 상관관계를 알아본 결과, 배아의 발달 단계에 따른 동결 - 해동 후 생존율에 있어서는 2세포기 배아가 4~8세포기 배아보다 유의하게 높았으나(p＜0.01), 배 발달율에 있어서는 4~8세포기 배아가 유의하게(p＜0.01), 높아 생존율과 배 발달율과는 상관관계가 없는 것으로 사료된다. 또한, 동결 보호제에 따른 배아의 생존율에 있어서는 2, 4~8세포기 배아 모두 DMSO에서 유의하게 높았으나(p＜0.01), 배 발달율에 있어서는 EG가 DMSO에 비해 더 유의하게 높은 성적을 나타내어(p＜0.01, p＜0.05) 동해로 인한 상해가 적은 것으로 생각되어 2, 4~8세포기 배아에서 EG가 더 효과적인 동결 보호제로 사료되며, 동결 프로그램으로는 완만 동결 - 급속 해동법이 더 우수한 프로그램으로 보인다.

생쥐 2세포기, 4세포기, 8세포기를 각 발생 단계에서 채취하여 동결 보호제를 첨가한 서로 다른 배양액에서 배양하고, 배아의 동결 보존 및 융해 시 급속 처리와 저속 처리 단계로 비교하여 이들 조건이 배아의 생존과 발현에 미치는 영향을 조사하여 다음의 결과를 얻었다. 동결 보호제로 처리하여 배양액을 달리한 경우, 급속 단계에서는 모든 배양액에서 비슷한 발생율을 보였고, 저속단계의 4세포기와 8세포기는 D-PBS에서 높은 발생율을 보였다(P＜0.05, P＜0.01). 배아의 발생 시기에 따른 동결 보존 후, 발생율은 2, 4, 8세포기로 넘어갈수록 발생율의 증가를 보여 8세포기에서 발생율이 가장 높았다(P＜0.01). 동결 보호제의 처리단계에 따른 발생율은 2세포기의 급속 단계에서는 유사하였으나, 4세포기와 8세포기는 저속단계에서 높은 발생율을 보였으며(P＜0.05), 특히 8세포기에서 가장 높았다(P＜0.01).

본 실험은 국화의 바이로이드 제거에 이용되는 초저온처리 시 국화 품종 'White ND'을 적합한 처리조건을 확립하기 위해 초저온처리의 단계별 요인을 실험하였다. 그 결과 생장점의 크기는 1 mm(엽원기 2~3매 포함)에서 높은 생존율과 신초 재생율을 나타내었고, vitirification 처리시 PVS3가 효과적이었으며, 처리 시간은 60분 처리 하였을 때 높은 생존율 및 정 상 신초 재생율을 보였다. 또한 vitrification을 위한 전처리 조건은 sucrose 농도를 88 mM 24시간, sucrose 0.3 M 16시간, sucrose 0.5 M 6시간, sucrose 0.7 M 3시간으로 처리하는 것이 초저온 처리 후 생존율 및 신초 재생율을 높이는데 효과적이었으며, 재생된 정상 식물체는 모본과 비교하여 ploidy level이 동일한 것으로 보아 식물체의 유전적 변이가 일어나지 않았다.

The main purpose of this study is to estimate the effect of adding Tea-N-Tris (TES) to the freezing buffer for miniature pig sperm. In particular, we attempted to identify the association between the MMPs expression and the fertility and viability of frozen sperm from each extender (LEY (Lactose Egg-Yolk), TLE (TES + LEY), TFGE (TES + Fructose + Glucose Egg-Yolk)). In accordance with this, Hypoosmotic Swelling Test (HOST) respond test was the lowest among sperms frozen in LEY while the highest HOST respond was observed among sperms frozen in TLE. Furthermore, we observed MMPs expression in all sperm groups, with pro-MMP showing lower expression than active MMPs. The expression of MMP-9 and MMP-2 was the highest in sperms frozen in LEY, Meanwhile, sperms from the TFGE and TLE group showed lower level of MMP-9 and MMP-2 expression in the order of TLE being the lowest. LEY group showed lower rate of blastocyst development than the TES supplement group, although the difference was not statistically significant. Meanwhile the rate of blastocyst development appeared similar when sperms from TLE and TFGE group were used for IVF. Together, these results indicate that adding Tea-N-Tris to the sperm freezing buffer only suppresses MMPs protein activation but also maximize in-vitro fertility, providing a means to improve the success rate in the in vitro manipulation of miniature pig sperm.

The purpose of this study is to improve production efficiency of vitrified-thawed transgenic bovine embryos. Transgenic bovine embryos were produced by injection of FIV-GFP lentiviral vector into perivitelline space of in vitro matured MІІ stage oocytes, and then in vitro fertilization. EGFP-expressing transgenic bovine blastocysts were cultured in serum-containing and serum–free medium. These blsatocysts were vitrified by pull and cut (PNC) container made with 0.25 cm plastic straw. Results indicate that total developmental rates of normal IVF embryo cultured in serumcontaining and–free medium into blastocyst were not significantly different (22.3 vs 21.5%) and those of GFPexpressing transgenic bovine embryo into blastocyst showed no significant difference between serum-containing (13.9%) and–free medium (13.1%). However, developmental rate of GFP transgenic embryo was significantly (P<0.05) lower than its of normal IVF embryos. In additional study, we vitrified GFP transgenic normal bovine blastocysts using PNC vitrification method. Survival rate of vitrified-thawed GFP transgenic blastocyst (23.1%) was significantly (P<0.05) lower than its of normal blastocysts (68.9%). Although, survival rate of vitrified-thawed GFP transgenic blastocyst was lower than its of normal blastocyst, our result may suggested that PNC vitrification method is feasible to cryopreserve transgenic embryos. Our next plan will be the production of GFP express transgenic bovine derived from vitrified-thawed embryos using PNC method.

The objective of this study was to develop of semen transport system for cryopreservation and fertility in bull sperm. The ejaculated semen were diluted with Triladyl containing 20% egg-yolk for transportation. Diluted semen was transported by three methods that there were wrapping tissue (Tissue), sinking under 30℃ water (Water) and sinking between warm water and air (Air) methods. Semen was transported within 2 hours in 0.3℃. For this study, the freezing of diluted semen were added with Triladyl containing 20% egg-yolk. And frozen-thawed sperm were estimated with SYBR14/PI double stain for viability, FITC-PNA/PI double stain for acrosome reaction analysis and Rhodamine123 double stain for mitochondrial intact assessment. In results, live sperm (SYBR+/PI-) in Air treatment group (43.3±4.7%) was significantly (p<0.05) higher than other treatment groups (Tissue: 16.3±2.7% and Water: 27.5± 3.1%), dying sperm (SYBR+/PI+) in Air treatment group (55.6±4.7%) was significantly lower than other treatment groups (Tissue: 77.6±3.2% and Water: 67.6±3.3%) (p<0.05). Acrosome reaction in Air treatment group (0.2±0.1%) within live sperm (PI negative region) was significantly (p<0.05) lower than other treatment groups (Tissue: 0.7±0.2% and Water: 0.5±0.1%), the acrosome reaction in Air treatment group (28.6±2.8%) within all sperm also was significantly lower than other treatment groups (Tissue: 44.2±1.8% and Water: 36.2±2.0%) (p<0.05). And mitochondrial intact in Air treatment group within live (97.1±0.4%) and all (61.9±3.3%) sperm were significantly higher than other treatment groups (Tissue: 85.2±3.3%, Water: 87.8±2.9% within live sperm and Tissue: 49.28±3.7%, Water: 42.0±3.1% within all sperm) (p<0.05). Therefore, we suggest that transportation by sinking method between warm water and air was beneficial to improvement of fertility in frozen-thawed in bull semen.

This study was carried out to effects of ethylene glycol concentration, sucrose and culture day of in vitro production embryo on slow-down freezing in Hanwoo. 6, 7, 8 and 9 day embryos produced in vitro were frozen using 1.8M EG+0.1M sucrose, 1.8M EG+0.5% BSA and 1.5M EG+0.1M sucrose media. Survivability was confirmed after frozen-thawed 24 and 48h and ICM, TE cell number were counted by Hoechst 33342 and PI staining after frozen-thawed 24h. As a result, 1.8M EG+0.1M sucrose group was most significantly (p<0.05) higher compared with the other treatment groups on survivability, TE and total cell number after frozen-thawed 24h (, and ). ICM number did not found significant (p<0.05) differences between the three treatment groups. in 6, 7, 8 and 9 day of embryos using three types of freezing media, frozen-thawed, 1.8M EG+0.1M sucrose groups with embryos cultured 8 day was significantly (p<0.05) highest survivability to after frozen-thawed 24h. 1.5M EG+0.1 sucrose group with embryos cultured 9 day was significantly higher survivability than group of embryos cultured 8 day after frozen-thawed 24 and 48h. In conclusion, 1.8M EG+0.1M sucrose media is considered to be effective to cryopreservation of embryos cultured 8 and 9 day.