The early-onset familial Alzheimer's disease (EOFAD/ FAD), the less common type of Alzheimer's disease (AD) currently affects a vast number of individuals worldwide. This type is being inherited as an autosomal dominant fashion. Missense mutations on Amyloid precursor protein (APP) and Presenilins 1 and 2 (PSEN1 & PSEN2) are known as major genetic factors in FAD. Conversely, missense mutations on microtubule-associated protein tau (MAPT) are also thought to involve. Up to date, several triple-transgenic animal models with muted forms of the human APP, PSENs and MAPT have been reported. Compared to other animals, canines are more emotional and their disease signs can be easily diagnosed. This attempt was to develop a triple transgenic canine model for the AD. We have obtained the coding sequences of APP, PSEN1 and MAPT from Dana-Farber/Harvard Cancer Center DNA resource core at HMS and incorporated several common AD mutations. The transgenic construct is composed of hNSE (ENO2) promoter-driven three AD genes fused together with modified 2A sequences. It was transfected into the canine fetal fibroblasts which were then used to perform somatic cell nuclear transfer (SCNT). The viable transgenic embryos were obtained after in vitro culture and the GFP was detected. In this study, we have successfully produced viable triple transgenic canine cloned embryos using SCNT technique. These transgenic canine embryos will be further developed into canines with FAD. The transgenic canines will be a good candidate in the AD research field.

Polo-like kinase 1 (Plk1) has multiple roles in somatic cell and mammalian oocyte division. In mice, Plk1 distributes to the centromeres from prophase to anaphase and compose spindle apparatus in mitosis stages. Somatic cell nuclear transfer (SCNT) has diverse advantages. However, low cloning efficiency of SCNT procedure causes difficulty to application. The causes of this low efficiency are still unclear. However, they are attributed to the cumulative results of several biological and technical factors. In this study, Plk1, a biological factor, was investigated. B6D2F1 mice (7 weeks old) were superovulated with 10 IU of pregnant mare’s serum gonadotropin and 9 U of human chorionic gonadotropin (HCG) 48 hr later. The oocytes were collected 14 hr after HCG injection and cultured on potassium simplex optimized medium. The BI2536, Plk1-specific inhibitor, was used to understand the influence of Plk1. Also, the embryos were assessed by immunofluorescence. All BI2536-treated embryos failed to the first mitotic division. It showed Plk1 has a critical role in the first mitotic division of the mouse embryo. Moreover, there were significant differences between the control and SCNT embryos in the patterns of Plk1. All SCNT embryos which failed 2-cell development presented incorrect positioning and low expression of Plk1. On the other hand, the control embryos which failed to 2-cell division showed only low expression of Plk1. Taken together, this results demonstrate that Plk1 is critical for successful mitotic division of mouse embryos. Also, correct localization of Plk1 has crucial effect in the development of murine SCNT embryos.

조직 플라스미노겐 활성제(Tissu-type plasminogen activator; tPA)는 혈전 용해제로 이용되고 있으며, 주로 심근경색증과 같은 급성 혈전후성 허혈증 등을 치료하는 데 유용하다. 본 연구에서는 혈관 질환 치료제를 생산하기 위하여 체세포 복제를 이용한 htPA 를 발현하는 형질전환 복제 돼지를 생산하였다. 형질전환 복제란 8,026 개를 25 두의 대리모에(321 개/1 두) 이식하였으며, 25 두 중 6 두의(24%) 대리모가 자연분만으로 19 두의(3.2 두/1 두) 산자를 생산하였다. 19 두 중 1 두가(1/19 두, 5.3%) 생존 중에 있으며, 8 두는 사산 개체였고(8/19 두, 42.1%), 10 두는 출생 후 2-3 일내에 폐사하였다(10/19 두, 52.6%). 폐사 개체들은 혀 기형, 구개열 등 안면기형이 관찰되었으며, 이와 함께 관절 혹은 발굽의 모양이 비정상적이라는 것을 확인하였다. 또한, 폐사개체들은 모두 하나 이상의 문제점을 동시에 가지고 있음을 확인하였다. 따라서, 이러한 문제점들이 형질전환에 의한 것인지, 아니면 복제에 의한 것인지를 규명하기 위해 연구를 더 진행해야 할 것으로 사료된다.조직 플라스미노겐 활성제(Tissu-type plasminogen activator; tPA)는 혈전 용해제로 이용되고 있으며, 주로 심근경색증과 같은 급성 혈전후성 허혈증 등을 치료하는 데 유용하다. 본 연구에서는 혈관 질환 치료제를 생산하기 위하여 체세포 복제를 이용한 htPA 를 발현하는 형질전환 복제 돼지를 생산하였다. 형질전환 복제란 8,026 개를 25 두의 대리모에(321 개/1 두) 이식하였으며, 25 두 중 6 두의(24%) 대리모가 자연분만으로 19 두의(3.2 두/1 두) 산자를 생산하였다. 19 두 중 1 두가(1/19 두, 5.3%) 생존 중에 있으며, 8 두는 사산 개체였고(8/19 두, 42.1%), 10 두는 출생 후 2-3 일내에 폐사하였다(10/19 두, 52.6%). 폐사 개체들은 혀 기형, 구개열 등 안면기형이 관찰되었으며, 이와 함께 관절 혹은 발굽의 모양이 비정상적이라는 것을 확인하였다. 또한, 폐사 개체들은 모두 하나 이상의 문제점을 동시에 가지고 있음을 확인하였다. 따라서, 이러한 문제점들이 형질전환에 의한 것인지, 아니면 복제에 의한 것인지를 규명하기 위해 연구를 더 진행해야 할 것으로 사료된다.

본 연구는 한우 체외 수정란의 동결보존에 hyaluronate(HA), ethylene glycol(EG), glycerol(G), sucrose(S)를 사용한 동결배양액의 효과를 검증하고자 수행되었다. 체외 수정란 생산을 위해 도축장에서 회수된 난소로부터 미성숙 난자를 채취하여 체외성숙, 수정, 발달 배양을 실시하였다. 성숙배양은 0.5 ug/ml FSH, 5 ug/ml LH, 0.125% BSA(v/v)가 첨가된 mTCM199 배양액에서 38.5 ℃, 5 % CO2 의 조건으로 22 h 동안 실시하였으며 체외수정은 mTALP 배양액에서 38.5 ℃, 5 % CO2 의 조건에서 22 h 동안 이루어졌고 발달배양은 mSOFaa 배양액을 사용하여 38.5 ℃, 5 % CO2, 5 % O2 의 조건에서 이루어졌다. 동결에 이용된 수정란은 수정배양 후 7 일차의 배반포를 대상으로 하였다. 실험에 사용된 동결배양액은 대조군으로써 vigro 의 1.8M EG 에 0.1 M S 가 첨가된 제품을 사용하였고 처리군으로써 1.8M EG 배양액, 1.8M EG 에 0.1M S 와 1.7M G 이 첨가된 배양액, 1.8M EG 에 0.1M S, 1.7M G 및 0.0007 g/ml HA 가 첨가된 배양액을 사용하였다. 모든 처리군은 0.01 g/ml Albumax 가 첨가된 CJ buffer 를 동결배양액의 base 로 사용하였다. 동결방법은 0.25 ml 스트로에 장착된 수정란을 CL-8800i(CryoLogic)에 의한 –0.3℃/min 의 완만동결로 –32℃까지 냉각시킨 후 액체질소에 침지하여 실시하였다. 동결과정 중, -6℃에서 스트로의 말단 부분에 식빙을 실시하였다. 동결융해 후 생존율 조사를 위해 동결보존 중인 스트로 32℃의 온수에서 25 초간 융해를 실시하여 발달배양액에서 48 시간 배양하여 총 동결융해 수정란에 대한 재확장율 및 부화율을 통해 생존율을 확인하였다. 실험결과, 동결융해 후 재확장율은 대조군(EG+S)과 EG, EG+S+G, EG+S+G+HA 에서 각 90.1±0.5, 76.0±1.0, ±85.4±2.1, 84.9±0.5 %로 관찰되었으며 부화율은 80.2±1.0, 70.8±4.2, 74.0±1.0, 82.8±1.6 %로 확인되었다. 재확장율은 대조군인 1.8M EG + 0.1M sucrose 배양액에서 90.1±0.5 %로 가장 유의적으로 높게 관찰되었으며 부화율은 대조군과 1.8M EG + 0.1M sucrose + 1.7M glycerol + 0.0007 g/ml HA 처리군에서 각 80.2±1.0 %와 82.8±1.6 %로 가장 유의적으로 높게 관찰되었다(p<0.05). 추가적으로 본 동결방법에 의해 생산된 동결수정란의 수정란이식을 실시하여 수태율을 조사하고 있지만 결과가 부족하여 본 연구결과에서 포함시키지 못하였다. 하지만 수정란 이식과정에서 EG+S+G 처리군의 경우 30 분이 경과된 시점부터 다른 처리군보다 수태율이 감소되는 경향이 뚜렷하게 관찰되었다. 반면, glycerol 과 HA 가 함께 첨가된 다른 동결배양액의 경우에는 이러한 경향이 관찰되지 않았다. 다른 체외수정란의 발달연구에서 HA 의 첨가는 부화율을 증가시킨다는 연구결과가 보고된 바 있는데 이러한 연구결과와 일치된다고 생각된다. 본 연구결과는 농촌진흥청 연구사업(세부과제번호:PJ012695032018)의 지원에 의해 이루어진 것임.

Cryopreservation is mainly used for preservation of boar sperm. However, this method stresses the sperm by reactive oxygen species (ROS), and the conception rate and the litter size are not more efficient than the liquid preservation of spermatozoa. Therefore, we use chitosan which is a natural product derived antioxidant compound. We used GnHA and GnHG as chitosan complexes to cryopreserve boar sperm for improve sperm metabolism and function. Sperm parameter (sperm motility, progressive motility, path velocity, straight-line velocity, curvilinear velocity) is measured by computer-assisted sperm analysis (CASA) using frozen sperm with GnHA or GnHG (0, 0.25, 0.5, 1 mg/mL). Also, lipid peroxidation analysis using malondialdehyde (MDA) is performed to confirm the antioxidative effect of chitosan in frozen spermatozoa. Sperm motility was higher in GnHA 0.25 mg/mL and GnHG 0.5 mg/mL compared to control. In addition, GnHG 0.5 mg/mL was significantly decreased in lipid peroxidation analysis. The results suggest that GnHA and GnHG are effective for boar sperm cryopreservation by antioxidant effect.

During the freezing and thawing process, fatty acids in the plasma membrane of sperm are released, which results in a functional damage of sperm. Sperm with functional loss due to cryo-damage result in a decrease in fertility. Previous studies have shown that the addition of one of the fatty acid alpha-linolenic (ALA) with carrier proteins improves the stability of plasma membrane and reduces the damage. In this experiment, we focused on the functional aspects of the plasma membrane of sperm and experimented with motility and morphology. For preparation of ALA-carrier protein complex, 3 ng/ml ALA was mixed with 0.7 μg/ml bovine serum albumin (BSA) or 14 ng/ml methyl-β-cyclodextrin (MBCD) in distilled water. The boar semen was purchased from GUMBO Company. Boar semen was cryo-preserved in 20% egg yolk freezing extender containing ALA, BSA, MBCD, ALA+BSA, ALA+MBCD. The frozen boar sperm was thawed at 37.5 ℃ for 45 sec in water-bath. The sperm motility and morphological abnormalities were evaluated under a phase-contrast microscope at 200 × magnification and randomly counts of 200 sperm each sample. In results, motility of frozen-thawed sperm was increased in all treatment groups. In particular, there has been significant improvement in ALA+BSA and ALA+MBCD treatment groups than control (p<0.05). However, there was no significant difference in ALA, BSA and MBCD treatment groups. Morphological normalities in frozen-thawed sperm was reduced in complex treatment groups (p<0.05). However, there was no significant difference in single treatment groups. In both motility and morphology characteristics, ALA+BSA and ALA+MBCD treatment group was higher than all treatment groups. In conclusion, the addition of ALA with carrier proteins during cryopreservation has a positive effect in its functional aspect. This work was supported by the National Research Foundation of Korea (NRF) grant funded by the Korea government (Ministry of Education) (2016R1D1A1B03931746).

To improve survival rates of vitrified pig oocytes, the treatment of cytoskeletal stabilizer on an appropriate time is one of the possible approaches. However, the exact treatment timing and effect of cytoskeletal stabilizer such as cytochalasin B (CB) is not well known during oocyte vitrification procedures. Thus, the present study was conducted to determine optimal treatment timing of CB during vitrification and warming procedures. In experiment 1, the survival rates of the post-warming pig oocytes were analyzed by fluorescein diacetate (FDA) assay with 4 classifications. In results, post-warming oocytes showed significantly (p<0.05) decreased number of alive oocytes (31.8% vs. 86.4%) compared to fresh control. In detail, the significant difference (p<0.05) was found only in strong fluorescence (18.2% vs. 70.5%) not in intermediate fluorescence groups (13.6% vs. 15.9%). In experiment 2, CB was treated before (CB-Vitri) and after (Vitri-CB) vitrification. In results, group of Vitri-CB showed significantly (p<0.05) higher (91.6%) survival rates compared to group of CB-Vitri (83.7%), significantly (p<0.05) and comparable with group of Vitri Control (88.7%) by morphological inspection. In FDA assay results, group of Vitri-CB showed significantly (p<0.05) higher (44.2%) survival rates compared to groups of CB-Vitri (36.7%) and Vitri Control (35.1%). In conclusion, the increased survival rates of post-warming pig oocyte treated with Vitri-CB method are firstly described here. The main finding of present study is that the CB treatment during recovery could be helpful to refresh the post-warming pig oocyte resulting its improved survival rates.

The purpose of this study was to establish an optimal storage temperature and characteristic analysis after frozen-thawed dairy goat sperm. Sperm was collected at Chojeong dairy goat farm using an electric stimulator and dilluted with semen washing media. The egg yolk-triladyl frozen solution was used for the freezing of Saanen dairy goat sperm and the freezing concentration was set to 1×108sperm/ml. The frozen sperm were thawed in water bath at 37.5℃ for 45 seconds and motility was measured after preservation for 0, 30, 60, 90 and 120 min at 4℃, 17℃ and 37℃, respectively. Sperm characteristic analysis was conducted by flow cytometry. In results, sperm motility at 30, 60, 90 and 120 min after thawing was significantly higher in 17℃ than 4℃ and 37℃ (p<0.05). On the other hand, the frozen-thawed sperm motility were gradually decreased with storage periods increased (30, 60, 90 and 120 min) at 4℃, 17℃ and 37℃. Viability(42%), acrosome damage(24%), mitochondrial damage(28%) and ROS level(4%) were analyzed by flow cytometry in frozen-thawed spermatozoa in 7 male dairy goats. In summary, the motility of frozen-thawed spermatozoa in Saanen dairy goat was more efficient for storage 17℃. The average of viability 42%(30%~54%), acrosome damage 24%(16%~33%), mitochondrial damage 28%(20%~54%) and ROS level 4%(3%~6%) were arranged as standard value by 7 male dairy goats. However, more researches are needed to establish the optimal conditions or proper supplementation for sperm preservation. This study was carried out with the support of research project on feasibility study of the research & development projects for activating the hillside livestock farming and the development of goat grazing program of Rural Development Administration by Korea government (2018PJ013546).

Successful cryopreservation of bovine oocytes is a very important technology for research and commercial applications. However, the survival and development rate of vitrified-thawed oocytes is lower than non-vitrified oocytes. Hydroxypropyl Cellulose supplementation (HPCs) has extremely high viscosity, which permits transitions to a glassy state at low temperatures. This characteristics of HPCs have been reported to help the survival of human oocytes. In this study, we investigated the survival rate, fertilization rate and ROS levels to confirm the effect of cryoprotectant solutions with HPC for oocyte vitrification in bovine. For vitrification, bovine MII oocytes were pretreated with EG10 (added 0, 10, 50 and 100 ㎍/㎖ HPC) for 5 min, exposed to EG30 (added 0, 10, 50 and 100 ㎍/㎖ HPC) for 30 sec, and then directly plunged into LN2. Thawing was taken by 4-step procedures [1 M sucrose and 10% FBS added D-PBS (SFD) -> 0.5 M SFD -> 0.25 M SFD -> 0.125 M SFD] for 1 min, respectively. After thawing, oocytes were washed with TL-HEPES, incubated in a droplet of previous cultured IVM medium for 1 h to recover. IVF drop (44 ㎕) contained 10 vitrified-thawed oocytes with sperm concentration of 1 × 106 cells ㎖, and then 2 ㎕ heparin and 2 ㎕ PHE were added. At 2 days after IVF, cleaved embryos were cultured in CR1aa + 3 mg/mL FAF-BSA for 48 h and cultured in CR1aa + 10% FBS for 4 days. In the results, in vitro survival rate of bovine vitrified-thawed MII oocyte was significantly higher in 50 (85.5%) and 100 ㎍/㎖ (80.2%) HPC groups than 0 (71.2%) and 10 ㎍/㎖ (71.3%) groups (p<0.05). The ROS level was lower in 50 ㎍/㎖ HPC group than in control group. After in vitro fertilization, cleavage rate and blastocyst development rate were not significantly different among treatment groups. Therefore, these results indicated that HPC treatment has a positive effect on the survival of vitrified-thawed bovine oocytes.

본 연구는 말 냉장정액의 정자 성상 향상을 위한 기초 연구로 국립축산과학원 난지축산연구소에서 한라마(더러브렛×제주마) 수말 정액의 보존시간대별 정자 성상을 분석하기 실시하였다. 본 연구의 공시마는 국립축산과학원 난지축산연구소에서 사육・보유하고 있는 한라마(더러브렛×제주마) 씨수마 2 두를 공시하였으며, 씨수마의 정액채취는 채취 시마다 같은 시간인 오전 9 시에서 10 시 사이에 실시하였다. 정액채취는 독성이 제거된 50ml 플라스틱 일회용 용기를 인공질(INRA model, IMV, French)에 장착하여 사용하였으며, 자외선 차단용 커버로 용기 주변을 보호하였다. 인공질은 45∼50℃의 온수를 이용하여 인공질 내부 온도가 38℃가 되도록 유지하였으며, 삽입을 원활하게 하기 위해 젤을 도포하였다. 정액은 채취 직후 10 분 이내 실험실로 운반하여 겔(gel) 제거 후 부피, 농도, 운동성 등을 조사하였다. 정장 물질 제거를 위하여 INRA96 희석제로 1:1 희석 후 50ml 튜브에 40ml 씩 분주한 후 400×g 에서 10 분 원심분리하여 seminal plasma 및 부유액을 제거하고 하층의 펠렛(정자괴)을 회수하였다(Cochran 등, 1984). 회수한 정자 펠렛은 최종 농도를 50×106/ml 로 희석하여 24h, 48, 72, 96 동안 혐기상태로 4℃냉장고에 보관하여 각각의 정자성상을 분석하였다. 말 정액은 INRA 96 희석제와 희석제에 Egg Yolk 3%가 처리 후 시간대별 정자성상을 분석하였다. 2 차 희석 후 INRA 96 과 Egg Yolk 3% 처리구에서 총 운동성은 각각 89.18%, 91.81%를 보였으며, 96 시간이 경과한 후에 정자 총 운동성은 INRA 96 이 80.71%, Egg Yolk 3% 처리구는 88.54%로 유의적인 차이를 보이지는 않았다. 본 연구의 기초 데이터를 바탕으로 정장제거 원심분리 조건, 정장제거 필터링 조건 등 정자 생존율 향상 연구 등 냉장정액의 시간대별 정자 성상분석을 통한 장기보존 방법 등의 연구가 수행되어야 할 것으로 생각된다.

Cryopreservation of bovine embryos is used to efficiently implant surrogate mothers. It has been widely accepted that high lipid content in the oocyte interrupts its survival during freeze-thaw cycles. Serum component in the culture medium is thought to increase the embryo`s lipid contents. Conversely, L-carnitine stimulates lipid metabolism by transporting long chain fatty acids into the mitochondria. Objective of this study was to analyze the effect of L-carnitine supplementation in IVM medium and defined IVC medium on the development, lipid contents and the cryosurvival of bovine IVF embryos. 0.0, 1.5, 3.0 and 6.0 mM L-carnitine was supplemented in IVM medium, respectively (IVM-LC 0.0, LC 1.5, LC 3.0 and LC 6.0). Development rate from the 2cell to the morula stages was higher in IVM-LC 3.0 groups than those of IVM-LC 6.0 (p<0.05). But there were no significant differences among the other groups in the blastocyst rates and lipid content results. When 0.0, 1.5, 3.0 and 6.0 mM L-carnitine were supplemented in IVC medium (IVC-LC 0.0, LC 1.5, LC 3.0 and LC 6.0), development competence was not significantly different between those embryos. Lipid contents of embryos treated L-carnitine (IVC-LC 1.5, 3.0 and 6.0) were significantly lower than embryos of non-treated group. L-carnitine was supplemented 0.0, 1.5, 3.0, 6.0 mM during IVM and 3.0 mM during IVC (LC 0.0 - 3.0, LC 1.5 – 3.0, LC 3.0 – 3.0, LC 6.0 – 3.0) and cryosurvival of blastocysts confirmed after freezing-thawing. There were no significant differences on development, but LC 3.0 – 3.0 was significantly lower lipid contents than other groups. And LC 3.0 – 3.0 had better survival rates and hatched rates of blastocysts than LC 0.0 – 0.0. In conclusion, supplementation of L-carnitine in defined IVC medium decreases lipid contents. And L-carnitine supplementation improves cryosurvival and developmental ability of bovine IVF embryos.

Sperm cryopreservation preserves genetic resources for animal breeding and for human patients who suffers from permanent testicular damage. Although the sperm cryopreservation has been used for many years, the addition of cryoprotective agent (CPA) during cryopreservation negatively affects sperm function and quality. Our previous study reported that the addition of CPA reduced bull sperm physiological functions. However, the sperm cells collected from individual bulls presented different sensitivity to the damage induced by CPA. In the present study, we examined if CPA affect sperm cells acquired from individual bulls. Individual bull spermatozoa were divided into two groups based on motility parameters; high CPA-tolerant sperm (HCS) and low CPA-tolerant sperm (LCS). Our results showed that the HCS group presented good physiological function after CPA exposure, whereas the LCS group showed a significant decrease in the sperm function. We also found differentially expressed five proteins between the HCS and LCS groups, which refer to cytosolic 5′-nucleotidase 1B (NT5C1B), fumarate hydratase (FH), F-actin-capping protein subunit beta (CAPZB), voltage-dependent anion-selective channel protein 2 (VDAC2), and cytochrome b-c1 complex subunit 1 (UQCRC1). NT5C1B and FH showed abundant expression in the HCS group, while the expression of CAPZB, VDAC2, and UQCRC1 was relatively lower in the HCS group than in the LCS group. The current results suggest that NT5C1B, FH, CAPZB, VDAC2, and UQCRC1 can be used as potential markers to predict CPA-tolerable spermatozoa. Those markers provide a reliable tool to select animals and breeds with CPA tolerance.

Sperm cryopreservation is well known as a valuable method to preserve the genetic traits. Although many studies have established semen cryopreservation protocols, lack of studies were conducted to discover the differences of sperm proteome and functions between ejaculated and epididymal spermatozoa following to cryopreservation. Therefore, the objective of this study was to (i) evaluate the effect of cryopreservation on bull epididymal spermatozoa and (ii) discover the potential biomarkers, which have highly tolerance to freezing on bull epididymal spermatozoa. Our preliminary study demonstrated that spermatozoa from each bulls have different resistance on freezing during cryopreservation. We divided spermatozoa into two groups according to sperm motility following to cryopreservation; high freezing-tolerant spermatozoa (HFS) and low freezing-tolerant spermatozoa (LFS). Several sperm functional parameters, i.e. sperm motility/motion kinematics, speed parameters, viability, mitochondrial membrane potential, and capacitation status. Our results showed that all parameters except for motion kinematics and capacitation status had significant differences between HFS and LFS. Subsequently, two dimensional electrophoresis were conducted to compare the expression levels of sperm proteome between both groups. Three proteins {glutathione s-transferase mu 5 (GSTM5), voltage-dependent anion-selective channel protein 2 (VDAC2), and ATP stynthase subunit beta (ATP1B1)} were differentially expressed. Based on these results, we propose that epididymal spermatozoa from individual bull have different freezability upon cryopreservation and three differentially expressed proteins might be selected as a biomarker to predict high freezing-tolerant epididymal spermatozoa.

In this study, we examined total number, motility and plasma membrane integrity of epididymal spermatozoa from cauda epididymis of bull after preservation at 4ºC. Totally, 23 testicles were castrated from 23 bulls (mean±standard error, age of days = 426.0±7.3, body weight (kg) = 379.7±8.4, scrotal circumference (cm) = 31.0±0.4) at Hanwoo Research Institute, NIAS, and transported to laboratory and preserved on 1, 4 and 6 days at 4 ºC. As control, epididymal spermatozoa recovery from 7 testicles was conducted after transportation to laboratory immediately. In experiment 1, we compared total number of spermatozoa among groups. Total number of spermatozoa from epididymis was not significantly on different preservation day of 0, 1, 4 and 6 which is 1778.0±304.7, 1824.8±343.9, 1228.4±91.7, 1201.8±178.6×106 cells/ml, respectively). In experiment 2, we examined spermatozoa motility and motility parameters (VCL (μm/s), VSL (μm/s), VAP (μm/s), LIN (%)) by computer assisted sperm analysis (SCA, MicroOptic) system. Percentage of motile on 0 and 1 day (88.9±5.2 and 85.8±6.1) was significantly higher than that on 4 and 6 days (32.6±6.5 and 34.3±8.25). Percentage of VCL (μm/s) on 0 and 1 day (93.5±7.6 and 83.0±14.9) was significantly higher than that on 4 and 6 days (36.6±5.1 and 39.5±5.5) (p<0.05). Percentage of VSL (μm/s) on 0 day (28.0±2.1) was significantly higher than that on 1, 4 and 6 days (20.2±3.0, 9.0±2.0 and 8.5±1.6, p<0.05). Percentage of VAP (μm/s) on 0 and 1 days (49.4±3.8 and 41.3±6.6) was significantly higher than that on 4 and 6 days (18.2±3.0 and 19.3±2.8, p<0.05). Percentage of LIN (%) on 0 day (30.7±2.6) was significantly higher than that on 4 and 6 days (23.4±2.7 and 21.1±1.0, p<0.05). Motility of spermatozoa was divided into 4 groups (fast progresive, slow progressive, non-progressive and immotile) based on WHO guideline. Percentage of fast progressive on day at 0 was significantly higher than that on 1, 4 and 6 days (0, 1, 4 and 6 days vs. 19.8±1.9, 10.2±1.1, 2.6±1.0 and 2.3±1.2%, respectively). In conclusion, cauda epididymal spermatozoa should be recovered within one day after preservation at 4 ºC to recover high quality of epididymal spermatozoa in Hanwoo bull

생체 유래 동해보호제인 난황 및 우유는 세균 및 바이러스에 의한 오염과 감염으로 동결융해 후 정액 성상(보존성)에 부정적 요소로 작용하여 왔다. 이에 본 연구는 반려견에 있어서 우수한 품종의 보존과 증식을 위하여 동결보존액이 융해 후 정액 성상에 미치는 영향을 조사하였다. 공시된 반려견은 경기도 김포에 위치한 애견 번식농장에서 보유 중인 포메라니언, 시츄 및 요크셔테리어 품종 각각 2 마리씩이었다. 정액채취는 맛사지법으로 실시하였으며 채취 직후 Androhep 희석액으로 35℃ 전후의 같은 온도로 1:1 로 희석시킨 다음 서울호서전문학교 브리딩전공 실습실로 2 시간이내에 옮겨졌다. 옮겨온 직후 개체별 정액성상을 평가하였으며 동결정액 제조방법은 지달영(2004)의 방법에 준하여 실시하였다. 동결보존액으로는 기본적으로 Androhep 희석액을 사용하였으며, 동결보존액이 융해 후 정액 성상에 미치는 영향을 조사하기 위하여 품종별 2 개체의 정액을 혼합하여 난황, 우유 및 코코넛 밀크가 각각 20%씩 포함된 1 차 동결보존액으로 1 차 희석 후 정자 농도를 4x107spermatozoa/ml 로 조정하였으며 1 시간에 걸쳐 4℃까지 냉각시켰다. 냉각된 정액은 1 시간에 걸쳐 8%의 glycerol 이 포함된 2 차 동결보존액의 10%, 20%, 30%, 및 40%씩 점등하여 1:1 로 희석시켰다. 최종 희석된 정액은 별도의 glycerol 평형 시간 없이 0.5ml straw 에 충진 시켜 포장하였다. 최종 포장된 정액은 액체질소 표면 5cm 위에서 10 분간 정치시켜 예비동결시킨 후 침지하여 동결시켰다. 동결과정 중 정자의 활력은 현미경 상의 혈구계산판을 이용하여 100 분율로 평가하였다. 반려견의 품종에 따른 채취 직후의 정자 활력은 시츄, 포메라니언 및 요크셔테리어 가 각각 87.5%, 40%, 및 60%로 시추의 정자 활력이 다른 품종 보다 높았다. 동결보존액의 주요 조성분인 난황, 우유 및 코코넛 밀크에 따른 동결융해 후 정자 활력은 각각 60%, 40% 및 20%로 난황이 가장 우수하였으나. 이들 주요 조성분의 오염도 수준은 난황, 우유 및 코코넛 순으로 나타났다. 식물성 동해보호제로 코코넛은 동결융해 후 활력이 가장 낮게 나타났으나 생체 유래 물질인 난황 및 우유보다는 오염의 정도가 매우 낮게 나타났다. 이상의 결과에서 동해보호제로서 식물성 코코넛 밀크가 반려견 정액의 동결 융해 후 정자 생존성(활력)에 있어서는 생체 유래물질인 난황, 우유보다 낮았으나 오염(세균 및 바이러스 등)을 줄일 수 있는 새로운 방향을 제시한 것으로 나타났다. 코코넛 밀크를 준비하는데 불편함이 많았던 관계로 대체할 수 있는 다른 식물성 동해보호제가 탐색되어야 할 것으로 사료되었다.

생체 유래 동해보호제인 난황 및 우유는 세균 및 바이러스에 의한 오염과 감염으로 동결융해 후 정액 성상(보존성)에 부정적 요소로 작용하여 왔다. 이에 본 연구는 렛트(rat)에 있어서 동결보존액이 융해 후 정액 성상에 미치는 영향을 조사하였다. 공시된 렛트는 18 주령 이상의 성숙된 수컷 6 마리였다. 정액채취는 렛트를 먼저 할로탄 흡입마취한 후 양측 정소의 중앙부를 1~2cm 절개하여 정소상체의 미부를 적출한 후 Androhep 희석액이 담긴 배양접시(⌀35mm)에 옮겨서 세절하여 정자를 채취하였다. 정액의 동결과정은 이장희(1993)의 방법에 준하여 실시하였다. 동결보존액으로는 기본적으로 Androhep 희석액을 사용하였으며, 동결보존액이 융해 후 정액 성상에 미치는 영향을 조사하기 위하여 2 개체의 정액을 혼합하여 난황, 두유 및 코코넛 밀크가 각각 20%씩 포함된 1 차 동결보존액으로 1 차 희석 후 정자 농도를 4x107spermatozoa/ml 로 조정하였으며 1 시간에 걸쳐 4℃까지 냉각시켰다. 냉각된 정액은 1 시간에 걸쳐 8%의 glycerol 이 포함된 2 차 동결보존액의 10%, 20%, 30%, 및 40%씩 점등하여 1:1 로 희석시켰다. 최종 희석된 정액은 별도의 glycerol 평형 시간 없이 0.5ml straw 에 충진 시켜 포장하였다. 최종 포장된 정액은 액체질소 표면 5cm 위에서 10 분간 정치시켜 예비 동결시킨 후 침지하여 동결시켰다. 동결과정 중 정자의 활력은 현미경 상의 혈구계산판을 이용하여 100 분율로 평가하였다. 2 개체로부터 채취하여 합쳐진 정액에 대해서 동결보존액의 주요 조성분인 난황, 두유 및 코코넛 밀크에 따른 동결융해 후 정자 활력은 각각 10%, 0% 및 0%로 난황이 다소 높게 나타났다. 이들 주요 조성분의 오염도 수준은 난황, 두유 및 코코넛 순으로 나타났다. 동결과정 중 활력 변화는 채취 직후 합친 상태의 활력은 평균 75% 였으며 냉각 후 2 차 희석이 완료되었을 때에는 40%, 예비동결 후에는 20% 수준, 동결 후 1 일 경과 후의 융해 후 정자 활력은 매우 저조하였다. 이상의 결과에서 동해보호제로서 식물성 동해보호제로써 두유와 코코넛 밀크의 이용 가능성이 없는 것으로 나타났다. 다만 동결 처리 과정 중 정자 활력의 급격한 손실은 정소상체 미부 정자의 미성숙 상태에 의한 내동성 부족으로 사료되었다.

본 연구는 렛트(rat)에 있어서 정액 보존액이 인공수정 후 수태율에 미치는 영향을 조사하였다. 시험에 공시한 렛트는 20 주령 이상의 성숙 완료된 암컷 18 마리, 수컷 6 마리였다. 렛트의 정액채취는 먼저 에테르로 흡입 마취시킨 후 양측 정소의 중앙부를 1~2cm 절개하여 정소상체의 미부를 적출한 후 PBS 용액이 담긴 배양접시(⌀35mm)에 옮겨서 수술용 미세 칼로 세절하여 정자를 채취하였다. 각 2 개체로부터 채취된 정액은 활력을 평가한 후 60% 이상의 개체의 정액을 혼합하여 Androhep, Modena 및 BTS 희석액으로 각각 희석하여 정자 농도를 1.5x108spermatozoa/ml 로 조정하였다. 보존액별 혼합된 정액은 17℃에서 6 시간 보관 후 인공수정에 사용하였다. 인공 수정할 암컷 렛트는 실리콘 탭형 progesterone(4-Pregnene-3,20-dione, Sigma) 방출 장치를 질 내에 6 일간 삽입시킨 다음 제거 직후 PMSG 25IU 를 근육주사하고 24 시간 후에 hCG 20IU 를 근육 주사하여 발정과 배란을 유도하였다. hCG 주사 후 6 시간 후에 정액을 주입하여 인공수정시켰다. 처리별 인공수정 직전의 정자 활력은 각각 80%, 60% 및 50% 수준이었다. 정액 주입 직전 미니 스포이드 내 생리식염수로 질 내 상피세포를 관류 흡입 한 후 슬라이드글라스에 도말시켜 표본을 제작하였다. 제작된 표본에서 유핵의 세포상(파편모양)을 관찰하여 정액 주입 시의 발정상태를 확인하였다. 상피세포의 용이한 관찰을 위해 10% giemsa 용액에 도말된 슬라이드글라스를 30 분간 침전하여 염색시킨 후 꺼내어 증류수로 세척하고 드라이기로 건조시킨 후 검경하였다. 인공수정으로 보존액별 정액을 처리별 6 마리의 암컷에게 마리당 0.2ml 의 정액을 주입하였다. 정액주입기는 1ml 주사기에 라운딩된 주사침을 연결한 주사기였다. 인공수정 후 10 일경에 복부촉진법으로 임신 여부를 진단한 결과 수태율은 Androhep, Modena 및 BTS 보존액별 수태율은 각각 83.3%, 66.7% 및 33.3%였다. 하였다. 인공수정 시 그룹별 발정발현율은 각각 83.3%(5/6), 100.0%(6/6) 및 66.7%(4/6)였다. 발정 발현된 개체에 대한 수태율은 Androhep, Modena 및 BTS 보존액의 수태율은 80.0%(4/5), 80.0(4/5)% 및 75.0%(3/4)였다. 이상의 결과에서 정액보존액에 따른 인공수정 후 수태율은 BTS 보존액이 낮게 나타났으나 Androhep 과 Modena 는 차이가 없었다. 또한 발정이 발현된 경우에는 인공수정으로도 거의 수태 된 것으로 나타났으므로 자연교배에 따른 인력과 시간낭비를 줄일 수 있을 것으로 사료되었다.

Until recently, there have been many researches about the freezing methods and several methods of cryopreservation. Hypothermic preservation has been used to complement the embryo freezing technology. There is a study to show the successful results for long-term hypothermic preservation. For that reason, FBS and BSA are commonly added to the culture medium to support embryo development. We investigated the effectiveness of hypothermic preservation method at 4℃ according to embryonic developmental stages for Hanwoo embryos and evaluated the effect of FBS and BSA on Hanwoo embryos as a supplemental reagent in hypothermic preservation medium after recovering preserved embryos from hypothermic preservation. The present study reported that survival and hatching rates of embryos at morula stage following storage at 4℃ is Day 7 group was significantly higher (p < 0.05) compared than those of other groups (p < 0.05). As a result, the survival and hatching rates of embryos at the blastocyst stage following storage at 4℃ result is showed that significantly higher (p < 0.05) survival rates than those of other groups an Day 6. The result showed that hatching rate at Day 6 and 7 were significantly lower (p < 0.05) compared with other groups. The result regarding the survival and hatching rates of bovine embryos following storage at 4℃ for 72 h in various concentrations of BSA are shown The results showed that survival rate of 1% BSA group was not significantly different (p < 0.05) compare with control (FBS) group. Also, the results showed that hatching rate of control (FBS) and 1% BSA were significantly different (p < 0.05) compared with other groups. In conclusion, our result demonstrated that the hypothermic preservation did not effect on the survival and hatching rates of embryos after recovering. In addition, the supplementation of BSA in preservation medium showed no difference in the embryo developmental competence after hypothermic preservation compared to FBS treatment. With that, BSA can be an alternative reagent for the hypothermic preservation medium as an energy source and pH buffer.

The aim of this study was to develop a chemically defined extender for dog sperm cryopreservation by supplementation of essential and non-essential amino acids solution in EY-free PVA extender. Spermatozoa collected from mature dogs (1 ｘ 108 cell/ml) were frozen with EY-free extender supplemented with 0 (control), 1, 2, 4 % essential amino acids (EAAs) or 1, 2, 4 % non-essential amino acids (NEAAs). Sperm progressive motility, viability and acrosome integrity were evaluated immediately after thawing at 37 ℃ for 25 s and post-thaw incubation at room temperature for 20 min. In addition, to evaluate the synergistic effect of EAAs and NEAAs, spermatozoa were frozen with 0, 0.5, 1 or 2 % EAAs-NEAAs mixture (v:v). Sperm progressive motility, viability and acrosome integrity were evaluated immediately after thawing and post-thaw incubation. Additionally, spermatozoa were frozen using EY-free PVA extender supplemented with 2 % EAAs, 2 % NEAAs or 0.5 % EAAs-NEAAs mixture. The ROS level and phosphatidylserine (PS) translocation (Annexin V-FITC assay) were assessed using flow cytometry. In addition, gene expression level for SMCP (motility-related), apoptosis-related BCL2 and BAX was measured after freezing-thawing. The progressive motility of spermatozoa cryopreserved in EAAs or NEAAs significantly increased (P < 0.05) in all groups compared to the control group regardless of thawing conditions. In addition, 1 % NEAAs significantly protected the acrosome membrane of spermatozoa after freezing-thawing (P < 0.05). However, EAAs has shown no significant effect on viability and acrosome membrane integrity of spermatozoa. On the other hand, addition of EAAs-NEAAs mixture to EY-free PVA extender significantly (P < 0.05) increased sperm progressive motility without any effect on viability. Supplementation of 0.5 % EAAs-NEAAs mixture significantly (P < 0.05) increased the expression level of SMCP, BCL2 and BAX compared to control without significant effect on PS translocation and ROS level. We conclude that essential and non-essential amino acids solution can be effectively used in EY-free extender to improve sperm motility, acrosome integrity and gene expression of SMCP and BCL2 in dog sperm cryopreservation.

The present study was undertaken to evaluate the effect of trisaccharides supplementation in glycerol-free tris (GFT) for the cryopreservation of dog spermatozoa. In the first experiment (E1), dog spermatozoa were resuspended with 50, 75, 100 or 125 mM of raffinose, melezitose or maltotriose and cooled at 4 ℃ for 10 min. To determine the effect of different cooling time, the spermatozoa resuspended with 100 mM of raffinose, melezitose or maltotriose were cooled during 10, 20, 30 or 40 min at 4 ℃ (second experiment; E2). The straws were then aligned horizontally for 10 min on the rack and then plunged into LN2. In the third experiment (E3), to determine the effect of different vapor freezing time, the spermatozoa resuspended with 100 mM raffinose were cooled at 4 ℃ for 20 min and frozen in LN2 for 5, 10, 15 or 20 min and then plunged into LN2. In the fourth experiment (E4), to compare different freezing methods [cooling plus vapor freezing (CV), cooling plus step-down freezing (CS) and direct step-down freezing (SD)], the spermatozoa resuspended with 100 mM raffinose were cooled for 20 min and frozen in LN2 vapor for 5 min in case of CV method. In case of CS method, spermatozoa were cooled for 20 min at 4℃ and then frozen by the step-down freezing method. The straws were then aligned horizontally at 18, 15, 5, and 2 cm respectively from the surface of LN2 for 1, 1, 1.4, and 5 min, respectively in an L shaped straw holder and then plunged into LN2. For SD method, the straws were directly aligned horizontally at the same levels as CS from the surface of LN2 for 1, 1, 1.9, and 5 min, respectively and then plunged into LN2. After thawing at 37℃ for 25 sec, the spermatozoa were then incubated for 30 min in the freezing extender (E1) or in the 50 mM sucrose supplemented GFT (E2, E3, and E4) at 24℃. Following post-thaw incubation, sperm progressive motility and viability were assessed in E1, E2, E3, and E4. In addition, acrosome integrity, and gene expression related to apoptosis (BAX, BCL2, and Caspase10) and sperm motility (SMCP) were evaluated in E4. The results demonstrated that, in E1, using 75 mM trisaccharides resulted in significantly (p<0.05) higher sperm motility in all sugar groups. Using 100 mM melezitose significantly (p<0.05) improved the post-thaw viability than the 100 mM raffinose. The viability in 100 mM maltotriose was similar with 100 mM raffinose and melezitose group. In E2, the different cooling time has no significant effect on post-thaw sperm progressive motility in all the sugar types. In addition, the viability was variable among the different groups. In E3, liquid nitrogen vapor freezing for 5 min resulted in improved motility and viability. The sperm progressive motility was significantly (p<0.05) higher in CV and SD group compared to CS group and the sperm viability was significantly (p<0.05) higher in CV group compared to the other groups in E4. However, the acrosomal integrity of spermatozoa in the group CV was significantly (p<0.05) higher than the group CS and SD. In addition, the expression of SMCP gene was significantly (p<0.05) higher in the CV group than the CS group. In contrast, the expression of Caspase10 significantly (p<0.05) lower in the group CV and SD than the group CS. Furthermore, the ratio of gene expression of BAX and BCL2 was significantly (p<0.05) lower in the group CV than the group CS. Therefore, cryopreservation of dog spermatozoa in 100 mM of raffinose supplemented GFT cooled for 20 min and vapor freezing for 5 min provides better progressive sperm motility, viability, and acrosome integrity with higher expression of SMCP gene and lower expression of caspase10 and BAX/BCL2 ratio following post-thaw incubation in 50 mM sucrose supplemented GFT for 30 min at 24℃.