Earticle

핵이식 방법을 이용하여 성공적인 복제를 이루기 위해서 인위적인 활성화 처리는 필수적인 요소이다. 본 연구는 전기자극에 의해 활성화된 난자를 chemical agent를 이용하여 추가적인 활성화 처리를 하였을 때 돼지 단위발생란의 발달에 미치는 영향을 알아보고자 수행되었다. 체외에서 40~44시간 동안 배양된 난자를 전기자극(E)으로 활성화 처리한 후 Thimerasol + Dithiothreitol(Thi+DTT), 6-Dimethylaminopurine(6-DMAP) 및 Cycloheximide(CH)를 사용하여 추가 활성화 처리를 하였다. 활성화 방법(E, E+Thi+DTT, E+6-DMAP 및 E+CH)에 따른 단위발생란의 배반포까지의 발달율을 조사한 결과, chemical agent에 의해 추가 활성화된 단위발생란이 전기자극만으로 처리된 구의 단위발생란보다 유의적으로 높은 발달율을 보였다(21.5~28.1% vs. 18.0%, P<0.05). 특히, E+Thi+DTT를 이용하였을 때 발달율이 유의적으로 높게 나타났다(28.1%, P<0.05). 활성화 처리별 전핵 형성율을 조사한 결과, chemical agent에 의해 추가 활성화 처리된 구에서 하나의 극체(1PN) 형성률은 처리별로 차이를 보이지 않았으나(59.9~64.7%), 2PN 형성율은 추가 활성화 처리구에서 전기자극만을 사용하였을 때보다 유의적으로 높게 나타났다(7.2~9.7% vs. 4.3%, P<0.05). 이상의 결과를 살펴볼 때, 전기자극 후 chemical agent를 이용한 추가 활성화는 단위발생란의 배반포까지의 발달능력을 증가시키는 것으로 생각된다.

Artificial activation of oocytes is a prerequisite for the successful cloning by nuclear transfer. This study investigated the effect of the different combination of activation agents such as electric pulse (E), thimerosal (Thi) + dithiothreitol (DTT), 6-dimethylaminopurine (6-DMAP) or cycloheximide (CH) on the developmental ability of porcine embryos derived from parthenogenetic activation (PA). PA embryos activated with chemicals showed significantly higher developmental rate to the blastocyst stage compared to the embryos activated with E alone (21.5~28.1% vs. 18.0%, respectively). Of chemicals, Thi + DTT supported higher development to the blastoryst stage (28.1%). There was no significant difference in 1 pronucleus (PN) formation rate (59.9~64.7%), but 2PN formation rate was significantly higher in PA embryos with additional activation using chemicals (7.2~9.7%). In conclusion, this study shows that chemical activation after electric pulse can increase the development of porcine PA embryos.

본 연구는 호르몬과 Na-pyruvate의 첨가가 개 미성숙난자의 체외성숙에 미치는 영향을 검토하였다. 개의 발정주기와 관계없이 암캐의 난소에서 회수한 난자를 NCSU-37 배양액에서 72시간 체외 배양하였다. 호르몬의 영향을 검토하기 위하여 배양액 중에 다양한 호르몬을 24{sim}72시간 첨가하여 성숙율을 검사하였으며, Na-pyruvate 첨가 농도의 영향을 검토하였다. E2 단독 첨가구에서는 제2감수분열 중기(MII) 단계까지 성숙이 관찰되었으나(5.7%), 타 처리구에서는 MII기까지의 성숙이 이루어지지 않았다(P<0.05), E2를 6~24시간만 처리하였을 때는 MII기로 성숙되는 난자가 없었으나 72시간 처리하였을 때는 6.0%로 유의적으로 높게 나타났다(P<0.05). Na-pyruvate의 농도를 0.25 mM 첨가한 구보다 2.5 mM 첨가한 구에서 제1감수분열 중기(MI) 단계까지의 성숙율이 증가하는 경향을 보였으나, MII 단계까지의 성숙율은 차이가 없었다. 본 실험의 결과는 개 난자의 체외성숙시 E2가 첨가된 배양액에서 72시간 배양시에 성숙율을 증진시킬 수 있으나, Na-pyruvate의 농도는 개 체외배양에 있어 큰 영향을 미치지 않는 것을 나타낸다.

This study was conducted to examine the effects of hormone and sodium pyruvate on in vitro maturation of canine oocytes. Canine oocytes were collected from the ovaries of dogs and cultured in NCSU-37 medium with hormones and sodium pyruvate for 72 hr. Oocytes matured to the metaphase II (MII) stage were observed only from estradiol 17β (E2), and the presence of gonadotropin did not improve the nuclear maturation. No oocytes were developed to the MII stage when E2 was added to medium during the first 6 and 24 hrs of culture period. The presence of E2 during the whole culture period enhanced the nuclear maturation to the MII stage (6.0%, P<0.05). High concentration of sodium pyruvate (2.5 mM) slightly enhanced the nuclear maturation to the metapahse I (HMI) stage, but not the MII stage. the result of the present study shows that the presence of E2 during the whole culture period of 72 hr enhances the maturation of canine oocytes to the M stage, but sodium pyruvate does not affect the nuclear maturation of the canine oocytes.

생물학적 자극통제 수단으로 활용하기 위한 돼지 웅성 페르몬성 분자를 탐색하고자 일련의 냄새 분자로서 2-(cyclo-hexyloxy) tetrahydrofurane 유도체들의 정량적인 구조와 수용체인 porcine odorant binding protein (pOBP)간의 결합 친화력 상수(p(Od)50)에 대한 비교 분자장 분석(CoMFA)을 실행하였다. 가장 양호한 CoMFA 모델 AIV (r2 cv.(q2)=0.886 및 r2 ncv.=0.984)은 기질 분자 내 입체 중심(chiral center)의 절대 배열이 C1(R),C2(S)인 분자를 atom based fit 방법으로 배열하였을 경우의 standard field와 indicator field가 조합된 CoMFA장의 조건에서 유도되었다. 이 CoMFA 모델은 입체장 40.8% 정전기장 14.6%및 소수성장 44.6%가 결합 친화력 상수에 영향을 미치는 요소임을 나타내었다. 등고도의 분석 결과로부터 효과적인 결합 친화력 냄새 분자를 수식하는 데 몇 가지 가치 있는 정보를 얻을 수 있었다.

To search of new porcine pheromonal odorants for biostimulation control system technologies to improve reproductive efficiency in livestock species, the comparative molecular field analysis (CoMFA) for binding affinity constant (p(Od)50) between porcine odorant binding protein (pOBP) and ligands of odorant 2-(cyclohexyloxy) tetrahydrofurane derivatives as substrate molecule was conducted and discussed. In the optimized CoMFA model AIV with chirality (C1'(R), C2(S)) in substrate molecule and atom based fit alignment (A) of odorants, the statistical results showed the best predictability of the binding affinities (p(Od)50) based on the LOO cross-validated value r2 cv.(q2=0.886) and non-cross-validated conventional coefficient (r2 ncv.=0.984). the binding affinity constants exhibited a good correlation with steric (40.8%), electrostatic (14.6%) and hydrophobic (44.6%) factors of the substrate molecules. from the analytical results of the contour maps, which may give us some valuable informations to the modification of odorants for effective binding affinity.

본 연구는 체외성숙 배지에 첨가하는 PVP의 농도, EGF, cysteine 및 PVP의 단독 또는 혼합첨가가 한우 체외수정란의 체외발생에 미치는 영향을 검토하였다. 체외성숙 배지에 PVP의 첨가농도(0.1~3.0%)에 따른 분할율은 차이가 없었으나, 배반포 발달율은 0.5% PVP 첨가군이 가장 높았다(P<0.05). PVP, EGF 및 cysteine의 단독 및 혼합 첨가에 따른 분할율은 cysteine 단독첨가군이 높았으나(P<0.05), 배반포 발달율은 차이가 없었다. Inner cell mass 수는 대조군과 cysteine 첨가군이 PVP 첨가군에 비하여 유의하게 높았고(P<0.05), 총 세포수도 cysteine 첨가군에서 가장 높았다. 수정란이식 결과는 대조군, EGF, cysteine 및 EGF+cysteine 군의 임신율은 46.1~63.6%로서 비슷하였으나, PVP 첨가군은 10%로서 다른 군에 비하여 유의하게 낮았다(P<0.05). 본 연구 결과는 체외성숙 배지에 PVP의 첨가로 배 발생은 가능하지만, 세포의 품질에는 악영향을 미치는 것을 보여준다.

This study was examined the effects of concentrations of polyvinylpyrrolidone(PVP) and supplementation of EGF, cysteine and PVP during in vitro maturation on the development of bovine embryos. In experiment 1, 0.1 to 3.0% PVP was supplemented to IVM medium before IVF. The development rates to the blastocyst stage was significantly higher in 0.5% PVP group than 3.0% PVP group (P<0.05). In experiment 2, EGF, rysteine and PVP were supplemented to IVM medium. The hight cleavage rate was obtained from cysteine group, but blastocyst formation rates did not differ among groups. The highest total cell number and inner cell mass (ICM) cell number were observed in cysteine group. In PVP group, ICM cell number was significantly low than those of cysteine and control groups (P<0.05). After embryo transfer, pregnancy rate was significantly low in PVP group compared to other groups (P<0.05). These results indicate that the supplementation of PVP in IVM medium support the embryo development, but has a deteriorate effect on the blastocyst quality.

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-α) 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.

본 연구는 OPS 기법에 의한 돼지 수정란의 동결-융해 시 수정란의 발달 단계와 superoxide dismutase (SOD)가 수정란의 생존능력에 미치는 영향을 검토하였다. 돼지 체외수정 배반포는 OPS방법에 의해 동결 후 융해하여 0~10unitsml의 SOD 존재 하에 48시간 체외배양하였다. 동결-융해 후 형태학적으로 정상적인 수정란의 비율은 초기, 중기 및 확장배반포간에 유의적인 차이는 인정되지 않았다(30.8~38.6%). 그러나 발육단계가 높을수록 형태적으로 정상인 수정란의 비율이 높은 경향을 나타냈다. 수정란의 융해 후 48시간 추가 배양했을 때, 발육이 진행된 수정란은 후기배반포기에 동결한 수정란이 38.7%로 유의적으로 높았으며(P<0.05), 1 unit/ml의 SOD를 첨가한 경우 비교적 높은 생존율을 나타내었다. 본 연구의 결과로부터, 수정란의 OPS방법에 의한 동결-융해 후 생존성의 향상을 위해서는 후기배반포기 단계에 동결하는 것이 유리하며, SOD의 첨가는 수정란의 손상을 어느 정도 방지할 수 있을 것으로 사료된다.

This study was performed to investigate the effects of embryo developmental stage and superoxide dismutase (SOD) on the survival of frozen-thawed porcine embryos by open pulled straw(OPS) method. Porcine IVF blastocysts were frozen-thawed by OPS method and cultured for 48 h under the existence of SOD. There are no significant differences in the proportions of normal morphology among the early, mid- and expanded blastoryst stages (30.8~38.6%). After culture of embryos, the developmental rates to the expanded blastocyst stage(38.7%) were significantly higher than those of other stages (P<0.05). The proportions of expanded and hatched embryos were higher in medium with 1 unit/ml SOD than 0 and 10 units/ml of SOD. The result indicates that OPS method can use for the pig embryo cryopreservation, especially for the late stage blastocysts. SOD may can reduce the demage of frozen-thawed porcine embryos.

Electrical treatment has been widely used for porcine oocytes activation. However, developmental rates following electrical activation of porcine oocytes is relatively inefficient compared to other domestic animals. To investigate the effects of porcine oocytes on combined activation by both chemical and electrical treatment, in-vitro matured oocytes were activated by combined cycloheximide and electrical pulses treatment. Cumulus-free oocytes were exposed with NCSU-23 medium containing cycloheximide (10μgml) for 0, 5, 10, 20, 30 min and then activated by electrical pulse treatment and cultured in PZM-3 for 8 days. Also effects of exposure to 6.25μM calcium ionophore for 2 min for cumulus-free oocytes were tested. The percentage of blastocyst formation in 10 min exposure to 10μgml cycloheximide and electrical pulse treatment was significantly increased (P<0.05) than in the control group. And exposure to 6.25μM calcium ionophore for 2 min with 10μgml cycloheximide for 10min and electrical pulse treatment significantly increased (P<0.05) the percentage of blastocyst developmental rates than the control group. In conclusion, activation by combined cycloheximide and electrical stimulation treatment promoted the subsequent development of porcine oocytes and improved the subsequence blastocyst development

본 연구는 단위발생유래 생쥐 배아줄기세포(P-mES)지가 체외수정유래 생쥐 배아줄기세포 (mES)와 마찬가지로 기능성 심근세포로 체외 분화되는지를 조사하였다. 각 세포주 P-mES04와 MES03를 4일간 부유 배양하여 배아체 (EB)를 형성한 다음 4일간 DMSO를 추가적으로 처리한 뒤 젤라틴이 코팅된 배양접시에 부착시켰다(4-/4+). P-mES04와 mES03으로부터 수축성 심근세포 생성 여부를 30일간 관찰한 결과, 각각 13일(69.83%)과 22일 (61.3%)에 누적 형성율이 가장 높았다. 면역 세포화학염색 결과, 수축성을 나타내는 P-mES04 세포는 수축성 mES03 세포에서와 같이 근육 특이적인 anti-sarcomeric a-actinin 항체와 심근 특이적인 anti-cardiac troponin I 항체에 염색되는 것을 확인하였다. 또한 RT-PCR 결과, 수축성을 나타내는 P-mES04 세포는 심근특이적인 L-type calcium channel, a1C, cardiac myosin heavy chain a, cardiac muscle heavy polypeptide 7β, GATA binding protein 4와 atrial natriuretic factor는 발현하나, 골격근 특이적인 L-type calcium channel, a1S는 발현하지 않아 웅성 성체의 심장세포와 유사한 양상을 보였다. 본 연구의 결과는 단위발생 유래 생쥐 배아 줄기세포를 배아줄기세포의 연구의 대체제로 이용할 수 있음을 보여준다.

This study was conducted to examine whether the parthenogenetic mouse embryonic stem (P-mES) cells can differentiate into functional cardiomyocytes in vitro similar to (mES) cells. p-mES04 and IVF-derived mES03 cells were cultured by suspension culture for 4 days. The formed embryoid bodies (EBs) were treated with 0.75% dimethyl-sulfoxide (DMSO) for further 4 days (4-/4+), and then plated onto gelatin coated culture dish. The appearance of contracting cardiomyocytes from the P-mES04 and mES03 cells was examined for 30 days. The highest cumulative frequency was detected at days 13 (69.83%) and 22 (61.3%), respectively. By immunocytochemistry, beating P-mES04 cells were positively stained with muscle specific anti-sarcomeric a-actinin Ab and cardiac specific anti-cardiac troponin I Ab similar to contracted mES03 cells. When the expression of cardiac muscle-specific genes was analyzed by RT-PCR, beating P-mES04 cells were expressed cardiac specific L-type calcium channel, a1C, cardiac myosin heavy chain a, cardiac muscle heavy polypeptide 7β, GATA binding protein 4 and atrial natriuretic factor, but not expressed skeletal muscle specific L-type calcium channel, a1S, which was similar to male adult heart cells and mES03-derived beating cardiomyocytes. The result demonstrates that the P-mES cells can be used as an alternative for the study on the characteristic analysis of in vitro cardiomyocyte differentiation from the ES cells.

본 연구는 한우 성체 유래 귀세포(Korean bovine ear skin fibroblasts, KbESF)와 태아 섬유아세포(Korean bovine fetal fibroblasts, KbFF)를 이용한 체세포 복제(SCNT) 시 세포종류, 배양기간 그리고 융합방법이 핵이식 수정란의 발달에 미치는 영향을 알아보기 위하여 실시하였다. 태아 섬유아세포는 임신 51일령의 한우태아에서 분리하였고, 귀세포는 28개월령의 성우의 귀에서 채취하였다. 세포는 15주 동안 체외에서 배양하며 체세포 핵이식(SCNT)에 공시하였다. 융합방법을 비교하기 위해 챔버방법과 전극 바늘을 이용한 방법으로 핵과 세포질을 융합하였다. 세포의 doubling time은 KbFF에서 17.3시간, KbESF에서 24.3시간으로 나타났다. 핵이식 후 융합과 분할율은 needle 방법에서 보다 유의적으로 높았으나(각 각 76.1과 81.2%, P<0.05), 배반포 발달율은 차이가 없었다. KbESF의 경우, 배반포 발달율은 passage 5~9(39.4%)와 13~15(40.4%)에서 passage 1~4에 비하여 유의적으로 높았다(P<0.05). KbFF의 경우, 융합율은 passage 5~8과 13~15에서 각각 75.0 및 76.8%로 passage 1~4(61.5%)보다 높았으나, 난분할율과 배반포 발달율은 차이가 없었다. 결론적으로, SCNT 수정란의 발달은 융합 방법에 의해 영향을 받을 수 있으나, 계대배양 15회까지 장기배양을 한 경우는 복제수정란의 발육에 영향을 주지 않는 것으로 판단된다.

The study was conducted to evaluate the effects of culture period and fusion method on the development of somatic cell nuclear transfer (SCNT) embryos reconstituted with Korean bovine fetal fibroblast cells (KbFF) and Korean bovine adult ear skin fibroblast cells (KbESF). KbFF were isolated from a day 51 Korean cattle (Hanwoo) fetus, and KbESF were isolated from a 28 month old Hanwoo calf. The cells were cultured up to 15 weeks (passage 15) in vitro for SCNT. Chamber and electrode needles were used for comparing fusion of reconstituted eggs. The doubling times of KbFF and KbESF were 17.3 hr and 24.3 hr, respectively. The fusion and cleavage rates were significantly higher in needle group (76.1 and 81.2% respectively, P<0.05) than those in chamber group. However, the blastocyst development rate was not different between both groups. Fusion and cleavage rates of NT eggs reconstituted with KbESF did not affected by passage number, however, blastocyst rates were lower in passage 1~4 group (21.3%) than passage 5~8 (39.4%) and 13~15 groups (40.4%, P<0.05). Whereas, fusion rate was lower in passage 1~4 group (61.5%) than those of passage 5~8(75.0%) and 13~15 (76.8%) groups, but cleavage and blastocyst rates were similar regardless of passage number in the KbFF. The results suggest that fusion method can affect the development of SCNT embryos, whereas the long term culture up to 15 passages may not affect the development of SCNT embryos.

본 연구는 돼지정액의 보다 간편하고 손쉽게 동결시킬 수 있은 방법을 확립하기 위하여 수행하였다. 돼지정액은 3시간에 걸쳐 5℃까지 냉각 후 Straw에 봉입하고 다양한 방법 및 step에 의해 스티로폼 용기 내에 들어있는 LN2 중에서 동결하였다. 정자의 생존성은 LN2 표면으로부터 10 cm 위에서 10분간 정치 후 침적할 경우 가장 높게 나타났다(54.0%). Straw를 -102℃에서 10분간 정치시킨 처리구가 여타구보다 높은 생존성이 얻어졌다(74.0%, P<0.05). 응해 방법에 따른 동결정액의 생존성 실험에서는 37℃의 융해구가 52℃ 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℃에서 10분간 정치시키는 1단계 동결방법이 간편하면서 유리한 동결 방법으로 활용될 수 있음을 보여준다.

This study was conducted to establish a convenient freezing method of boar semen. Boar semen was cooled until 5℃ for 3 hrs using cell freezer and loaded into straws. Semen straws were frozen in different steps in strofoam box filled with LN2. Highest sperm viability (54.0%) was obtained by 1-step freezing(holding at 10 cm height from the surface of LN2 for 10 min). Sperm viability increased by holding at -102℃ for 10min (74.0%, P<0.05). In thawing regime, sperm viability was significantly higher in 37℃ group than in 52℃ group. The sperm characteristics did not differ between 1-step and 3-step. After IVF using frozen-thawed boar semen, developmental rate of embryos to the morula+blastocyst stage was in 1-step freezing group than that of 3-step freezing group (27.5 vs 14.7%, P<0.05). The result shows that the 1-step freezing with holding at -102℃ for 10min before plunging into LN2 is a convenient and easy freezing method for boar semen.

본 연구는 활성화처리 방법 및 배양 조건이 돼지 단위발생란의 체외발달 및 apoptosis에 미치는 영향을 알아보기 위해 실시되었다. 도축장 유래 난소로부터 채취된 미성숙 난자를 42~44시간 동안 성숙배양한 후 사용하였다. Apoptosis는 TUNEL 방법을 사용하여 조사하였다. 실험 1에서는 성숙배양된 난자들을 electric pulse(1.2 kV/cm for 30μsec 2회, E), E + 6-dimethylaminopurine(6-DMAP) 또는 E + cycloheximide(CH) 방법으로 활성화 처리하여 PZM-3를 이용하여 5% CO2, 38.5℃에서 배양하였다. 실험 2에서는 전기자극을 이용하여 활성화처리된 난자들을 각각 PZM-3 또는 NCSU-23 배양액 내에서 배양하였다. 각 배양액 내의 난자들은 각각 20% O2 조건으로 나뉘어 배양하였다. E + 6-DMAP(36.5%) 또는 E + CH 구(32.5%)에서 E 구(27.7%)보다 유의적으로 높은 배반포 형성율을 보였다(P<0.05). 처리별 apoptosis 발생율은 각각 5.3%(E), 7.7%(6-DMAP) 및 7.1%(CH)였다. 실험 2에서는 PZM-3 구의 배반포 형성율이 NCSU-23 구에 비하여 산소분압조건과 관계없이 다소 높았다(28.2{sim}29.7% vs. 22.6~24.4%). PZM-3 및 20% O2 조건하에서 유의적으로 낮은 apoptosis 발생 비율을 나타냈다(9.2%, P<0.05). 그러므로 돼지 단위발생란을 chemical agent를 이용한 추가 활성화처리 후 PZM-3, 20% O2, 조건으로 배양하면 더 나은 배반포 발생율을 얻을 수 있다고 생각된다.

This study investigated apoptosis and in vitro development of parthenogenetic preimplantation porcine embryos. In vitro matured oocytes for 42~44h were used. Apoptotic cell death was analyzed by using a terminal deoxynucleatidyl transferase mediated deoxyuridine 5-triphosphate nick-end tabling (TUNEL) assay. In experiment 1, oocytes were activated with two electric pulses (CH) of 1.2 kV/cm for 30μsec (E), E + 6-dimethylaminopurine (6-DMAP) or E + cycloheximide (CH) and cultured in PZM-3 under 5% CO2 in air at 38.5℃. In experiment 2, oocytes were activated by E and cultured in PZM-3 or NCSU-23 under a gas atmosphere of 20% O2 (5% CO2, in air) or 5% O2 (5% CO2, 5% O2 90% N2) at 38.5℃. Oocytes activated with E+6-DMAP or E+CH showed higher blastocyst rates (36.3% and 32.5%) compared to E alone (27.7%). The frequency of apoptosis according to treatments were 5.3%, 7.7% and 7.1% respectively. Oocytes activated with E alone showed lower (P<0.05) frequency of apoptosis compared to other groups. In experiment 2, parthenotes cultured in PZM-3 showed slightly higher blastocyte rates (28.2% and 29.7%) compared to NCSU-23 (22.6% and 24.4%) regardless of atmosphere. Blastocysts generated in PZM-3 showed lower (P<0.05) apoptosis rate under 20% O2 (9.2% vs 16.9%), whereas those in NCSU-23 had slightly lower apoptosis rate under 5% O2 (14.0% vs 18.4%). This result represents that activation method and culture condition could affect the frequency of apoptosis as well as in vitro developmental rate.

Hypoosmotic swelling test (HOST) is used for evaluating the plasma membrane function and fertilizing ability in mammal spermatozoa. However, HOS solutions and experimental conditions have not been determined clearly for assessing canine spermatozoa. This study was conducted to examine the HOS solutions and assay conditions, including incubation time (30 to 120 min), storage temperature (4, 17 and 20℃), semen status (fresh and frozen). Maximum spermatozoal plasma membrane swelling was obtained in an 150 mOsm Na-citrate/Fructose solutions with an incubation time for 45 min. The storage temperature and semen status affected the percentage of HOS positive spermatozoa. The HOS test adapted to canine spermatozoa in this study was simple and highly consistent assay with good repeatability. The optimal condition of HOST in canine spermatozoa is an 150 mOsm Na-citrate/Fructose solutions with an incubation time for 45 min regardless of semen storage temperature and semen status.