• Title/Summary/Keyword: 미니돼지

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Maxillary sinus floor elevation using autogenous skin-derived mesenchymal stem cells in miniature pigs (미니돼지에서 자가 피부유래 간엽성 줄기세포를 이용한 상악동저 거상술)

  • Byun, June-Ho;Kang, Eun-Ju;Maeng, Geun-Ho;Rho, Gyu-Jin;Kang, Dong-Ho;Lee, Jong-Sil;Park, Bong-Wook
    • Journal of the Korean Association of Oral and Maxillofacial Surgeons
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    • v.36 no.2
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    • pp.87-93
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    • 2010
  • Introduction: In our previous studies, we isolated porcine skin-derived mesenchymal stem cells (pSDMSCs) from the ears of adult miniature pigs and evaluated the pluripotency of these pSDMSCs based on expressions of transcription factors, such as Oct-4, Sox-2, and Nanog. Moreover, the characteristic of mesenchymal stem cells was revealed by the expression of various mesenchymal stem cell markers, including CD29, CD44, CD90, and vimentin. The aim of this study was to evaluate in vivo osteogenesis after maxillary sinus lift procedures with autogenous pSDMSCs and scaffold. Materials and Methods: The autogenous pSDMSCs were isolated from the 4 miniature pigs, and cultured to 3rd passage with same methods of our previous studies. After cell membranes were labeled using a PKH26, $1{\times}10^{7}$ cells/$100{\mu}L$ of autogenous pSDMSCs were grafted into the maxillary sinus with a demineralized bone matrix (DBM) and fibrin glue scaffold. In the contralateral control side, only a scaffold was grafted, without SDMSCs. After two animals each were euthanized at 2 and 4 weeks after grafting, the in vivo osteogenesis was evaluated with histolomorphometric and osteocalcin immunohistochemical studies. Results: In vivo PKH26 expression was detected in all specimens at 2 and 4 weeks after grafting. Trabecular bone formation and osteocalcin expression were more pronounced around the grafted materials in the autogenous pSDMSCs-grafted group compared to the control group. Newly generated bone was observed growing from the periphery to the center of the grafted material. Conclusion: The results of the present study suggest that autogenous skin-derived mesenchymal stem cells grafting with a DBM and fibrin glue scaffold can be a predictable method in the maxillary sinus floor elevation technique for implant surgery.

Analysis of Frozen-Thawed Sperm Characteristic in Alpha 1,3-Galactosyltransferase(GalT) Knock-out Cloned Miniature Pig (Alpha 1,3-Galactosyltransferase (GalT) Knock-out 복제 미니돼지 정액의 동결-융해 후 정액 성상 분석)

  • Woo, Jea-Seok;Lee, Yong-Seung;Yoo, Han-Jun;Hwang, Seong-Soo;Oh, Keon-Bong;Cheong, Hee-Tae;Yang, Boo-Keun;Park, Soo-Bong;Park, Choon-Keun
    • Reproductive and Developmental Biology
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    • v.35 no.3
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    • pp.385-390
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    • 2011
  • The purpose of this study was undertaken to evaluate of cryopreservation efficiency in ${\alpha}$ 1,3-galactosyltransferase knock-out(GalT KO) cloned miniature pig sperm. To compare ability of frozen-thawed sperm characteristics, three different pig strains (GalT KO) cloned miniature pig, PWG miniature pig and Duroc were used. The ejaculated semen from the three pig species was diluted with same volume extender and added to LEY solution for freezing. The diluted semen was placed in 0.5 ml straws, and freezing was initiated by exposing the straws to liquid nitrogen ($LN_2$) vapours for 10 min before placing them into $LN_2$ for cryopreservation. After thawing, the sperm ability were assessed for viability (SYBR-14/PI staining), abnormality (Rose Bengal staining), and acrosome status (intactness, intensity and capacitation) (chlorotetracycline, CTC staining). The viability of frozen-thawed GalT KO pig sperm had no significant difference as compared with Duroc and PWG miniature pig sperm. However, The CTC pattern of frozen-thawed GalT KO cloned miniature pig spermatozoa showed significantly lower rates in F pattern and AR pattern (p<0.05) and significantly higher rates in B pattern than Duroc and PWG miniature pig (p<0.05). The abnormality of GalT KO cloned miniature pig sperm was significantly lower as compared to Duroc and PWG miniature pig sperm (p<0.05). In conclusion, GalT KO cloned miniature pig semen can be cryopreserved successfully and used for artificial insemination reasonably.

Establishment of Freezing Conditions for Improving Cryosurvival in Miniature Pig Spermatozoa (미니돼지 정액의 동결-융해 후 생존율 향상을 위한 동결 조건 확립)

  • Lee, Yong-Seung;Yoo, Han-Jun;Cheong, Hee-Tae;Yang, Boo-Keun;Woo, Jea-Seok;Park, Choon-Keun
    • Reproductive and Developmental Biology
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    • v.35 no.3
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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.

Effect of Bacteria Eliminated Sperm by Percoll Method on Sperm Quality and Embryo Cleavage in Miniature Pig (Percoll에 의한 미니돼지 정액내 세균 제거가 정자 성상과 수정란 분할에 미치는 영향)

  • Yoo, Han-Jun;Jeon, Jun-Myeong;Lee, Yong-Seung;Cheong, Hee-Tae;Yang, Boo-Keun;Kim, Dae-Young;Park, Choon-Keun
    • Reproductive and Developmental Biology
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    • v.33 no.1
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    • pp.35-40
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    • 2009
  • The objectives of this study was to evaluate the efficiency of the bacteria eliminated sperm by percoll gradient method on sperm quality and embryo cleavage in vitro in pig. The semen of miniature pig collected by gloved-hand method pre-warmed ($37^{\circ}C$) in thermos bottle, and separated by 65% percoll. Analysis of sperm ability was estimated by examining viability, capacitation and acrosome reaction using chlortetracycline (CTC) and the abnormality. Also, fertility of sperm was monitored with cleavage rate of embryo after IVF using separated and un-separated sperm by percoll. The result, viability of separated sperm was significantly(p<0.05) higher($83.6{\pm}$2.0 vs $59.0{\pm}4.4%$) than un-separated sperm. The results of CTC analysis showed the percentage of F- and B-patterned separated sperm was higher in separated that than un-separated sperm. On the contrary, the percentage of AR-patterned form unseparaed sperm was significantly(p<0.05) higher($13.6{\pm}0.8$ vs $8.1{\pm}0.6%$) than separated sperm. Also, abnormality of un-separated sperm was significantly(p<0.05) higher($2.2{\pm}0.4$ vs $16.8{\pm}2.8%$) than separated sperm. However, the cleavage rates of embryo using separated sperm by percoll and un-separated sperm had not significantly difference on 2 cell stage(9.25 vs 11.88%), 4 cell stage(26.76 vs 24.51%) and >4 cell stage(63.99 vs 63.61%) at 48h of IVF. Therefore, the sperm separated by percoll method showed improvement in sperm quality than un-separated sperm in miniature pig.

Initial Diagnosis of Acute Renal Failure Induced by Ischemia in Miniature Pig (미니돼지에서 허혈성 신장 손상의 조기진단)

  • Kim, Se-Eun;Ko, A-Ra;Bae, Chun-Sik;Park, Soo-Hyun;Han, Ho-Jae;Shim, Kyung-Mi;Kang, Seong-Soo
    • Journal of Veterinary Clinics
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    • v.28 no.1
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    • pp.52-56
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    • 2011
  • Acute renal injury induced by ischemia is a major cause of high morbidity and mortality in hospitalized patients and a common complication in hospitalized patients. Thus, the work with acute renal failure and renal ischemia has been studied for many years. Although serum creatinine concentration that is widely used as an index of renal function performs fairly well for estimating kidney function in patients with stable chronic kidney disease, it performs poorly in the setting of acute disease. Thus, an ideal biomarker for acute kidney injury would help clinicians and scientists diagnose the most common form of acute kidney injury in hospitalized patients, acute tubular necrosis, early and accurately, and may aid to risk-stratify patients with acute kidney injury by predicting the need for renal replacement therapy, the duration of acute kidney injury, the length of stay and mortality. In this study, renal ischemia and reperfusion were performed by clapming and un-clamping right renal artery in miniature pigs. Plasma blood urea nitrogen (BUN) and creatinine were examined at pre- clamping, after-clamping at 0, 1 and 3 hours. And we searched initial indicators in these samples. Also, renal tissue was collected and searched the initial indicator by PCR and western blotting. As a result, hypoxia inducible factor $1{\alpha}$ ($HIF1{\alpha}$), nuclear factor kappa-B ($NF{\kappa}B$), $I{\kappa}B$, erythropoietin (EPO), erythropoietin receptor (EPOR), angiopoietin-1 and vascular endothelial growth factor (VEGF) were showed significant changes among the renal protein. $HIF1{\alpha}$, EPO, and EPOR were showed significant changes among the renal gene. Thus, these markers will be used as initial diagnosis of acute renal failure.

Physiological Evaluation of Cloned Mini-pigs in a Transportable Isolator for the Study of Xenotransplantation (바이오장기 연구를 위한 이동식 아이솔레이터 내 복제 미니 돼지의 생리 활성 평가)

  • Kim, Hae-Sung;Jeon, Yu-Byeol;Kwak, Seong-Sung;Jeong, Seung-A;Jung, Eui-Man;Hyun, Sang-Hwan;Jeung, Eui-Bae
    • Journal of Embryo Transfer
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    • v.26 no.3
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    • pp.165-169
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    • 2011
  • The present study investigated the physiological evaluation of cloned mini-pigs in a transportable isolator. Transportable isolator was designed and manufactured by our research team for transporting gnotobiotic pig. Until now, no previous reports are available regarding the physiological activities and harmful effects when pigs were transported in this isolator. Five cloned mini-pigs of 1~2 year (s) old female with a body weight between 80~90 kg were used. The effects of transportable isolator on stress-related hormone, adrenocorticotrophic hormone (ACTH) and cortisol levels, and heart rate were evaluated. In addition, it was also examined the effects of transportable isolator on blood chemistry factors (alanine aminotransferase: ALT, aspartate aminotransferase: AST, blood urea nitrogen: BUN, glucose, and creatinine). Blood was sampled just before the beginning of transport (T0), at the end of transport (30min after the transport; T1), and 30 min after the end of transport (T2). At the same time, heart rate was also evaluated. As a result, heart rate had no significant (p>0.05) differences at the various-time points of study (T0, T1, T2). However, heart rate was slightly higher than normal range in T1 and T2. The ACTH level was higher than normal range. Whereas, the cortisol level was lower than normal range. There were no statistical significant differences both ACTH and cortisol level between different time groups. Also, there were no significant differences in blood chemistry factors. Therefore, our present study shows that transportable isolator has no harmful effect on stress and physiological condition in cloned mini-pigs.

Effect of Cholesterol and Serum Albumin on Sperm Ability and Lipid Peroxidation during the Storage of Miniature Pig Sperm (미니돼지정액의 보존 시 콜레스테롤과 혈청 알부민이 정자 성상과 지질 과산화에 미치는 영향)

  • Kim, Dong-Woo;Lee, Yong-Seung;Yoo, Han-Jun;Cheong, Hee-Tae;Yang, Boo-Keun;Park, Choon-Keun
    • Journal of Embryo Transfer
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    • v.26 no.1
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    • pp.71-78
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    • 2011
  • This study was undertaken to find out the effect of cholesterol and serum albumin on sperm ability and lipid peroxidation levels period to the liquid storage of miniature pig sperm. Ejaculated semen from miniature pigs was collected by gloved-hand method into a pre-warmed ($37^{\circ}C$) thermos bottle, and extended with Modena solution {with and without BSA, methyl-beta-cyclodextrin (-cholesterol) and cholesterol loaded cyclodextrin (+cholesterol)}. Each semen was assessed for viability (SYBR-14/PI staining) and acrosome intactness, intensity and capacitation status by chlorotetracycline (CTC) staining at 1, 3, 5, 7 and 10 days of storage. At for the effects of cholesterol and serum albumin on lipid peroxidation, semen were incubated with $H_2O_2$ ($10\;{\mu}M$), and lipid peroxidation level were measured by flow cytometry using the lipid peroxidation reporter probe $C_{11}-BODIPY^{581/591}$. The result, lipid peroxidation level in sperm added with cholesterol were lower in $10\;{\mu}M$ $H_2O_2$ compared to the added sperm with serum albumin. Also, added cholesterol to sperm had significant (p<0.05) higher viability when storage for 7 and 10 days and lower when 10 days of storage percentage of acrosome-reacted sperm (AR pattern) in acrosome state as say result compared to other treated groups. In conclusion, role of cholesterol during lipid storage in miniature pig spermatozoa was protected boar spermatozoa from lipid peroxidation prior to lipid storage. Addition serum albumin during lipid storage in sperm may be induce sperm membrane damage by lipid peroxidation. Therefore, addition of cholesterol to miniature pig sperm will be lead to extension of liquid storage periods.

Production of Cloned Miniature Pig by Surrogate Mother Conditions (대리모의 준비 조건 변화를 통한 복제미니돼지의 생산)

  • Hur, Chang-Gi;Yang, Hae-Young;Lee, Eun-Kyeong;Han, Joo-Hee;Park, Chun-Gyu;Shin, Teak-Soon;Lee, Hong-Gu;Kang, Han-Seok;Ahn, Jong-Deok;Cho, Seong-Keun
    • Journal of Embryo Transfer
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    • v.27 no.1
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    • pp.1-7
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    • 2012
  • Somatic cell nuclear transfer (SCNT) for miniature pig has been developed for xenotransplantation and many other biomedical experiments. However, the efficiency of SCNT is still very low due to many factors. To optimize the surrogate mother condition for improvement of cloned miniature pigs efficiency, we investigated the effect of the status of surrogate mother on pregnancy, farrowed rate in SCNT pigs. After SCNT with mesenchymal stem cells as donor cells, the SCNT embryos were surgically transferred into the oviduct of surrogated pigs. To compare the effects of status of surrogate pigs on pregnancy, surrogate pigs were prepared by artificial abortion at day 20~29 (Group 1), 30~39 (Group 2), and 40~45 (Group 3) of gestation. After SCNT embryos transfer in three different status of surrogate pigs, Group 2 (56.3%) and 3 (55.6%) had significantly ($p$ <0.05) higher the pregnancy rate than group 1 (0%) at day 30 of gestation. The status of ovulation in surrogate pig also was investigated. Post-ovulation status (54.8%) had higher proportion than pre-ovulation status (38.7%) and ovulation status (6.5%). We obtained 19 cloned miniature piglets from seven surrogate gilts and five piglets are living healthy but fourteen piglets died soon after birth or stillbirth. The weights of piglets greatly differ from 254 to 1,296 g. Microsatellite analysis showed that cloned piglets were genetically different from the surrogate mother and cloned piglets were genetically equal to the donor cell. In conclusion, the present result indicates that artificially abortion method can improve the efficiency of pregnancy after SCNT in pigs. This study will provide available method for the further study and application in the field of xenotransplantation.

Changes of Frozen-Thawed Semen Characteristics in Miniature Pig and Duroc (Miniature Pig와 Duroc 종간의 동결-융해 후 정액 성상 비교)

  • Lee, Y.S.;Choi, W.C.;Lee, S.H.;Cheong, H.T.;Lee, S.Y.;Yang, B.K.;Park, C.K.
    • Journal of Embryo Transfer
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    • v.21 no.3
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    • pp.263-271
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    • 2006
  • The purpose of this study was undertaken to compare ability of frozen-thawed sperm characteristics between two strains (miniature pig and Duroc). The semen was collected by gloved-hand method into a pre-warmed ($37^{\circ}C$) thermos bottle. The semen was diluted with same volume extender and added to LEY solution for freezing. The diluted semen was placed in 0.5 ml straws, and freezing was initiated by exposing the straws to liquid nitrogen ($LN_2$) vapours for 10 min before placing them into $LN_2$ for cryopreservation. The frozen-semen straw were thawed at 20, 37 and $50^{\circ}C$ for 1 min, 45 sec and 10 sec within water-bath. The semen sample were evaluated at 0, 3, 6, 9, and 12 h after incubation at $37^{\circ}C$ for analysis of sperm ability. Abnormality of spermatozoa in miniature pig was significantly (p<0.05) higher than that in Duroc at 0, 9 and 12 h of post-thawing incubation after frozen-thawing. The percentage of F-patterned spermatozoa in miniature pig was significantly (p<0.05) lower, while the percentage of AR (acrosome reacted spermatozoa) pattern was higher in the miniature than in the Duroc. On the other hand, there was no significant difference in the viability of spermatozoa thawed at different temperature ($20^{\circ}C\;and\;37^{\circ}C$) between two species, but the viability in miniature pig was higher (p<0.05) than in Duroc when sperm was thawed at $50^{\circ}C$. In conclusion, this study suggest that suitable freezing method for miniature pig semen is required for increasing post-thawing viability and fertilization capacity.