Selection of pre- versus postpubertal pig oocytes for parthenogenetic activation and somatic cell nuclear transfer

Pig oocytes have been used increasingly for in vitro production techniques in recent years. The slaughterhouse-derived oocytes that are often used are mostly of prepubertal origin. The aims of the present study were to compare the developmental competence between pre- and postpubertal pig oocytes, and to develop a simple and practical method for the selection of prepubertal pig oocytes for parthenogenetic activation (PA) and somatic cell nuclear transfer (SCNT) based on oocyte morphology after IVM and oocyte inside zona pellucida (ZP) diameter (‘small’ ≤110 µm; ‘medium’ >110 µm; ‘large’ ≥120 µm). Meiotic competence and blastocyst rates after PA and SCNT of prepubertal oocytes increased with oocyte size, with the large prepubertal oocytes reaching a level similar to postpubertal oocytes after SCNT. Blastocyst cell number was not related to oocyte inside ZP diameter and oocyte donor to the same extent as blastocyst rate. Very low blastocyst rates were obtained after PA of morphologically bad pre- and postpubertal oocytes. In conclusion, measurement of inside ZP diameter combined with morphological selection is useful to remove incompetent oocytes. Further studies are needed to clarify the relative importance of cytoplasmic volume and stage in oocyte growth phase.

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185 DEVELOPMENT CAPACITY OF PRE- AND POSTPUBERTAL PIG OOCYTES EVALUATED BY SOMATIC CELL NUCLEAR TRANSFER AND PARTHENOGENETIC ACTIVATION

Reproduction Fertility and Development ◽

10.1071/rdv25n1ab185 ◽

2013 ◽

Vol 25 (1) ◽

pp. 241 ◽

Cited By ~ 1

Author(s):

H. S. Pedersen ◽

R. Li ◽

Y. Liu ◽

P. Løvendahl ◽

P. Holm ◽

...

Keyword(s):

Somatic Cell ◽

Somatic Cell Nuclear Transfer ◽

Nuclear Transfer ◽

Cell Number ◽

Time Interval ◽

Cell Stage ◽

Parthenogenetic Activation ◽

Development Capacity ◽

Developmental Capacity

Most of the porcine oocytes used for in vitro studies are collected from gilts. Our aims were to study development capacity of gilt v. sow oocytes (pre- and postpubertal respectively) using 2 techniques illustrating development competence [parthenogenetic activation (PA) and somatic cell nuclear transfer (SCNT)], and to describe a simple method to select the most competent oocytes. Inside-ZP diameter of in vitro-matured gilt oocytes was measured (µm; small ≤110; medium >110; large ≥120). Gilt and sow oocytes were morphologically grouped as good (even cytoplasm, smooth cell membrane, visible perivitelline space) or bad before used for PA (good and bad) or SCNT (good). The PA and SCNT were performed as before with minor modifications (Cryobiol. 64, 60; Cell. Reprogr. 13, 521) before culture for 6 days in a standard or timelapse incubator. Rates of cleavage (CL%, Day 2), blastocyst (BL%, Day 6), and blastocyst cell number (Hoechst 33342) were recorded. For PA embryos in a timelapse incubator (26 oocytes/group; 2 replicates), the first appearance of 2-cell stage was recorded. Between groups, CL% and BL% were analysed by chi-square and cell number by t-test. Results are presented in the table for the development of good oocytes after PA. The results show a low CL% of small-gilts compared with the other groups. The BL% increased with gilt-oocyte-diameter; however, sow oocytes reached the highest BL%. Total cell number was higher in sow than in gilt blastocysts. The SCNT experiments showed no differences in CL% (90–96) and blastocyst cell number (51–59) between groups. The BL% was higher in medium gilts and sows (41; 45) compared with large gilts (21). The BL% of bad oocytes was 1% from all 4 groups (176 oocytes, 25 replicates). Time interval for appearance of 2-cell stage for embryos developing into blastocysts showed no differences between groups (19–20 h). Within groups, this time interval showed a larger standard deviation for embryos not developing v. embryos developing into blastocysts. It is concluded that (a) sow oocytes have higher developmental capacity compared to gilts, (b) small gilt oocytes are not developmentally competent, (c) measurement of inside-ZP diameter, combined with morphological selection, is useful to remove non-competent oocytes. Further studies are needed to dissect the developmental capacity of medium and large gilt oocytes. Also, further timelapse studies may reveal a time interval in which the first cleavage of embryos with high developmental capacity takes place. Table 1.Rates of cleavage (CL%), blastocyst (BL%), and total no. of cells (mean ± SEM) in blastocysts of PA embryos from gilts and sows1

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Effect of glycosaminoglycans on the preimplantation development of embryos derived from in vitro fertilization and somatic cell nuclear transfer

Reproduction Fertility and Development ◽

10.1071/rd02054 ◽

2003 ◽

Vol 15 (3) ◽

pp. 179 ◽

Cited By ~ 25

Author(s):

Goo Jang ◽

Byeong Chun Lee ◽

Sung Keun Kang ◽

Woo Suk Hwang

Keyword(s):

Hyaluronic Acid ◽

In Vitro Fertilization ◽

Chondroitin Sulfate ◽

Somatic Cell ◽

Somatic Cell Nuclear Transfer ◽

Nuclear Transfer ◽

Cell Number ◽

Developmental Competence ◽

Vitro Fertilization

The purpose of this study was to evaluate the effect of glycosaminoglycans (GAGs) added to the culture medium on the developmental competence of bovine embryos derived from in vitro fertilization (IVF) and from somatic cell nuclear transfer (SCNT). In vitro-matured oocytes were either inseminated with 1 × 106 spermatozoa mL−1 or enucleated and reconstructed with bovine adult ear fibroblasts by SCNT. The embryos were then cultured in modified synthetic oviduct fluid (mSOF) containing 8 mg mL−1 bovine serum albumin (BSA) (control mSOF) or control mSOF supplemented with various GAGs (hyaluronic acid, heparin or chondroitin sulfate) in a dose-dependent manner (0.1, 0.5 or 1.0 mg mL−1). Developmental competence was evaluated by monitoring the numbers of 2-cell embryos, 8–16-cell embryos and blastocysts. The mean cell number of flattened blastocysts stained with 5 μ M bisbenzimide on Day 8 was counted. The percentage of blastocyst formation (IVF and SCNT embryos) from cleaved embryos was significantly higher (P < 0.05) in control mSOF supplemented with 0.5 mg mL−1 hyaluronic acid (45% and 47%), heparin (40% and 47%) or chondroitin sulfate (38% and 44%) compared with control mSOF (30–31% and 30–33%). When compared with the efficacy of 0.5 mg mL−1 GAGs, no significant differences were observed in the developmental competence of both IVF and SCNT embryos. Supplementing control mSOF with 0.5 mg mL−1 GAGs had no effect on the cell number of IVF embryos. In contrast, supplementing 0.5 mg mL−1 of hyaluronic acid, heparin or chondroitin sulfate to control mSOF significantly (P < 0.05) increased the numbers of total cells (93–98 v. 88 cells) and trophectoderm (TE) cells (64–66 v. 55 cells), and decreased the inner cell mass (ICM) to TE cell ratio (48.2–49.8 v. 61.3) in SCNT blastocysts compared with embryos in control mSOF. In conclusion, supplementation of culture media with GAGs may improve the development of bovine IVM–IVF and SCNT embryos to the blastocyst stage. The GAGs increased the quality of blastocysts by increasing total cell numbers in the SCNT embryos.

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79 DEVELOPMENTAL COMPETENCE OF OVINE OOCYTES VITRIFIED AT GERMINAL VESICLE STAGE: IN VITRO FERTILIZATION, PARTHENOGENETIC ACTIVATION, AND SOMATIC CELL NUCLEAR TRANSFER

Reproduction Fertility and Development ◽

10.1071/rdv23n1ab79 ◽

2011 ◽

Vol 23 (1) ◽

pp. 145

Author(s):

A. R. Moawad ◽

I. Choi ◽

J. Zhu ◽

K. H. S. Campbell

Keyword(s):

Somatic Cell ◽

Somatic Cell Nuclear Transfer ◽

Nuclear Transfer ◽

Germinal Vesicle ◽

Developmental Competence ◽

Control Groups ◽

High Frequencies ◽

Parthenogenetic Activation ◽

And Control

Oocyte cryopreservation represents an important development in the field of assisted reproductive technologies. This study investigated the effects of vitrification on spindle morphology following subsequent in vitro maturation (IVM), cleavage, and development following IVF and parthenogenetic activation. The developmental competence of ovine oocytes vitrified at the germinal vesicle (GV) stage, matured, and used as cytoplast recipients for somatic cell nuclear transfer (SCNT) was also determined. Cumulus–oocyte complexes obtained at slaughter were divided into 3 groups: 1) untreated (control), 2) toxicity (exposed to vitrification solutions without freezing), and 3) vitrified (2008 Reprod. Fertil. Dev. 20, 122). At 24 hpm (hours post onset of maturation), oocytes were subjected to 1) immunostaining, 2) IVF, or 3) activation by 2 different protocols [calcium ionophore, cycloheximide, and cytochalasin B (CA+CHX/CB), or strontium and CB (Sr/CB)]. The SCNT was performed as previously described (2010 Reprod. Fertil. Dev. 22, 1000–1014). Presumptive zygotes were cultured in vitro for 7 days. No significant differences (P > 0.05; chi-square) were observed in the frequencies of oocytes with normal spindle configuration between vitrified, toxicity, and control groups (50.0, 54.9, and 70.4%, respectively). Cleavage 24, 48 hpi, and morula development (5 days pi) were significantly decreased (P < 0.01) in the vitrified group (17.3, 42.9, and 36.4%) compared with toxicity (47.0, 85.3, and 60.7%) and control (68.9, 89.7, and 62.6%) groups. Blastocyst development significantly decreased (P < 0.01) in the vitrified group (12.3%) compared with toxicity (42.7%) and control (40.4%) groups. Based on cleaved embryos, no significant difference was observed between vitrified and control groups (29.4 v. 45.1%). Post-activation, cleavage 24 hpa (hours post-activation, 6.2 v. 3.8%) and 48 hpa (28.4 v. 27.5%) was significantly lower (P < 0.05) in vitrified oocytes activated by (CA+CHX/CB and Sr/CB) than other groups. No blastocyst developed from vitrified oocytes activated by CA+CHX/CB; however, 3.8% developed from Sr/CB oocytes. This was significantly (P < 0.05) lower than toxicity and control (20.0 and 27.3%) groups. Following SCNT, high frequencies of enucleation (99%) and fusion (98%) were achieved in vitrified and control groups. Cleavage 24 and 48 hpa significantly decreased (P < 0.05) in the vitrified group (31.0 and 48.0%) compared with the control (55.1 and 85.0%). No significant differences were observed in morula (38.0 v. 46.7%) and blastocyst (13.0 v. 23.4%) development. The proportion of cleaved embryos that developed to blastocyst stages was similar in both groups (27.0%). No significant differences (t-test) were observed in total cell numbers, apoptotic nuclei, and proportion of diploid embryos. In conclusion, ovine oocytes vitrified at GV stage can be matured, fertilized, and develop in vitro with high developmental potential. Strontium can be used effectively for activation of vitrified/thawed ovine oocytes. Vitrified/thawed ovine oocytes were used successfully for the first time as recipient cytoplasts for SCNT and produced high frequencies of good-quality blastocyst stage embryos.

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Parthenogenetic activation and somatic cell nuclear transfer of porcine oocytes activated by an electric pulse and AZD5438 treatment

Zygote ◽

10.1017/s0967199417000272 ◽

2017 ◽

Vol 25 (4) ◽

pp. 453-461 ◽

Cited By ~ 5

Author(s):

Xiao-Chen Li ◽

Qing Guo ◽

Hai-Ying Zhu ◽

Long Jin ◽

Yu-Chen Zhang ◽

...

Keyword(s):

Somatic Cell ◽

Somatic Cell Nuclear Transfer ◽

Nuclear Transfer ◽

Electric Pulse ◽

Formation Rate ◽

Developmental Competence ◽

Oocyte Activation ◽

Blastocyst Formation ◽

Parthenogenetic Activation

SummaryWe examined the in vitro developmental competence of parthenogenetic activation (PA) oocytes activated by an electric pulse (EP) and treated with various concentrations of AZD5438 for 4 h. Treatment with 10 µM AZD5438 for 4 h significantly improved the blastocyst formation rate of PA oocytes in comparison with 0, 20, or 50 µM AZD5438 treatment (46.4% vs. 34.5%, 32.3%, and 24.0%, respectively; P < 0.05). The blastocyst formation rate was higher in the group treated with AZD5438 for 4 h than in the groups treated with AZD5438 for 2 or 6 h (42.8% vs. 38.6% and 37.2%, respectively; P > 0.05). Furthermore, 66.67% of blastocysts derived from these AZD5438-treated PA oocytes had a diploid karyotype. The blastocyst formation rate of PA and somatic cell nuclear transfer (SCNT) embryos was similar between oocytes activated by an EP and treated with 2 mM 6-dimethylaminopurine for 4 h and those activated by an EP and treated with 10 µM AZD5438 for 4 h (11.11% vs. 13.40%, P > 0.05). In addition, the level of maturation-promoting factor (MPF) was significantly decreased in oocytes activated by an EP and treated with 10 µM AZD5438 for 4 h. Finally, the mRNA expression levels of apoptosis-related genes (Bax and Bcl-2) and pluripotency-related genes (Oct4, Nanog, and Sox2) were checked by RT-PCR; however, there were no differences between the AZD5438-treated and non-treated control groups. Our results demonstrate that porcine oocyte activation via an EP in combination with AZD5438 treatment can lead to a high blastocyst formation rate in PA and SCNT experiments.

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46 EFFECT OF SHORT-TERM TRICHOSTATIN A TREATMENT ON BOVINE SOMATIC CELL NUCLEAR TRANSFER EMBRYOS

Reproduction Fertility and Development ◽

10.1071/rdv21n1ab46 ◽

2009 ◽

Vol 21 (1) ◽

pp. 123

Author(s):

H. J. Oh ◽

J. E. Park ◽

M. J. Kim ◽

S. G. Hong ◽

J. T. Kang ◽

...

Keyword(s):

Somatic Cell ◽

Somatic Cell Nuclear Transfer ◽

Nuclear Transfer ◽

Trichostatin A ◽

Cell Number ◽

Total Cell ◽

Developmental Competence ◽

Short Term ◽

Total Cell Number

Epigenetic reprogramming such as acetylation in somatic cell nuclear transfer (SCNT) has been known as one of problems in cloned embryos. For resolving this acetylation reprogramming, many investigators recently have reported the effect of long-term culture of post-activated SCNT embryos using trichostatin A (TSA), a histone deacetylase inhibitor (HDACi). The objective of this study is to investigate the effect of short-term TSA treatment on in vitro developmental ability and the quality of bovine SCNT embryos. Immature oocytes were aspirated from abattoir-derived ovaries, matured in vitro for 22 h, and enucleated. A bovine fetal fibroblast was placed into the enucleated oocyte and fused by electrical stimulation. The fused couplets were activated by 4-min incubation in 10 μm ionomycin, followed by 4 h of culture in 1.9 mm 6-dimethylaminopurine with or without TSA (0, 50, or 100 nm). The SCNT embryos were subsequently cultured in modified synthetic oviduct fluid medium for 8 days. Developmental competence was assessed by blastocyst formation and total cell number. Total cell numbers were determined by staining with bisbenzimide 33342. As results, developmental competence to blastocysts was higher in 100 nm than control (36.7 v. 27.9%, P < 0.05). In blastocyst hatching rate, TSA 100 nm group (19.5%) at 8 days showed an increased pattern as opposed to control and TSA 50 nm group (11.1 and 12.7%; P < 0.05). No significant differences in two cell and morula stage were observed among treatment groups. In terms of development to hatching stage of blastocysts, TSA 100 nm group (19.5%) at 8 days has a significant effect compared to control and TSA 50 nm group (11.1 and 12.7%; P < 0.05). Total cell number of blastocysts derived from TSA 100 nm was significantly higher (P < 0.05) than that in TSA 50 nm (116 v. 100), whereas there was not significant difference between control and TSA 100 nm. In conclusion, short-term culture with high concentration of TSA improved the blastocysts formation however total cell number of blastocysts showed contradictory result. The epigenetic modification by TSA treatment on bovine SCNT needs further investigation. This study was financially supported by KOSEF (grant # M10625030005-08N250300510) and the Korean MEST, through the BK21 program for Veterinary Science.

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Cotreatment with RepSox and LBH589 improves the in vitro developmental competence of porcine somatic cell nuclear transfer embryos

Reproduction Fertility and Development ◽

10.1071/rd17543 ◽

2018 ◽

Vol 30 (10) ◽

pp. 1342 ◽

Cited By ~ 4

Author(s):

Zhao-Bo Luo ◽

Long Jin ◽

Qing Guo ◽

Jun-Xia Wang ◽

Xiao-Xu Xing ◽

...

Keyword(s):

Somatic Cell ◽

Somatic Cell Nuclear Transfer ◽

Nuclear Transfer ◽

Formation Rate ◽

Epigenetic Reprogramming ◽

Developmental Competence ◽

Abnormal Development ◽

Control Group ◽

Blastocyst Formation

Accumulating evidence suggests that aberrant epigenetic reprogramming and low pluripotency of donor nuclei lead to abnormal development of cloned embryos and underlie the inefficiency of mammalian somatic cell nuclear transfer (SCNT). The present study demonstrates that treatment with the small molecule RepSox alone upregulates the expression of pluripotency-related genes in porcine SCNT embryos. Treatment with the histone deacetylase inhibitor LBH589 significantly increased the blastocyst formation rate, whereas treatment with RepSox did not. Cotreatment with 12.5 μM RepSox and 50 nM LBH589 (RepSox + LBH589) for 24 h significantly increased the blastocyst formation rate compared with that of untreated embryos (26.9% vs 8.5% respectively; P < 0.05). Furthermore, the expression of pluripotency-related genes octamer-binding transcription factor 4 (NANOG) and SRY (sex determining region Y)-box 2 (SOX2) were found to significantly increased in the RepSox + LBH589 compared with control group at both the 4-cell and blastocyst stages. In particular, the expression of NANOG was 135-fold higher at the blastocyst stage in the RepSox + LBH589 group. Moreover, RepSox + LBH589 improved epigenetic reprogramming. In summary, RepSox + LBH589 increases the expression of developmentally important genes, optimises epigenetic reprogramming and improves the in vitro development of porcine SCNT embryos.

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Overexpression of MicroRNA-29b Decreases Expression of DNA Methyltransferases and Improves Quality of the Blastocysts Derived from Somatic Cell Nuclear Transfer in Cattle

Microscopy and Microanalysis ◽

10.1017/s1431927618000016 ◽

2018 ◽

Vol 24 (1) ◽

pp. 29-37 ◽

Cited By ~ 4

Author(s):

Shuang Liang ◽

Zheng-Wen Nie ◽

Jing Guo ◽

Ying-Jie Niu ◽

Kyung-Tae Shin ◽

...

Keyword(s):

Somatic Cell ◽

Somatic Cell Nuclear Transfer ◽

Nuclear Transfer ◽

Cellular Proliferation ◽

Dna Methyltransferases ◽

Blastocyst Stage ◽

Developmental Competence ◽

Somatic Cell Reprogramming

AbstractMicroRNA (miR)-29b plays a crucial role during somatic cell reprogramming. The aim of the current study was to explore the effects of miR-29b on the developmental competence of bovine somatic cell nuclear transfer (SCNT) embryos, as well as the underlying mechanisms of action. The expression level of miR-29b was lower in bovine SCNT embryos at the pronuclear, 8-cell, and blastocyst stages compared within vitrofertilized embryos. In addition, miR-29b regulates the expression of DNA methyltransferases (Dnmt3a/3bandDnmt1) in bovine SCNT embryos. We further investigated SCNT embryo developmental competence and found that miR-29b overexpression during bovine SCNT embryonic development does not improve developmental potency and downregulation inhibits developmental potency. Nevertheless, the quality of bovine SCNT embryos at the blastocyst stage improved significantly. The expression of pluripotency factors and cellular proliferation were significantly higher in blastocysts from the miR-29b overexpression group than the control and downregulation groups. In addition, outgrowth potential in blastocysts after miR-29b overexpression was also significantly greater in the miR-29b overexpression group than in the control and downregulation groups. Taken together, these results demonstrated that miR-29b plays an important role in bovine SCNT embryo development.

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41 STUDY ON THE ESTABLISHMENT OF SOMATIC CELL NUCLEAR TRANSFER USING IN VIVO-MATURED OOCYTES IN DOGS

Reproduction Fertility and Development ◽

10.1071/rdv18n2ab41 ◽

2006 ◽

Vol 18 (2) ◽

pp. 129 ◽

Cited By ~ 2

Author(s):

G. Jang ◽

M. Kim ◽

H. J. Oh ◽

F. Y. Heru ◽

M. S. Hossein ◽

...

Keyword(s):

Electrical Stimulation ◽

Somatic Cell Nuclear Transfer ◽

Nuclear Transfer ◽

Chemical Activation ◽

Developmental Competence ◽

Perivitelline Space ◽

Parthenogenetic Activation ◽

Vaginal Cytology

The present study was performed to collect in vivo matured canine oocytes for somatic cell nuclear transfer (SCNT) and to investigate the developmental competence of canine parthenogenetic and SCNT embryos as the preliminary research for producing cloned dog. The day of ovulation as described by Hase et al. (2000 J. Vet. Med. Sci. 62, 243-248) was determined by serum progesterone levels and at that time vaginal cytology was performed to assess the cornified index. In vivo-matured oocytes were recovered by retrograde flushing of the oviducts at around 48 h (n = 20) or 72 h (n = 25) after the estimated time of ovulation. Overall size of each oocyte, as well as ooplasmic diameter, zona pellucida thickness, and perivitelline space width, was determined after removing the cumulus cells by pipetting (Exp. 1). To determine activation protocols, two treatments, (1) chemical activation (10 �M Ca ionophore for 4 min, followed by incubation for 4 h with 1.9 mM 6-dimethylaminopurine) and (2) electrical stimulation (3.1?3.4 kV/cm in 0.25M mannitol solution), were evaluated to induce parthenogenetic activation of oocytes (Exp. 2). Donor cells were obtained from the primary cell culture of a canine ear skin biopsy, and SCNT was performed according to our laboratory procedures (Jang et al. 2004 Theriogenology 62, 512-521). Three voltages (1.7?2.0 kV/cm, 2.1-2.4 kV/cm, and 3.1-3.4 kV/cm) were tested for fusion. The fused couplets were subjected to chemical or electrical stimulation as in parthenogenetic activation and in vitro developmental competence was monitored (Exp. 3). As a result, more in vivo-matured canine oocytes were obtained at 72 h (92%) than at 48 h (15%) after ovulation; the 72-h occytes had progesterone concentrations of 4-8 ng/mL and a cornified index (vaginal cytology) of 83.34. The average number of oocytes recovered was 12 and sizes of ooplasmic diameter, cytoplasm, zona pellucida, and perivitelline space in in vivo canine-matured oocytes (n = 120) were 178.8 � 9.3 �m, 125.0 � 8.2 �m, 21.7 � 3.7 �m, and 12.7 � 3.5 �m, respectively. Parthenogenetically activated oocytes developed to the 16-cell and morula stages, but failed to develop to the blastocyst stage. Among the three voltages, in the highest voltage (75.2%) the number of fused couplets was increased compared to either of the other voltages (33.3% and 44.0%). Cleavage rates (60.9% vs. 58.0%) of cloned embryos were not significantly affected by method of activation. In terms of in vitro developmental competence, cloned embryos developed to the 16-cell or morula stage in vitro after electrical or chemical activation, respectively. In conclusion, in the present study we demonstrated that measurement of progesterone levels, in combination with evaluation of vaginal cytology, can be used to determine the estimated time of ovulation in bitches. In addition, we determined fusion/activation protocols that resulted in in vitro development of a portion of parthenogenetically activated and cloned embryos to the 16-cell and morula stages. This study was supported by grants from the Biogreen 21-1000520030100000.

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59 HIGHER BLASTOCYST FORMATION OF SOMATIC CELL NUCLEAR TRANSFER EMBRYOS DOES NOT GUARANTEE BETTER PREGNANCY IN PIG

Reproduction Fertility and Development ◽

10.1071/rdv19n1ab59 ◽

2007 ◽

Vol 19 (1) ◽

pp. 147

Author(s):

E. Lee ◽

K. Song ◽

Y. Jeong ◽

S. Hyun

Keyword(s):

Somatic Cell ◽

Somatic Cell Nuclear Transfer ◽

Nuclear Transfer ◽

Cell Number ◽

Skin Fibroblasts ◽

Miniature Pig ◽

Donor Cells ◽

Fetal Fibroblasts

Generally, blastocyst (BL) formation and embryo cell number are used as main parameters to evaluate the viability and quality of in vitro-produced somatic cell nuclear transfer (SCNT) embryos. We investigated whether in vitro development of SCNT pig embryos correlates with in vivo viability after transfer to surrogates. For SCNT, cumulus–oocyte complexes (COCs) were matured in TCM-199 supplemented with follicular fluid, hormones, EGF, cysteine, and insulin for the first 22 h and in a hormone-free medium for 18 h. Three sources of pig skin cells were used as nuclear donor: (1) skin fibroblasts of a cloned piglet that were produced by SCNT of fetal fibroblasts from a Landrace × Yorkshire × Duroc F1 hybrid (LYD), (2) skin fibroblasts of a miniature pig having the human decay accelerating factor gene (hDAF-MP), and (3) skin fibroblasts of a miniature pig with a different strain (MP). MII oocytes were enucleated, subjected to nuclear transfer from a donor cell, electrically fused, and activated 1 h after fusion. SCNT embryos were cultured in a modified NCSU-23 (Park Y et al. 2005 Zygote 13, 269–275) for 6 days or surgically transferred (110–150 fused embryos) into the oviduct of a surrogate that showed standing estrus on the same day as SCNT. Embryos were examined for cleavage and BL formation on Days 2 and 6, respectively (Day 0 = the day of SCNT). BLs were examined for their cell number after staining with Hoechst 33342. Pregnancy was diagnosed by ultrasound 30 and 60 days after embryo transfer. Embryo cleavage was not affected by donor cells (82, 81, and 72% for LYD, hDAF-MP, and MP, respectively), but BL formation was higher (P < 0.05) in hDAF-MP (16%) than in LYD (9%) and MP (6%). MP showed higher (P < 0.05) BL cell number (46 cells/BL) than hDAF-MP (34 cells) but did not show a difference from LYD (37 cells). LYD and MP showed higher pregnancy rates (Table 1) on Days 30 and 60, even though they showed lower BL formation in vitro. Due to a relatively small number of embryo transfers through a limited period, we could not exclude any possible effects by seasonal or operational differences. These results indicated that pregnancy did not correlate with in vitro BL formation of SCNT pig embryos but rather were affected by the source of donor cells. Table 1.In vivo development of somatic cell nuclear transfer pig embryos derived from different sources of donor cells This work was supported by the Research Project on the Production of Bio-organs (No. 200506020601), Ministry of Agriculture and Forestry, Republic of Korea.

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