During in-vitro handling, oocytes and embryos are always exposed to light. This has been shown to affect embryonic development and quality in different species, i.e. hamster and mouse (Takenaka et al. 2007 PNAS, 104, 14 289–14 293) and human (Tatsuo et al. 2010 J. Assist. Reprod. Genet. 27, 93–96). However, similar experiments have not been made on porcine embryos, so our aim was to test effects of different types of light on the development and quality of porcine parthenote embryos. Cumulus–oocyte complexes from slaughterhouse-derived sow ovaries were aspirated and matured (38.5°C, 5% CO2, maximum humidity, 42 h). Parthenogenetic activation was made (Day 0) first by an electric pulse (1.26 kV cm–1, 80 µs) and then by incubation with 5 µg mL–1 cytochalasin B and 10 µg mL–1 cycloheximide in porcine zygote medium-3 (PZM-3) for 4 h. During these processes, the oocytes would be exposed to ~30 min of light. After activation, the oocytes were either directly cultured in incubator (0 h), or experimentally exposed to two types of light (DAY: near window, no direct sunlight; LAB: app. 40 cm from warm white lamps (12 V, 40 W) in PZM-3 while placed on a heating plate (38.5°C) with the culture dish covered by a crystal plastic foil filled with the appropriate gas (5% O2, 5% CO2) for different periods (1 h, 4 h, 24 h), and then cultured in PZM-3 in incubator. On Day 6, total and good blastocysts were counted. Good blastocysts were defined as blastocysts having expanded to 1.5 times the oocytes’ size, having cells of uniform color and distribution, and having formed a regular blastocyst cavity. The total number of cells and the apoptotic cells were detected on Day 6 blastocysts with TUNEL assay to evaluate embryonic quality. All statistics analysis were performed by ANOVA test (R). The results are summarized in Table 1. The developmental rates were decreased with both types of light: the decrease appeared earlier for good blastocyst rates, only after 1 h exposure; however, a clear adverse effect was also found on total blastocyst rates after 24 h exposure. The total cell number decreased after 4 h light exposure for both types of light. The rate of apoptotic cells tended to increase with both types of light (from approximately 7 to 9.5%) when embryos were exposed during 24 h, but no significant difference was found between groups. We conclude that the blastocyst morphology would be altered already after 1 h extra exposure to both light types, and their quality would further decrease after 4 h exposure. However, under normal working conditions, this should not represent a real problem. Table 1.Effect of light exposure on development of porcine parthenote embryos

The need for zona pellucida (ZP) during pre-implantation embryo development is still debated. In porcine parthenogenetically activated (PA) embryos, we have previously shown a different distribution in cell numbers on Day 6 blastocysts cultured with or without ZP (Li et al. 2010 Reprod. Fertil. Dev. 22, 234). In the present study, we expanded this study to include also the timing of early development and the resulting quality and robustness (for vitrification) of porcine PA embryos. Parthenogenetic activation was made first by an electric pulse (1.26 kV cm–1, 80 μs) and then by incubation with 5 μg mL–1 cytochalasin B and 10 μg mL–1 cycloheximide in PZM-3 medium for 4 h. ZP was removed by 3.3 mg mL–1 pronase. Both zona-intact (PAZI) and zona-free (PAZF) embryos were cultured individually for 6 days either in time-lapse incubator (Embryoscope D, Unisense A/S, Aarhus, Denmark) for 15-min observations (Exp. 1; 60 oocytes, 2 replicates) or in standard a incubator for blastocyst quality studies on Day 6 (Exp. 2; 524 oocytes, 11 replicates) or for cryo-tolerance studies with vitrification using Cryotop on Day 4, followed by warming and 2 days further culture (Exp. 3; 449 oocytes, 4 replicates). The timing of morulae was recorded when they completed compaction. Good blastocysts were defined when they expanded to 1.5 times larger than oocytes and formed regular blastocoel cavity with uniform colour and distribution of cells. Timing data were analysed by Student's t-test, while development rates and survival rates were analysed by chi-squared test. Exp. 1: after activation, 42 blastocysts formed on Day 6, during which the timing of development was monitored (Table 1). PAZF embryos developed faster than PAZI, especially during the first 3 cell cycles. Exp. 2: after activation, 212 and 197 blastocysts formed on Day 6 with or without ZP, respectively. Both rates of total blastocysts and good blastocysts of PAZI embryos were significantly higher than those of PAZF embryos (80.1 ± 2.7% vs 69.9 ± 1.1%, 61.9 ± 3.3% vs 49.5 ± 2.5%, respectively), but no difference was found in all blastocyst's cell numbers between PAZI and PAZF (48.2 ± 2.3 and 47.9 ± 3.2, respectively). Exp. 3: after activation, 107 PAZI and 44 PAZF embryos were vitrified on Day 4. More PAZI than PAZF embryos survived (60.8 ± 8.3% vs 30.4 ± 11.9%; P < 0.05). In conclusion, removal of ZP can increase the speed of development of porcine PA embryos, especially at the timing of embryonic genome activation (5-cell stage). Furthermore, the zona pellucida can benefit the blastocyst formation and cryo-tolerance for PA embryos, perhaps by creating a more stable microenvironement. Table 1.Timing of developmental stages of porcine PA embryos with (PAZI) or without (PAZF) zona pellucida*

Viability of cloned and transgenic piglets is seriously compromised and one obvious reason could be malformations. The aim of the present study was therefore to describe gross pathological conditions in dead pre-weaned piglets born after transfer to Large White (LW) recipients of cloned (LW donor cells) or transgenic (Yucatan or Göttingen donor cells) embryos. Donor cells were fibroblasts and the Göttingen and Yucatan cells were made transgenic with 1 of 5 genes known to dispose for different human diseases. Handmade cloning was used to produce embryos that after 5 to 6 days of in vitro culture were transferred surgically to 108 LW sows 4 days after their natural heat. Of these, 21 sows delivered cloned LW piglets, whereas 17 and 16 sows, respectively, delivered transgenic Göttingen and Yucatan piglets. Stillborn and dead pre-weaned piglets were necropsied and malformations registered. Data were analysed by Fisher's exact test with a significance level of P < 0.05. In the 54 litters, total litter size ranged from 1 to 22 piglets (mean 5.4 ± 0.5) and the overall mortality rate until weaning on day 28 was 59%. Malformations were found in piglets from 38 litters where an average of 35% of the piglets showed malformations (between 8 and 100%). In those litters, 1 to 7 piglets had 1, 2, or several malformations (Table 1). The malformation rate in the autopsied transgenic Göttingen was 58% and in Yucatan 46%; these were significantly higher than in the autopsied cloned LW piglets with 18%. Some of the malformations seemed to be related to breed and/or transgene; for instance, heart malformations were most frequent in Yucatan litters (70%) independent of the transgene, whereas gallbladder and gonad malformations were more frequent in various litters with the same transgene. These results show that the use of cloning in pigs results in a considerable loss of piglets due to malformations and transgenic transformation of the cells used for cloning superimpose on this problem. In combination, these elements could seriously compromise the use of pigs as a model for human diseases and the choice of breeds and also transgenes for this kind of work should be considered carefully. However, further improvements in production of cloned/transgenic embryos may ultimately reduce the incidence of malformations. Table 1.Number of malformations in 54 litters of cloned or transgenic piglets

Extract from Xenopus eggs can induce reprogramming in somatic cells. In our previous study, cell colony formation was induced during culture of porcine fetal fibroblasts after a single treatment with Xenopus egg extract and culture for several passages and using these long-term cultured cells for cloning increased the resulting blastocyst rate (Liu et al. 2011 Reprod. Fertil. Dev. 23, 130). However, both colony number and cloned blastocyst rate decreased after Passage 15 and no colonies formed after Passage 18. Therefore, in this study we investigated the effect of a second extract treatment on colony formation and cloned blastocyst formation. Extract-treated (ExT) porcine fetal fibroblasts at Passage 13 (P13) grown on poly-L-lysine-coated coverslips were permeabilized by digitonin (7 μg mL–1, 2 min, 4°C) and incubated in extract at 37°C for 30 min. After resealing the membrane in DMEM supplemented with 2 mM CaCl2, the remaining cells were cultured in ES medium (Vejlsted et al. 2005 Mol. Reprod. Dev. 70, 445). The treated cells were split onto 2 coverslips on Day 7 after the second extract treatment (2ExT), defined as Passage 1 (2ExT P1, comparable with ExT P14). New subcultures were made every 7 to 8 days when 70 to 80% clusters became colonies (i.e. 2ExT P8). Colony cells from both ExT (P14 and P16) and 2ExT (P1, P3 and P6) were used for handmade cloning and nontreated cells were used as control (Day 0). Blastocyst rates were analysed by chi-square test and colony numbers were analysed by 1-way ANOVA (SAS version 9.2). Colony numbers and cloned blastocyst rates on Day 6 are summarised in Table 1. Colonies continued to form in treated cells from 2ExT P1 to P8. The colony number maintained at a high level (60 to 80) from 2ExT P4 to P8 and it was significantly higher than that of ExT cells at the comparable passage numbers. No colonies formed in control cells. When using 2ExT colony cells at P3 and P6 for cloning, the blastocyst rates increased compared with controls and they were also higher than in the ExT group. Cloned blastocyst rates were not different between 2ExT P1 and ExT P14 groups. In conclusion, a second extract treatment can induce colony formation and increase cloned blastocyst rates, indicating that this repeated extract treatment again could activate the extract-treated cells to an activity level similar to that achieved after the first treatment. Table 1.Summary of colony number and cloned blastocyst rate with ExT and 2ExT colony cells

Extracts from eggs of Xenopus laevis frogs can induce nuclear remodelling or increase transcriptional reprogramming in somatic cells. However, it is not known if this effect is passed on from one cell generation to another, or how it affects somatic cell nuclear transfer in porcine cells. This study aimed to investigate the effect of extract-treated cells over several generations on porcine cloning. Extracts were prepared from 2 frogs (B1 and B2) by the same protocol (Higa et al. 2006 Methods 39, 284–290). Fetal fibroblasts grown on poly-L-lysine coated coverslips were permeabilized by digitonin (7 μg mL–1, 2 min, 4°C) and incubated with 1 extract batch at 37°C for 30 min. After resealing the membrane in DMEM supplemented with 2 mM CaCl2 at 37°C for 2 h, the remaining cells were cultured in ES medium (Vejlsted et al. 2005 Mol. Reprod. Dev. 70, 445–454) for 7 to 8 days when they formed colonies. The colonies were trypsinized and divided onto 2 coverslips for subculture, defined as Experimental Passage 1 (XP1). New subcultures were made every 7 to 8 days when 70 to 80% clusters become colonies until XP15. Colonies from XP3, 8 and 15 were isolated and trypsinized before being used in handmade cloning. Nontreated cells grown in DMEM were used as controls (no colony formation was observed). On each cloning day, cells from different XP number and controls were used. Rates of cleavage (Day 2) and blastocyst development (Day 6) were analysed with chi-square test (SAS version 9.2, SAS Institute Inc., Cary, NC, USA). Results are summarised in Table 1. No difference was observed in cleavage rate between groups. Blastocyst rates of all XP colony cells were significantly higher than their controls. For the same XP number and their controls, blastocyst rates were similar between the colony cells from the 2 extract batches, and there was no difference between their controls, either. In conclusion, the cloning efficiency in porcine cells could be increased with extract-treated cells used for several generations, and this effect was present at XP3, 8, and 15. Table 1.Developmental competence of cloned porcine embryos with extract-treated cells from different batches of extract (B1 and B2) and Experimental Passage (XP) numbers

The perinatal mortality of cloned animals is a well-known problem. In the present retrospective study, we report on mortality of cloned transgenic or non-transgenic piglets produced as part of several investigations. Large White (LW) sows (n = 105) received hand-made cloned LW or minipig blastocysts and delivered either spontaneously or after prostaglandin induction followed by either Caesarean section or vaginal birth. The overall pregnancy rate was 62%, with 26% of pregnancies terminating before term. This resulted in 48 deliveries. The terminated pregnancies consisted of 12 abortions that occurred at 35 ± 2 days gestation and five sows that went to term without returning to heat and then by surgery showed the uterus without fetal content. The gestation length was for sows with LW piglets that delivered by Caesarean section or vaginally was 115.7 ± 0.3 and 117.6 ± 0.4 days, respectively. In sows with minipiglets, the gestation length for those delivered by Caesarean section or vaginally 114.4 ± 0.2 and 115.5 ± 0.3 days, respectively. Of the 34 sows that delivered vaginally, 28 gave birth after induction, whereas 6 farrowed spontaneously. Of the 14 sows that delivered after Caesarean section and in the five empty sows, the endometrium and placenta showed severe oedema. Piglet mortality following vaginal delivery was higher than after Caesarean section (31% v. 10%, respectively; P < 0.001). When vaginal delivery occurred spontaneously, the stillborn rate was greater than after induced delivery (56% v. 24%, respectively; P < 0.0001). Internal organ weights were recorded for seven cloned LW piglets and six normal piglets. The relative weight of the heart, liver, kidneys and small intestine was found to be reduced in the cloned piglets (P < 0.05). The present study demonstrates extensive endometrial oedema in sows pregnant with cloned and transgenic piglets, as well as in empty recipients, at term. The growth of certain organs in some of the cloned piglets was reduced and the rate of stillborn piglets was greater in cloned and transgenic piglets delivered vaginally, possibly because of oedema of the fetal-maternal interface.

The aim of this study was to report from a larger study with pregnancy and delivery results after transfer of cloned transgenic/non-transgenic Large White or minipig embryos to Large White sow recipients. The effect of both total numbers of transferred embryos as well as site of their deposition (uni- vs. bi-lateral) was studied. Four to five days after natural heat, 85 Large White (LW) sows received Day 5 or 6 handmade cloned embryos. Large White embryos were non-transgenic and were transferred to 36 recipients, while 49 recipients each received Minipig embryos, either non-transgenic or with 1 of 4 types of transgenes. Furthermore, the number of embryos transferred was in two categories, as 46 recipients received 40-60 embryos while 39 received 60-120 embryos. Finally, in 59 of the recipients embryos were transferred to one of the uterine horns (unicornual) while 26 other recipients had embryos transferred to both uterine horns (bicornual). The overall pregnancy rate was 55% with an abortion rate of 26% resulting in 41% deliveries with no difference between LW and Minipig embryos and no difference between transgenic and non-transgenic Minipig embryos. Transfer of 60-120 embryos resulted in more pregnancies and deliveries (62%) than <60 embryos (24%). The mean litter size was 5.1 ± 0.5 and after transfer of 60-120 embryos significantly higher (6.0 ± 0.5) than after transfer of <60 embryos (3.5 ± 0.8). Also, the bicornual transfer resulted in significantly higher delivery rate (74% vs. 44%) and mean litter size (6.1 ± 0.7 vs. 4.2 ± 0.6) than the unicornual. The mean rate of piglets/transferred embryos was 7.3 ± 0.6% while the mean rate of piglets/reconstructed embryos was 179/18,000 = 1% with no difference between breeds or number of embryos transferred. The overall perinatal mortality rate was 49%, and it was significantly lower in LW piglets (20/59 = 34%) than in Minipiglets (67/120 = 56%) (vs. 10-15% in normal piglets at the farm) and the total rate of piglets with one or more malformation was 22%, and lower in LW (12%) than in Minipiglets (28%). This study demonstrate that although the perinatal mortality was rather high, an acceptable birth rate can be achieved after transfer to LW recipients of cloned LW embryos as well as cloned, transgenic/non-transgenic Minipig embryos. Furthermore, the pregnancy rate and litter size were correlated to the number of embryos transferred and to bicornual transfer.

Production of cloned piglets, using normal or transgenic donor cells, has been of limited efficiency. One reason could be breed differences between recipient and embryos. The aim of this study was to compare pregnancies after transfer to Large White (LW) sow recipients of cloned LW embryos or cloned, transgenic minipig embryos. Large White donor cells were from LYxD, and minipig cells were transgenic with 1 of 5 genes related to different human diseases and from either Göttingen or Yucatan. The cells were used on Day 0 for handmade cloning (Du et al. 2005 Cloning Stem Cells 7, 199-205). The reconstructed embryos were cultured in vitro until transfer on Day 5 to 6. As recipients, 70 LW sows were weaned and anesthetized 4 days after natural heat. Through an abdominal incision the ovaries were controlled (CL formation, absence of cysts) and the embryos slowly introduced into the uterus via a catheter inserted 5 to 6 cm into the tip of the upper horn. To each of 33 recipients, 40 to 60 LW embryos were transferred, and 37 recipients each received 50 to 100 transgenic minipig embryos. Pregnancies were examined by ultrasound scanning every second week. Abortions were defined as absence of earlier confirmed scanning or delivery of aborted fetuses. Caesarean sections were performed on Day 114 (minipig) or Day 116 (LW) 24 h after injection of a prostaglandin analogue. At delivery, placental gross morphology was recorded with samples taken for later histology. The piglets were fed every 3 h with colostrum for the first 24 h and then by the recipient LW sow. Data were analyzed by Fisher’s Exact test with a significance level of P < 0.05. The overall pregnancy rate was 49% (34/70) with an abortion rate of 29% (8 aborted + 2 resorbed/34) from Day 30 to 45 giving 24/70 deliveries (34% of the transfers). There was no significant difference between minipig (54%, 20/37) and LW pregnancy results (42%, 14/33), although there tended to be more abortions with minipig pregnancies (8/20 v. 2/14; P = 0.14) resulting in 12 minipig and 12 LW litters of which 4 and 9, respectively, have grown up to adulthood. In almost every recipient the placenta and fetal membranes showed abnormal thick and edematous morphology. The total litter sizes ranged from 1 to 10 piglets (mean 4.4 ± 0.6), and in 13 of 24 litters there were 1 to 5 stillborn piglets. Except for one litter of 9 transgenic Yucatan piglets that all died within their first 2 weeks, the postnatal mortality of both LW- and mini-piglets seemed similar to that of farm piglets of the same age, and the piglets appeared normal with respect to weight gain, gross morphology, and behavior. These results demonstrate that, in spite of a rather high abortion rate and some fetal mortality, an acceptable birth rate can be achieved after transfer to LW recipients of cloned LW embryos (36%) as well as cloned, transgenic minipig embryos (32%). Therefore, a breed difference between the embryos and their recipient seems not to influence the pregnancy results. The authors thank B. Synnestvedt, H. Kristiansen, S. Starsig, A. Pedersen, J. Adamsen, R. Kristiansen, and K. Villemoes for invaluable technical assistance.

Pretreatment of somatic cells to promote subsequent reprogramming during somatic cell nuclear transfer (SCNT) may significantly improve efficiency of the technique. The aim of this study was to evaluate the effect of Xenopus laevis egg extract pretreatment of porcine fetal fibroblast cells using different permeabilization agents prior to SCNT. Fibroblasts were permeabilized using streptolysin O (SLO; 300 ng mL-1, 30 min, 37°C) or digitonin (7 μg mL-1, 2 min, 4°C), and exposed to egg extract for 1 h or 0.5 h, respectively. Cell membranes were resealed in DMEM supplemented with 2 mM CaCl2 for 2 h. After culture for 1, 3, and 5 days (for SLO) or 3 and 5 days (for digitonin), the SLO extract-treated cells (SETC) and digitonin extract-treated cells (DETC) were used as donor karyoplasts for handmade cloning. Controls were SCNT with nontreated cells. Embryos were evaluated for cleavage rate (Day 2), blastocyst rate (Day 6), and total cell numbers of blastocysts. Statistical differences were analyzed by ANOVA. Results are summarized in Table 1. When SETC were used as donors, blastocyst rates were significantly lower compared with the controls, except when the donor cells were cultured for 3 days after treatment. Blastocysts of the latter group also had higher total cell number. With DETC as donors, blastocyst rates and total cell number of embryos at Day 6 reconstructed with cells cultured for 5 days were higher than those in other groups. Results indicate that extract treatment of the donor cells after SLO-permeabilization can give higher number of cells in cloned blastocysts but not improve overall embryo development. However, digitonin treatment for donor cell permeabilization improved both embryo development and cell number of blastocyst. The latter effect was detected only 5 days after the treatment. In conclusion, qualitative efficiency of porcine SCNT could be improved with a combined donor cell permeabilization and extract treatment. Table 1.Effect of different permeabilization agents prior to SCNT

The purpose of our work was to establish an efficient-oriented enucleation method to produce transgenic embryos with handmade cloning (HMC). After 41-42 h oocytes maturation, the oocytes were further cultured with or without 0.4 microg/ml demecolcine for 45 min [chemically assisted handmade enucleation (CAHE) group vs polar body (PB) oriented handmade enucleation (OHE) group respectively]. After removal of the cumulus cells and partial digestion of the zona pellucida, oocytes with visible extrusion cones and/or polar bodies attached to the surface were subjected to oriented bisection. Putative cytoplasts without extrusion cones or PB were selected as recipients. Two cytoplasts were electrofused with one transgenic fibroblasts expressing green fluorescent protein (GFP), while non-transgenic fibroblasts were used as controls. Reconstructed embryos were cultured in Well of Wells (WOWs) with porcine zygote medium 3 (PZM-3) after activation. Cleavage and blastocyst rates were registered on day 2 and day 7 of in vitro culture respectively. Meanwhile, the total blastocyst cell number was counted on day 7. We found that the difference was only observed between blastocyst rates (38.6 +/- 2% vs 48.1 +/- 3%) of cloned embryos with GFP transgenic fibroblast cells after CAHE vs OHE. With adjusted time-lapse for zonae-free cloned embryos cultured in WOWs with PZM-3, it was obvious that in vitro developmental competence after CAHE was compromised when compared with the OHE method. OHE enucleation method seems to be a potential superior alternative method used for somatic cell nuclear transfer (SCNT) with transgenic fibroblast cells.

Modified environmental stress was reported to improve the developmental competence and cryotolerance of porcine oocytes, such as high hydrostatic pressure (HHP; Du et al. 2008 Cloning Stem Cells, Epub ahead of print) and osmotic stress (Lin et al. 2008 Reprod. Biomed. Online, in press). HHP also improved the cryotolerance of bovine and murine blastocysts (Pribenszky et al. 2005a Reprod. Dom. Anim. 40, 338–344; Pribenszky et al. 2005b Anim. Reprod. Sci. 87, 143–150). In the present study we compared the effects of NaCl with that of concentrated solutions of two non-permeable osmotic agents, sucrose and trehalose on in vitro maturated oocytes. A total of 2050 slaughterhouse-derived porcine cumulus–oocyte complexes (COCs) were matured for 41–42 h, and then put into 800 μL T2 (HEPES-buffered TCM-199 [Earle’s salts] with 2% cattle serum) supplemented with additional NaCl, sucrose or trehalose with the same osmotic level (588 mOsmol) in 4-well dishes and incubated for 1 h at 38.5°C in air. COCs incubated in T2 under the same conditions without supplementation were used as controls. Subsequently COCs were incubated in IVM medium for 1 h at 38.5°C in 5% CO2 with maximum humidity. After this recovery period cumulus cells were removed with 1 mg mL–1 hyaluronidase and pipetting, and oocytes were used as recipients for somatic nuclear transfer with handmade cloning (HMC) method. Porcine fetal fibroblasts were used as nuclear donor cells. Embryo culture was performed in PZM-3 medium (Yoshioka et al. 2002 Biol. Reprod. 66, 112–119) in 5% CO2, 5% O2 and 90% N2 and maximum humidity. Cleavage and blastocyst rates were checked on Day 1 and Day 6, respectively. Cell numbers were counted after fixation in glycerol containing 20 μg mL–1 Hoechst 33342 fluorochrome on Day 6. t-test was performed for statistical calculations with SPSS 11.0 program (SPSS, Chicago, IL, USA). Results are shown in Table 1. Osmotic stress with both permeable and non-permeable agents increased developmental competence of porcine IVM oocytes. NaCl seems to be more appropriate for the purpose, as the other two components resulted in decreased cell number in blastocysts after somatic cell nuclear transfer (SCNT). In conclusion, a simple NaCl pre-treatment of oocytes has improved the in vitro efficiency of porcine SCNT. Table 1.Developmental competence of porcine HMC embryos derived from oocytes treated with different agents The authors thank Ruth Kristensen, Anette Pedersen, Janne Adamsen and Klaus Villemoes for their help and excellent technical assistance.

Abnormal epigenetic modification is supposed to be one of factors accounting for inefficient reprogramming of the donor cell nuclei in ooplasm after somatic cell nuclear transfer (SCNT). Trichostatin A (TSA) is an inhibitor of histone deacetylase, potentially enhancing cloning efficiency. The aim of our present study was to establish the optimal TSA treatment in order to improve the development of handmade cloned (HMC) porcine embryos and examine the effect of TSA on their development. The blastocyst percentage of HMC embryos treated with 37.5 nM TSA for 22-24 h after activation increased up to 80% (control group-54%; P<0.05). TSA mediated increase in histone acetylation was proved by immunofluorescence analysis of acH3K9 and acH4K16. 2-cell stage embryos derived from TSA treatment displayed significant increase in histone acetylation compared to control embryos, whereas no significant differences were observed at blastocyst stage. During time-lapse monitoring, no difference was observed in the kinetics of 2-cell stage embryos. Compact morula (CM) stage was reached 15 h later in TSA treated embryos compared to the control. Blastocysts (Day 5 and 6) from HMC embryos treated with TSA were transferred to 2 recipients resulting in one pregnancy and birth of one live and five dead piglets. Our data demonstrate that TSA treatment after HMC in pigs may affect reprogramming of the somatic genome resulting in higher in vitro embryo development, and enable full-term in vivo development.

High hydrostatic pressure (HHP) has been introduced into the field of embryology recently, with the possible mechanism that a sublethal HHP could induce the synthesis of molecular chaperons to protect the embryos from further stresses. Improved cryotolerance has been achieved successfully in HHP-treated mouse (Pribenszky 2005 Anim. Reprod. Sci. 87, 143–150) and bovine (Pribenszky 2005 Reprod. Domest. Anim. 40, 338) embryos, and the semen of bull (Pribenszky 2007 Reprod. Fertil. Dev. 19, 181–182) and boar (Pribenszky 2005 Reprod. Fertil. Dev. 18, 162–163). The objective of the present study was to apply this new technique to in vitro-matured (IVM) porcine oocytes and further investigate its effect in the procedure of handmade cloning (HMC). After 40 h IVM, cumulus–oocyte complexes (COCs) were loaded in 0.5-mL straws by a 2-mL syringe, with HEPES-buffered TCM199 as the loading medium. COCs were then treated with 20 MPa (200 times greater than atmospheric pressure) for 60 min by a pressurizing device (Cryo-Innovation Inc., Budapest, Hungary), with an interval of 120 min between HHP treatment and subsequent HMC. Two different cell lines (from Day 40 fetuses of Yucatan and Danish Landrace breeds (LW1-2)) were used as donor cells for nuclear transfer. A total of 592 reconstructed embryos were produced from both HHP-treated and control groups and were in vitro cultured for 6 days to evaluate the developmental competence through to blastocyst formation. The effect of donor cells on blastocyst development was also investigated. SPSS 11.0 program (SPSS, Inc., Chicago, IL, USA) was used for statistical analysis; values with P < 0.05 were regarded as significant. Blastocyst rates of the different groups are shown in Table 1. Our results indicated that COCs treated with HHP had a much higher blastocyst rate than those untreated (P < 0.01) and this improvement was not affected by using different donor cells for nuclear transfer. In conclusion, the sublethal HHP treatment could improve the in vitro developmental competence of porcine IVM oocytes when they are used for HMC. Further in vivo experiments are required to investigate the long-term effect of HHP on embryo development. Table 1. Day 6 blastocyst rates of HHP-treated and control groups with different donor cells for nuclear transfer The authors thank Ruth Kristensen and Janne Adamsen for their help and excellent technical assistance.