Maternal endometrial oedema may increase perinatal mortality of cloned and transgenic piglets.

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.

Efficiency of two enucleation methods connected to handmade cloning to produce transgenic porcine embryos.

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.

High in vitro development after somatic cell nuclear transfer and trichostatin A treatment of reconstructed porcine embryos.

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.

Piglets born from handmade cloning, an innovative cloning method without micromanipulation.

Porcine handmade cloning (HMC), a simplified alternative of micromanipulation based traditional cloning (TC) has been developed in multiple phases during the past years, but the final evidence of its biological value, births of piglets was missing. Here we report the first births of healthy piglets after transfer of blastocysts produced by HMC. As a cumulative effect of technical optimization, 64.3+/-2.3 (mean+/-S.E.M.) reconstructed embryos from 151.3+/-4.8 oocytes could be obtained after 3-4h manual work, including 1h pause between fusion and activation. About half (50.1+/-2.8%, n=16) of HMC reconstructed embryos developed to blastocysts with an average cell number of 77+/-3 (n=26) after 7 days in vitro culture (IVC). According to our knowledge, this is the highest in vitro developmental rate after porcine somatic cell nuclear transfer (SCNT). A total of 416 blastocysts from HMC, mixed with 150 blastocysts from TC using a cell line from a different breed were transferred surgically to nine synchronized recipients. Out of the four pregnancies (44.4%) two were lost, while two pregnancies went to term and litters of 3 and 10 piglets were delivered by Caesarean section, with live birth/transferred embryo efficiency of 17.2% (10/58) for HMC. Although more in vivo experiments are still needed to further stabilize the system, our data proves that porcine HMC may result in birth of healthy offspring. Future comparative examinations are required to prove the value of the new technique for large-scale application.

Chemically assisted handmade enucleation of porcine oocytes.

The purpose of our work was to find an efficient and reliable chemically assisted procedure for enucleation of porcine oocytes connected to the handmade cloning (HMC) technique without the potentially harmful chromatin staining and ultraviolet (UV) irradiation for cytoplast selection. After 41-42 h in vitro maturation, porcine oocytes were incubated with 0.4 microg/mL demecolcine for 45 min. Subsequently, the cumulus cells were removed and zonae pellucidae were partially digested. Oocytes with extrusion cones or oocytes only with polar body (PB) were subjected to oriented bisection. Less than half of the cytoplasm with the extrusion cone or adjacent to the PB was removed with a microblade. The remaining putative cytoplasts, containing the major part of the cytoplasm, were used as recipients for reconstruction with porcine fetal fibroblasts as nuclear donors. The overall efficiency achieved with chemically assisted enucleation was higher compared to oriented bisection without demecolcine incubation (90 +/- 3% vs. 81 +/- 4%, respectively; mean +/- absolute deviation [AD]). Reconstructed and activated embryos were cultured in vitro for 7 days. Fusion, cleavage and blastocyst rates were 87 +/- 7%, 97 +/- 6%, and 28 +/- 9%, respectively. These rates are at least as good as those achieved with normal HMC (81 +/- 4%, 87 +/- 8%, and 21 +/- 9%, respectively). For traditional, micromanipulator-based cloning, fusion and blastocyst rates were similar (81 +/- 10% and 21 +/- 6%, respectively), but the cleavage rate was lower (69 +/- 9%). In conclusion, chemically assisted handmade enucleation seems to be a simpler and potentially superior alternative to more conventional methods used for somatic cell nuclear transfer in pigs.

High overall in vitro efficiency of porcine handmade cloning (HMC) combining partial zona digestion and oocyte trisection with sequential culture.

We investigated the in vitro developmental competence of porcine embryos produced from in vitro matured (IVM) oocytes by improved HMC and parthenogenetic activation (PA). Embryos were cultured in a modified North Carolina State University (NCSU37) medium. Firstly, we compared the developmental competence between oocytes from sows and gilts by zona-intact (ZI) and zona-free (ZF) PA. Significantly higher (p < 0.05) blastocyst rates were obtained from sow oocytes (42 +/- 4% for ZF and 55 +/- 6% for ZI) than gilt oocytes (20 +/- 2% for ZF and 26 +/- 5% for ZI). Secondly, sow oocytes were used to establish the modified HMC that was based on a modified enucleation with partial zona digestion and trisection of porcine oocytes and the use of three cytoplasts and one somatic cell for embryo reconstruction. In vitro fertilization (IVF) and in parallel ZF PA were used as the control systems. After oocyte trisection, >90% of oocyte fragments were recovered, resulting in an average of 37 reconstructed embryos from 100 oocytes. Blastocyst rates of HMC, IVF, and ZF PA embryos were 17 +/- 4%, 30 +/- 6%, and 47 +/- 4%, respectively. Our results prove that HMC in pigs may result in high in vitro efficiency up until the blastocyst stage. In vivo developmental competence will be confirmed in embryo transfer experiments.

Hand-made cloning approach: potentials and limitations.

Two major drawbacks hamper the advancement of somatic cell nuclear transfer in domestic animals. The first is a biological problem that has been studied extensively by many scientists and from many viewpoints, including the cell, molecular and developmental biology, morphology, biochemistry and tissue culture. The second is a technical problem that may be responsible for 50% or more of quantitative and/or qualitative failures of routine cloning experiments and is partially the result of the demanding and complicated procedure. However, even the relatively rare documented efforts focusing on technique are usually restricted to details and accept the principles of the micromanipulator-based approach, with its inherent limitations. Over the past decade, a small alternative group of procedures, called hand-made cloning (HMC), has emerged that has the common feature of removal of the zona pellucida prior to enucleation and fusion, resulting in a limited (or no) requirement for micromanipulators. The benefits of HMC are low equipment costs, a simple and rapid procedure and an in vitro efficiency comparable with or higher than that of traditional nuclear transfer. Embryos created by the zona-free techniques can be cryopreserved and, although data are still sparse, are capable of establishing pregnancies and resulting in the birth of calves. Hand-made cloning may also open the way to partial or full automation of somatic cell nuclear transfer. Consequently, the zona- and micromanipulator-free approach may become a useful alternative to traditional cloning, either in special situations or generally for the standardisation and widespread application of somatic cell nuclear transfer.

Efficient in vitro production of porcine blastocysts by handmade cloning with a combined electrical and chemical activation.

The purpose of our work was to establish an efficient protocol for activation of porcine cytoplast-fibroblast constructs produced by the handmade cloning technique. Firstly, we investigated a combined electrical and chemical activation protocol for parthenogenetic development of in vitro matured zona-free oocytes. Oocytes were activated by one 80 micros pulse and subsequently cultured in cytochalasin B and cycloheximide. Developmental rates of blastocysts from activated oocytes were 49+/-1 and 40+/-2%, when using one 80 micros pulse of 0.85 or 1.25 kV/cm, respectively. The activation procedure was further confirmed by a simultaneous re-fusion and activation of bisected oocytes, resulting in a blastocyst rate of 41+/-8%. Secondly, the activation protocol was applied in the handmade cloning technique. In vitro matured zona-free porcine oocytes were bisected and halves containing no chromatin, i.e. the cytoplasts, were selected. Reconstructed embryos were produced by a two-step fusion procedure. At the first step, one cytoplast was fused to one fibroblast by one 80 micros pulse of 1.25 kV/cm. After 1h, the cytoplast-fibroblast pair and another cytoplast were fused and activated simultaneously by one 80 micros pulse of 0.85 kV/cm, and subsequently cultured in cytochalasin B and cycloheximide. The development of reconstructed embryos to the blastocyst stage was in average 21+/-4%, and total blastocyst cell counts were in average 48+/-3. Thus, the combined electrical and chemical activation procedure resulted in efficient blastocyst development in the handmade cloning technique.

Production of transgenic porcine blastocysts by hand-made cloning.

Recently, a zona-free technique for bovine somatic cell nuclear transfer (NT) with no requirement for micromanipulation (i.e. hand-made cloning (HMC)) has been described. The present study demonstrates the application of the HMC technique in the production of transgenic porcine blastocysts. In vitro-matured zona-free porcine oocytes were bisected manually using a microblade and halves containing no chromatin (i.e. the cytoplasts) were selected. Two cytoplasts were electrofused with one transgenic fibroblast expressing enhanced green fluorescent protein and reconstructed embryos were activated in calcium ionophore (A23187) followed by 6-dimethylaminopurine. Subsequently, embryos were cultured in NCSU-23 medium supplemented with 4 mg mL(-1) bovine serum albumin for 7 days. In five replicates, 93.0 +/- 7.0% (mean +/- s.e.m.) of attempted reconstructed embryos fused and survived activation (31/31, 15/23, 28/28, 37/37 and 28/28). On Day 7 after activation, the respective blastocyst rates (per successfully reconstructed embryos) were 6% (2/31), 7% (1/15), 7% (2/28), 3% (1/37) and 7% (2/28), resulting in an average of 6.0 +/- 0.8%. Enhanced green fluorescent protein was expressed in all cells of all eight developing blastocysts. Efforts are now directed towards the production of offspring from such transgenic NT blastocysts.

Pregnancies and piglets from large white sow recipients after two transfer methods of cloned and transgenic embryos of different pig breeds.

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.

Simplified cryopreservation of porcine cloned blastocysts.

Recently, a non-invasive delipation (lipid removal) method combined with ultrarapid vitrification has been used successfully for in vitro produced (IVP) porcine embryos. In the present study, this method was combined with parthenogenesis and a recent form of somatic cell nuclear transfer (SCNT) - handmade cloning (HMC) - to establish a simplified and efficient cryopreservation system for porcine cloned embryos. In Experiment 1, zonae pellucidae of oocytes were partially digested with pronase, followed by centrifugation to polarize lipid particles. Ninety percent (173/192) oocytes were successfully delipated in this way. Parthenogenetic activation (PA) after complete removal of zona resulted in similar blastocyst rates in delipated vs. control oocytes (28+/-7% vs. 28+/-5%, respectively). Subsequent vitrification of produced blastocysts with the Cryotop technique resulted in higher survival rates in the delipated group compared to the control group (85+/-6% vs. 32+/-7%, respectively; P<0.01). In Experiment 2, delipated oocytes were used for HMC with normal oocytes as control. Partial zona digestion was further applied before enucleation both in delipated and control groups, to bisect oocyte successfully. Although the blastocyst rate of reconstructed embryos was similar between groups derived from delipated vs. control oocytes (21+/-6% and 23+/-6%, respectively), after vitrification higher survival rates were achieved in the delipated groups than in controls (79+/-6% vs. 32+/-8%, respectively). Our results prove that porcine embryos produced from delipated oocytes by PA or HMC can be cryopreserved effectively by ultrarapid vitrification. Further experiments are required to assess the in vivo developmental competence of the cloned-vitrified embryos.