The NIH miniature pig was developed specifically for xenotransplantation and has been extensively used as a large animal model in many other biomedical experiments. However the cloning efficiency of this pig is very low (less than 0.2%) and this has been an obstacle to the promising application of these inbred swine genetics for biomedical research. It has been demonstrated that increased histone acetylation in somatic cell nuclear transfer (SCNT) embryos, by applying histone deacetylase inhibitors (HDACi) such as trichostatin A (TSA), significantly enhances the developmental competence in several species. However some researchers also reported that TSA treatment had various detrimental effects on the in vitro and in vivo development of the SCNT embryos. Here we report that treatment with 500 nM Scriptaid, a novel HDACi, significantly enhanced the development SCNT embryos to the blastocyst stage when NIH inbred fetal fibroblast cells (FFCs) (21% vs. 9%, P < 0.05) were used as donors compared to the untreated group. Scriptaid treatment resulted in 8 pregnancies from 10 embryo transfers (ET) and 14 healthy NIH miniature pigs from 8 litters while no viable piglets (only 3 mummies) were obtained from 9 ETs in the untreated group. Thus Scriptaid dramatically increased the cloning efficiency when using inbred genetics from zero to 1.3%. In contrast, Scriptaid treatment decreased the blastocyst rate in IVF embryos (from 37% to 26%, P < 0.05). In conclusion, the extreme low cloning efficiency in the NIH miniature pig may be caused by its inbred genetic background and can be improved by alteration of genomic histone acetylation patterns.

The National Institutes of Health (NIH) miniature pig was developed specifically for xenotransplantation and has been extensively used as a large-animal model in many other biomedical experiments. However, the cloning efficiency of this pig is very low (<0.2%), and this has been an obstacle to the promising application of these inbred swine genetics for biomedical research. It has been demonstrated that increased histone acetylation in somatic cell nuclear transfer (SCNT) embryos, by applying a histone deacetylase (HDAC) inhibitor such as trichostatin A (TSA), significantly enhances the developmental competence in several species. However, some researchers also reported that TSA treatment had various detrimental effects on the in vitro and in vivo development of the SCNT embryos. Herein, we report that treatment with 500 nM 6-(1,3-dioxo-1H, 3H-benzo[de]isoquinolin-2-yl)-hexanoic acid hydroxyamide (termed scriptaid), a novel HDAC inhibitor, significantly enhanced the development of SCNT embryos to the blastocyst stage when NIH inbred fetal fibroblast cells (FFCs) were used as donors compared with the untreated group (21% vs. 9%, P < 0.05). Scriptaid treatment resulted in eight pregnancies from 10 embryo transfers (ETs) and 14 healthy NIH miniature pigs from eight litters, while no viable piglets (only three mummies) were obtained from nine ETs in the untreated group. Thus, scriptaid dramatically increased the cloning efficiency when using inbred genetics from 0.0% to 1.3%. In contrast, scriptaid treatment decreased the blastocyst rate in in vitro fertilization embryos (from 37% to 26%, P < 0.05). In conclusion, the extremely low cloning efficiency in the NIH miniature pig may be caused by its inbred genetic background and can be improved by alteration of genomic histone acetylation patterns.

Histone deacetylase inhibitor treatment improves developmental competence of nuclear transfer NIH miniature pig embryos, but not IVF embryos.

It is still difficult to successfully cryopreserve in vitro produced (IVP) swine embryos, as they are sensitive to chilling due to the abundance of intracellular lipids. Mechanical delipation through micromanipulation is successful, but this increases the potential of pathogen transmission because of the damage inflicted upon the zona pellucida during micromanipulation, and is labor intensive. Reported here is a method to remove the lipid of IVP porcine embryos without significantly compromising the zona pellucida by trypsin treating the embryos or exposing the embryo to a high osmolality solution to enlarge the perivitelline space so that the lipid could be polarized and separated completely after subsequent centrifugation without micromanipulation. The procedures work both for nuclear transfer derived embryos as well as in vitro fertilized embryos. Both methods provide a high throughput process that leaves the zona pellucida intact (or relatively intact as for the trypsin treatment) to aid in preventing disease transmission. It is also demonstrated that this procedure results in viable piglets, a claim not able to be made by many previous reports. Although the efficiencies of cryopreservation have not been dramatically improved, these procedures allow a single person to process very large numbers of embryos without the necessity of manipulating each individual embryo on a micromanipulator. Such high throughput processing overcomes the lack of high efficiency, i.e. the system can be overloaded with embryos for transfer to surrogates.

It is still difficult to successfully cryopreserve in vitro-produced (IVP) swine embryos, as they are sensitive to chilling due to the abundance of intracellular lipids. Mechanical delipation through micromanipulation is successful, but this method increases the potential of pathogen transmission because of the damage inflicted upon the zona pellucida during micromanipulation, and it is labor intensive. Reported here is a method to remove the lipid of IVP porcine embryos, without significantly compromising the zona pellucida, by trypsin treating the embryos or exposing the embryo to a high-osmolality solution to enlarge the perivitelline space so that the lipid could be polarized and separated completely after subsequent centrifugation without micromanipulation. The procedures work both for nuclear transfer-derived embryos and in vitro-fertilized embryos. Both methods provide a high-throughput process that leaves the zona pellucida intact (or relatively intact for the trypsin treatment) to aid in preventing disease transmission. It is also demonstrated that this procedure results in viable piglets, a claim that could not be made in many previous reports. Although the efficiencies of cryopreservation have not been dramatically improved, these procedures allow a single person to process very large numbers of embryos without the necessity of manipulating each individual embryo on a micromanipulator. Such high-throughput processing overcomes the lack of high efficiency (i.e., the system can be overloaded with embryos for transfer to surrogates).

Centrifugation in a high osmolality solution without micromanipulation can efficiently separate the lipids in swine embryos to facilitate cryopreservation.