Livestock induced pluripotent stem cells.

Chimeric animals generated from livestock-induced pluripotent stem cells (iPSCs) have opened the door of opportunity to genetically manipulate species for the production of biomedical models, improving traits of agricultural importance and potentially providing a system to test novel iPSC therapies. The potential of pluripotent stem cells in livestock has long been recognized, with many attempts being chronicled to isolate, culture and characterize pluripotent cells from embryos. However, in most cases, livestock stem cells derived from embryonic sources have failed to reach a pluripotent state marked by the inability to form chimeric animals. The in-depth understanding of core pluripotency factors and the realization of how these factors can be harnessed to reprogram adult cells into an induced pluripotent state has changed the paradigm of livestock stem cells. In this review, we will examine the advancements in iPSC technology in mammalian and avian livestock species.

Somatic cell cloned transgenic bovine neurons for transplantation in parkinsonian rats.

Parkinson's disease symptoms can be improved by transplanting fetal dopamine cells into the putamen of parkinsonian patients. Because the supply of human donor tissue is limited and variable, an alternative and genetically modifiable non-human source of tissue would be valuable. We have generated cloned transgenic bovine embryos, 42% of which developed beyond 40 days. Dopamine cells collected from the ventral mesencephalon of the cloned fetuses 42 to 50 days post-conception survived transplantation into immunosuppressed parkinsonian rats and cells from cloned and wild-type embryos improved motor performance. Somatic cell cloning can efficiently produce transgenic animal tissue for treating parkinsonism.

KIT ligand and bone morphogenetic protein signaling enhances human embryonic stem cell to germ-like cell differentiation.

BACKGROUND: Signaling mechanisms involved in early human germ cell development are largely unknown and believed to be similar to mouse germ cell development; however, there may be species specific differences. KIT ligand (KITL) and Bone morphogenetic protein 4 (BMP4) are necessary in mouse germ cell development and may play an important role in human germ cell development. METHODS: KITL signaling studies were conducted by differentiating human embryonic stem cells (hESCs) on KITL wild-type, hetero- or homozygous knockout feeders for 10 days, and the effects of BMP signaling was determined by differentiation in the presence of BMP4 or its antagonist, Noggin. The formation of germ-like cells was ascertained by immunocytochemistry, flow cytometry and quantitative RT-PCR for germ cell markers. RESULTS: The loss of KITL in enrichment and differentiation cultures resulted in significant down-regulation of germ cell genes and a 70.5% decrease in germ-like (DDX4+ POU5F1+) cells, indicating that KITL is involved in human germ cell development. Moreover, endogenous BMP signaling caused germ-like (DDX4+ POU5F1+) cell differentiation, and the inhibition of this pathway caused a significant decrease in germ cell gene expression and in the number of DDX4+ POU5F1+ cells. Further, we demonstrated that eliminating feeders but maintaining their secreted extracellular matrix is sufficient to sustain the increased numbers of DDX4+ POU5F1+ cells in culture. However, this resulted in decreased germ cell gene expression. CONCLUSIONS: From these studies, we establish that KITL and BMP4 germ cell signaling affects in vitro formation of hESC derived germ-like cells and we suggest that they may play an important role in normal human germ cell development.

Cloned transgenic calves produced from nonquiescent fetal fibroblasts.

An efficient system for genetic modification and large-scale cloning of cattle is of importance for agriculture, biotechnology, and human medicine. Here, actively dividing fetal fibroblasts were genetically modified with a marker gene, a clonal line was selected, and the cells were fused to enucleated mature oocytes. Out of 28 embryos transferred to 11 recipient cows, three healthy, identical, transgenic calves were generated. Furthermore, the life-span of near senescent fibroblasts could be extended by nuclear transfer, as indicated by population doublings in fibroblast lines derived from a 40-day-old fetal clone. With the ability to extend the life-span of these primary cultured cells, this system would be useful for inducing complex genetic modifications in cattle.

BMP4 promotes formation of primitive vascular networks in human embryonic stem cell-derived embryoid bodies.

The vasculature develops primarily through two processes, vasculogenesis and angiogenesis. Although much work has been published on angiogenesis, less is known of the mechanisms regulating the de novo formation of the vasculature commonly called vasculogenesis. Human embryonic stem cells (hESC) have the capability to produce all of the cells of the body and have been used as in vitro models to study the molecular signals controlling differentiation and vessel assembly. One such regulatory molecule is bone morphogenetic protein-4 (BMP4), which is required for mesoderm formation and vascular/hematopoietic specification in several species. However, hESC grown in feeder-free conditions and treated with BMP4 differentiate into a cellular phenotype highly expressing a trophoblast gene profile. Therefore, it is unclear what role, if any, BMP4 plays in regulating vascular development in hESC. Here we show in two National Institutes of Health-registered hESC lines (BG02 and WA09) cultured on a 3D substrate of Matrigel in endothelial cell growth medium-2 that the addition of BMP4 (100 ng/ml) for 3 days significantly increases the formation and outgrowth of a network of cells reminiscent of capillary-like structures formed by mature endothelial cells (P<0.05). Analysis of the expression of 45 genes by quantitative real time-polymerase chain reaction on a low-density array of the entire culture indicates a rapid and significant downregulation of pluripotent and most ectodermal markers with a general upregulation of endoderm, mesoderm, and endothelial markers. Of the genes assayed, BMPR2 and RUNX1 were differentially affected by exposure to BMP4 in both cell lines. Immunocytochemistry indicates the morphological structures formed were negative for the mature endothelial markers CD31 and CD146 as well as the neural marker SOX2, yet positive for the early vascular markers of endothelium (KDR, NESTIN) and smooth muscle cells (alpha-smooth muscle actin [alpha SMA]). Together, these data suggest BMP4 can enhance the formation and outgrowth of an immature vascular system.

Transgenic bovine chimeric offspring produced from somatic cell-derived stem-like cells.

We have developed a method, using nuclear transplantation, to produce transgenic embryonic stem (ES)-like cells from fetal bovine fibroblasts. These cells, when reintroduced into preimplantation embryos, differentiated into derivatives from the three embryonic germ layers, ectoderm, mesoderm, and endoderm, in 5-month-old animals. Six out of seven (86%) calves born were found to be chimeric for at least one tissue. These experiments demonstrate that somatic cells can be genetically modified and then de-differentiated by nuclear transfer into ES-like cells, opening the possibility of using them in differentiation studies and human cell therapy.

Development of porcine embryos reconstituted with somatic cells and enucleated metaphase I and II oocytes matured in a protein-free medium.

BACKGROUND: Many cloned animals have been created by transfer of differentiated cells at G0/G1 or M phase of the cell cycle into enucleated M II oocytes having high maturation/meiosis/mitosis-promoting factor activity. Because maturation/meiosis/mitosis-promoting factor activity during oocyte maturation is maximal at both M I and M II, M I oocytes may reprogram differentiated cell nuclei as well. The present study was conducted to examine the developmental ability in vitro of porcine embryos reconstructed by transferring somatic cells (ear fibroblasts) into enucleated M I or M II oocytes. RESULTS: Analysis of the cell cycle stages revealed that 91.2 +/- 0.2% of confluent cells were at the G0/G1 phase and 54.1 +/- 4.4% of nocodazole-treated cells were at the G2/M phase, respectively. At 6 h after activation, nuclear swelling was observed in 50.0-88.9% and 34.4-39.5% of embryos reconstituted with confluent cells and nocodazole-treated cells regardless of the recipient oocytes, respectively. The incidence of both a swollen nucleus and polar body was low (6.3-10.5%) for all nocodazole-treated donor cell regardless of the recipient oocyte. When embryos reconstituted with confluent cells and M I oocytes were cultured, 2 (1.5%) blastocysts were obtained and this was significantly (P < 0.05) lower than that (7.6%) of embryos produced by transferring confluent cells into M II oocytes. No reconstructed embryos developed to the blastocyst stage when nocodazole-treated cells were used as donors. CONCLUSIONS: Porcine M I oocytes have a potential to develop into blastocysts after nuclear transfer of somatic cells.

In vitro culture of bovine IVM-IVF embryos: Cooperative interaction among embryos and the role of growth factors.

The objective of this study was to determine whether there is a cooperative interaction among bovine embryos during in vitro culture. Furthermore, culture medium was supplemented with the growth factors, epidermal growth factor (EGF) and transforming growth factor-beta1 (TGF-beta1), to determine if these factors had a stimulatory effect on bovine embryo development similar to that seen in mouse development. In vitro matured - in vitro fertilized bovine embryos (2- to 8-cell) were cultured singly and in groups of five in 25 mul of medium (CR1 + amino acids + fatty acid-free bovine serum albumin) with or without EGF and TGF-beta1. Bovine embryos cultured in groups had a significantly higher rate of development to the blastocyst stage than embryos cultured singly. Neither EGF (10 ng/ml) nor TGF-beta1 (2 ng/ml) affected blastocyst development, hatching or the cell number of the embryos cultured in groups. Epidermal growth factor stimulated hatching of embryos cultured singly from the 8-cell stage, but did not significantly affect blastocyst development.

Cloning: new breakthroughs leading to commercial opportunities.

Research on cloning animals, again, came to the forefront of public attention in 1997. Most scientists involved in biomedical and agricultural research have emphasized the benefits, of which there are many, of cloning to the public. Basic studies on nuclear transfer have and will continue to contribute to our understanding of how genomic activation and cell cycle synchrony affect nuclear reprogramming and cloning efficiencies, specifically. Also, more basic information on actual mechanisms and specific factors in the oocyte causing nuclear reprogramming is forthcoming. As new molecular approaches in functional genomics are combined with nuclear transfer experiments, new genes involved in nuclear reprogramming will be found. The commercial potentials of products stemming from discoveries in cloning are vast. Cloning will be a more efficient, faster and more useful way of making transgenic fetuses for cell therapies, adult animals for protein production and organs for xenotransplantation. Clearly there are new opportunities in animal cloning technology that will produce many benefits to society.

Clinical and pathologic features of cloned transgenic calves and fetuses (13 case studies).

The neonatal abnormalities, treatments and outcomes in a group of 13 cloned transgenic calves and fetuses that progressed into the third trimester of pregnancy are described. From these 13 fetuses, 8 calves were born live, 4 stillborn fetuses were recovered from 3 cows that died 7 d to 2 mo before term, and 1 aborted fetus was recovered at 8 mo gestation. All fetuses and calves were derived from the same male fetal Holstein fibroblast cell line transfected with a beta-galactosidase marker gene. Six calves were delivered by Cesarian section and two by vaginal delivery between 278 and 288 d of gestation. Birth weights ranged from 44 to 58.6 kg. Five of the 8 live born calves were judged to be normal within 4 h of birth based on clinical signs and blood gas measurements. One of these 5 calves died at 6 wk of age from a suspected dilated cardiomyopathy. Three of the 8 calves were diagnosed with neonatal respiratory distress immediately following birth, one of which died (at 4 d of age) as a result of pulmonary surfactant deficiency coupled with pulmonary hypertension and elevated systemic venous pressures. Similar findings of chronic pulmonary hypertension were also observed in 2 of 5 fetuses. Placental edema was present in both calves that later died and in the 2 fetuses with cardiopulmonary abnormalities. Hydrallantois occurred with or without placental edema in 6 cows, and only 1 calf from this group survived. The 6 cows without hydrallantois or placental edema produced 5 live calves and 1 aborted fetus. The cardiopulmonary abnormalities observed in the calves and fetuses occurred in utero in conjunction with placental abnormalities, and it is likely that the cloning technique and/or in vitro embryo culture conditions contributed to these abnormalities, although the mechanism remains to be determined.

Ion channels and ionotropic receptors in human embryonic stem cell derived neural progenitors.

Human neural progenitor cells differentiated from human embryonic stem cells offer a potential cell source for studying neurodegenerative diseases and for drug screening assays. Previously, we demonstrated that human neural progenitors could be maintained in a proliferative state with the addition of leukemia inhibitory factor and basic fibroblast growth factor. Here we demonstrate that 96 h after removal of basic fibroblast growth factor the neural progenitor cell culture was significantly altered and cell replication halted. Fourteen days after the removal of basic fibroblast growth factor, most cells expressed microtubule-associated protein 2 and TUJ1, markers characterizing a post-mitotic neuronal phenotype as well as neural developmental markers Cdh2 and Gbx2. Real-time PCR was performed to determine the ionotropic receptor subunit expression profile. Differentiated neural progenitors express subunits of glutamatergic, GABAergic, nicotinic, purinergic and transient receptor potential receptors. In addition, sodium and calcium channel subunits were also expressed. Functionally, virtually all the hNP cells tested under whole-cell voltage clamp exhibited delayed rectifier potassium channel currents and some differentiated cells exhibited tetrodotoxin-sensitive, voltage-dependent sodium channel current. Action potentials could also be elicited by currents injection under whole-cell current clamp in a minority of cells. These results indicate that removing basic fibroblast growth factor from the neural progenitor cell cultures leads to a post-mitotic state, and has the capability to produce excitable cells that can generate action potentials, a landmark characteristic of a neuronal phenotype. This is the first report of an efficient and simple means of generating human neuronal cells for ionotropic receptor assays and ultimately for electrically active human neural cell assays for drug discovery.

Factors influencing the commercialisation of cloning in the pork industry.

Production of cloned pigs using somatic cell nuclear transfer (SCNT) is a repeatable and predictable procedure and multiple labs around the world have generated cloned pigs and genetically modified cloned pigs. Due to the integrated nature of the pork production industry, pork producers are the most likely to benefit and are in the best position to introduce cloning in to production systems. Cloning can be used to amplify superior genetics or be used in conjunction with genetic modifications to produce animals with superior economic traits. Though unproven, cloning could add value by reducing pig-to-pig variability in economically significant traits such as growth rate, feed efficiency, and carcass characteristics. However, cloning efficiencies using SCNT are low, but predictable. The inefficiencies are due to the intrusive nature of the procedure, the quality of oocytes and/or the somatic cells used in the procedure, the quality of the nuclear transfer embryos transferred into recipients, pregnancy rates of the recipients, and neonatal survival of the clones. Furthermore, in commercial animal agriculture, clones produced must be able to grow and thrive under normal management conditions, which include attainment of puberty and subsequent capability to reproduce. To integrate SCNT into the pork industry, inefficiencies at each step of the procedure must be overcome. In addition, it is likely that non-surgical embryo transfer will be required to deliver cloned embryos, and/or additional methods to generate high health clones will need to be developed. This review will focus on the state-of-the-art for SCNT in pigs and the steps required for practical implementation of pig cloning in animal agriculture.

Efficient adenoviral-mediated gene delivery into porcine mesenchymal stem cells.

Mesenchymal stem cell (MSC) mediated gene therapy research has been conducted predominantly on rodents. Appropriate large animal models may provide additional safety and efficacy information prior to human clinical trials. The objectives of this study were: (a) to optimize adenoviral transduction efficiency of porcine bone marrow MSCs using a commercial polyamine-based transfection reagent (GeneJammer, Stratagene, La Jolla, CA), and (b) to determine whether transduced MSCs retain the ability to differentiate into mesodermal lineages. Porcine MSCs (pMSCs) were infected under varying conditions, with replication-defective adenoviral vectors carrying the GFP gene and GFP expression analyzed. Transduced cells were induced to differentiate in vitro into adipogenic, chondrogenic, and osteogenic lineages. We observed a 5.5-fold increase in the percentage of GFP-expressing pMSCs when adenovirus type 5 carrying the adenovirus type 35 fiber (Ad5F35eGFP) was used in conjunction with GeneJammer. Transduction of pMSCs at 10.3-13.8 MOI (1,500-2,000 vp/cell) in the presence of Gene Jammer yielded the highest percentage of GFP-expressing cells ( approximately 90%) without affecting cell viability. A similar positive effect was detected when pMSCs were infected with an Ad5eGFP vector. Presence of fetal bovine serum (FBS) during adenoviral transduction enhanced vector-encoded transgene expression in both GeneJammer-treated and control groups. pMSCs transduced with adenovirus vector in the presence of GeneJammer underwent lipogenic, chondrogenic, and osteogenic differentiation. Addition of GeneJammer during adenoviral infection of pMSCs can revert the poor transduction efficiency of pMSCs while retaining their pluripotent differentiation capacity. GeneJammer-enhanced transduction will facilitate the use of adenoviral vectors in MSC-mediated gene therapy models and therapies.

Knockdown of the Dnmt1s transcript using small interfering RNA in primary murine and bovine fibroblast cells.

RNA interference (RNAi) has rapidly developed into one of the most widely applied technologies in molecular and cellular research, and although young, is now an essential experimental tool. The versatility of RNAi, especially in mammalian species, lends to its potential applications in a wide array of fields. Without having to genetically manipulate the genome, the ability to selectively reduce the level of a specific transcript using small interfering RNA (siRNA) molecules has great appeal in studying reprogramming issues in somatic cell nuclear transfer (SCNT) embryos. In such embryos, the aberrant expression of the somatic isoform of Dnmt1 (Dnmt1s), the enzyme responsible for maintaining DNA methylation in all somatic cells, has been implicated as one factor in the improper reprogramming of the donor genome. In the present study, the ability to develop a method allowing for the knockdown, or reduction, of Dnmt1s in primary fibroblast cells, like those commonly used as karyoplast donors in SCNT studies, was investigated in primary murine and bovine fibroblast cells as well as in a compromised cell line (NIH/3T3). Two Dnmt1s-specific siRNA candidates were designed and tested. Using optimized conditions, these siRNAs were transiently transfected into the cells with total RNA and nuclear protein being collected. A 56.5% knockdown in Dnmt1s was achieved in the compromised and primary murine cells whereas Dnmt1s was reduced by 15.4% in the primary bovine cells. A reduction in Dnmt1s mRNA did not correspond to a reduction in protein as determined by immunodetection of Western blots. Overall, this study demonstrated the ability of siRNA to knockdown Dnmt1s mRNA in primary fibroblast donor cells. In order to substantially increase the efficiency while decreasing the anomalies seen in SCNT, novel techniques, like the one proposed, are needed to assist the oocyte's ability to reprogram a differentiated genome.

Nuclear reprogramming in nuclear transplant rabbit embryos.

The first six genetically verified nuclear transplant rabbits have been produced in this study. Individual eight-cell stage embryo blastomeres were transferred and fused with enucleated mature oocytes of which six full-term offspring were produced out of 164 manipulated eggs. The following efficiency rates were determined for the nuclear transplantation procedure: chromosomal removal from oocytes, 92%; fusion rate, 84%; activation rate, 46%; embryo transfer rate, 27%. Additional reasons for the low efficiency rate of nuclear transplant embryos may include limited development due to aging in recipient oocytes and asynchronous transfers of manipulated embryos to recipient females. The successful development to term may have been due to the ability of the mature oocyte to reprogram the eight-cell stage nuclei. The number of cells in blastocysts derived from isolated eight-cell blastomeres (18 +/- .08) was lower than that of nonmanipulated pronuclear (106 +/- 5.1) and nuclear transplant embryos derived from eight-cell stage nuclei (91 +/- 10.2) (p less than 0.001). This evidence along with the significant amount of nuclear swelling in nuclear transplant embryos and a delay in the time of blastocyst formation indicate that nuclear reprogramming had taken place in these embryos. Successful nuclear reprogramming indicates that serial transfers could result in the expanded multiplication of mammalian embryos.

Activation of mammalian oocytes by a factor obtained from rabbit sperm.

In this study a fraction was prepared from rabbit sperm that activated rabbit and mouse oocytes following injection into the cytoplasm. The sperm factor activated oocytes exhibited cortical granule exocytosis, pronuclear formation, and cleavage. The sperm factor was soluble in aqueous solution and was not active extracellularly. Unlike most artificial activation methods that are only effective with aged oocytes, the sperm factor activated recently ovulated oocytes. The factor appears to be a protein or associated with a protein but not an acrosomal protein. Fractions from both mouse and bull sperm did not activate rabbit or mouse oocytes. Their inactivity may be owing to the techniques used to recover the fractions or differences between species in sperm morphology and fertilization processes. These observations support the hypothesis that oocyte activation is induced by a factor within sperm that is released into the cytoplasm of the oocyte at the time of sperm-oocyte fusion.

Effects of the ovary and conceptus on uterine blood flow in the pig.

Changes in uterine blood flow throughout pregnancy appear to be due to steroid-induced alterations in uterine arterial tone and contractility. Arterial contractility is a transient reduction in luminal diameter in response to nerve stimulation or to an alpha-1 adrenergic agonist, leading to short-term reduction in uterine blood flow. Tone is the pressure exerted by an arterial segment against an intraluminal flow (distensibility) and is considered to set the baseline rate of flow. These phenomena appear to be regulated individually, with tone changes predominating during pregnancy. In pregnancy, tone is markedly depressed as oestrogen concentrations rise, and the vessel is distended and flaccid. Arterial tone is a function of the amount of calcium available to the contractile proteins of the arterial smooth muscle, which is derived from extracellular sources. Calcium availability is regulated by the opening and closing of calcium channels in the surface membrane in response to changes in the membrane potential. The loss of uterine arterial tone associated with oestrogen results from a markedly depressed uptake of calcium by the vessels. A significant negative correlation (P less than 0.001; r = - 0.93) is observed between uterine arterial uptake of calcium and the concentrations of oestrogens in systemic blood of pigs throughout gestation. Several lines of evidence suggest that the blockade of potential-sensitive calcium channels associated with uterine hyperaemia is produced by catechol oestrogens, short-lived metabolites of oestrogens that are found in the circulation when oestrogen levels are high. Synthesis of catechol oestrogens from the parent oestrogens has been shown to occur in the placenta, endometrium and uterine arteries of pigs.(ABSTRACT TRUNCATED AT 250 WORDS)

Multiple generational bovine embryo cloning.

Repeated cycles (multiple generations) of nuclear transfer procedures have the potential of producing a large number of identical offspring from the micromanipulation of one mammalian embryo. This paper outlines the differences in nuclear transfer procedure efficiencies and developmental rates for the various stages and generations of embryos. We report the production of 54 genetically identical embryos that developed to the morula stage from one parent embryo. When a limited number of nuclear transfer embryos (clones) were transferred to recipient females, first, second, and third generation calves were born. Clones cultured for 5 days in the sheep oviduct had a greater number of cells (28.1 +/- 1.7) than clones cultured for 4 days (17.8 +/- 1.7). However, fusion rates were lower for Day 5 clones (Day 5, 57% vs. Day 4, 68%; p < 0.001) used as donors for the next generation of cloning. Different generations of clones also varied in fusion rates. Fusion rates generally declined with each additional generation and ranged from 66% for the parent-donor embryos to 52% for second generation donors (p < 0.01). Developmental rates were highest for first and third generation donors (20% and 19%) and lowest for second generation donors (10%; p < 0.001). Further understanding of the factors that are conducive to high developmental rates and nuclear reprogramming may make it possible to produce an unlimited number of genetically identical animals.

Effect of follicle-stimulating hormone and luteinizing hormone during bovine in vitro maturation on development following in vitro fertilization and nuclear transfer.

The effects of luteinizing hormone (LH) (0, 100, 10,000 IU/ml) and follicle-stimulating hormone (FSH) (20 micrograms/ml) supplementation during in vitro maturation of slaughterhouse-derived oocytes on polar body formation and embryo development subsequent to in vitro fertilization and nuclear transfer were evaluated. Gonadotropin supplementation of maturation medium in the presence of serum neither enhanced the proportion of oocytes forming a polar body nor significantly affected development following in vitro fertilization or nuclear transfer, except at the highest LH concentration. A very high concentration of LH (10,000 IU/ml) significantly decreased polar body formation, initial cleavage, and blastocyst development (P < 0.05).

Bovine nuclear transfer embryos: oocyte activation prior to blastomere fusion.

Successful bovine nuclear transfer (NT) embryo production requires proper oocyte activation and transfer of a nucleus into this oocyte. However, the temporal relationship between these two events is unclear. The current study examined whether activation of the oocyte prior to fusion would induce nuclear swelling while also affecting development to morula and blastocyst stage and finally development to offspring. Aged oocytes can be activated by a number of techniques including exposure to room temperature. In this study oocyte activation was induced through three different means: reduced temperature culture alone, reduced temperature culture and calcium ionophore, and naturally, through the fertilization process. Electrofusion was carried out after the activation stimulus. When used in the NT procedure, activation of oocytes prior to fusion resulted in NT embryos that underwent nuclear swelling and had a high developmental rate to morula and blastocyst stages. Also, these NT embryos developed to normal offspring when transferred to recipient animals. The addition of a calcium ionophore treatment to the reduced temperature culture was not beneficial and resulted in less nuclear swelling. The use of enucleated fertilized oocytes as recipient cytoplasm for the new nucleus resulted in NT embryos developing to morula and blastocyst stages at the same rate as room temperature activated NT embryos. Therefore, improved embryo development can be obtained from NT embryos if the aged recipient oocyte is activated prior to the time of fusion. Also, offspring were obtained from these pre-activated NT embryos.