Advanced genetic and nutritional strategies aimed at modulating fat deposition can significantly reduce production costs and enhance profitability in the poultry industry. Melanophilin (MLPH) is recognized as a key gene regulating pigmentation as shown by diluted hair and feather coloration in MLPH mutant animals, including avian models. However, the effects of MLPH during fat accretion have not been studied yet. Therefore, the objectives of the current study are to measure the temporal expression of the MLPH gene during the adipocyte differentiation in vitro and in vivo and to investigate the effect of MLPH loss on fat accretion and adipocyte sizes in vivo using MLPH knockout quail model. The current in vitro studies reveal that MLPH gene expression levels were considerably elevated during adipogenesis in avian cells [101-fold in DF-1, 28.5-fold in chicken embryonic fibroblasts (CEF) and 4-fold in quail embryonic fibroblasts (QEF), compared to the undifferentiated cells of each cell type, p < 0.05]. In addition, fractionated fat cells (FC) showed increased expression levels of MLPH (5.7-fold, p < 0.05) compared to stromal-vascular cells (SVC). Using the MLPH knockout quail, disruption of the MLPH gene resulted in significantly reduced body weight (BW) and subcutaneous fat (S. Fat) pad weights compared to the wild type (WT) (p < 0.05). Further analysis through sectioning and staining of the fat tissues revealed that the mutation in Rab binding domain (RBD) of quail MLPH resulted in decreased fat cell sizes (p < 0.01). Overall, our data clearly demonstrated that MLPH can be a potential adipogenic marker gene, and MLPH may be associated with fat accretion in the gene edited quail model, highlighting the important role of MLPH in adipogenesis.

The woody breast (WB) myopathy poses significant economic and welfare concerns to the poultry industry, however, there is no effective strategy to mitigate this pathology due to its unknown etiology. After showing previously that hypoxia is a key factor in WB progression, we used here various techniques demonstrating dysregulated mitochondria (morphology, biogenesis, tethering, function, and bioenergetics) in WB-affected muscles and in hypoxic myoblasts compared to healthy tissues and normoxic cells, respectively. The increased levels of calcium (Ca2+) in both WB-affected tissues and hypoxic myoblasts suggested that mitochondrial Ca2+ overload is likely a leading cause for mitochondrial dysfunction that merits further in-depth investigation. These findings are the first, to the best of our knowledge, to provide fundamental insights into the underlying molecular mechanisms of WB and open new vistas for understanding the interplay between calcium, mitochondrial (dys)function, and avian muscle health for subsequent development of effective preventative/corrective strategies.

In the broiler industry, intensive genetic selection has been placed on muscle growth which has undesirably led to increased fat accretion. Models of chicken preadipocyte differentiation in vitro have conventionally used incubators without the ability to control oxygen (O2) tension; thus, the cells are exposed to atmospheric (∼20-21%) O2, which is supraphysiological compared to the O2 tension within adipose tissue. The objective of this study was to investigate embryonic broiler preadipocyte differentiation at different O2 tensions, including atmospheric (20%), physiological (5%), and hypoxic (1%). Culture at 1% O2 resulted in increased abundance of HIF1α, a canonical protein stabilized during hypoxia, thus confirming effectiveness of the treatment. Increased accumulation of lipid was observed in preadipocytes cultured in adipogenic differentiation medium compared to the control medium. When considering oxygen tension, lipid accumulation was increased in preadipocytes that were cultured in differentiation medium at 20% O2 compared to 5% or 1% O2. Furthermore, abundance of transcripts related to fatty acid transport and adipogenesis, fatty acid binding protein 4 (FABP4) and peroxisome proliferator-activated receptor gamma (PPARγ), were increased in differentiated preadipocytes cultured at 20% O2 compared to 5% or 1% O2. Abundance of transcripts related to lipid synthesis and oxidation, acyl-CoA synthetase long chain family member 1 (ACSL1) and carnitine palmitoyltransferase 1A (CPT1A), were increased in the differentiation cultures compared to the control cultures. Abundance of glutathione peroxidase 4 (GPX4) was increased in all the differentiation cultures compared to the controls, regardless of oxygen tension; however, differences in the abundance of other antioxidant enzymes were not observed. Overall, exposure to atmospheric oxygen tension promotes lipid accumulation within chicken preadipocytes, which may need to be considered when developing in vitro models of this process.

Genetic modification of genes such as recombination activating gene 2 (RAG2) or interleukin-2 receptor-γ (IL2RG) results in pigs exhibiting severe combined immunodeficiency (SCID). Pigs presenting a SCID phenotype are important animal models that can be used to establish xenografts and to study immune system development and various immune-related pathologies. However, due to their immunocompromised nature, SCID pigs have shortened lifespans and are notoriously difficult to maintain. The failure-to-thrive phenotype makes the establishment of a breeding population of RAG2/IL2RG double-knockout pigs virtually impossible. Here, to overcome this limitation, we investigated whether reconstituting the immune system of SCID piglets with a fetal bone allograft would extend their lifespan. Following intramuscular transplantation, allografts gave rise to lymphocytes expressing T cell (CD3, CD4 and CD8), B cell (CD79α) and natural killer cell (CD335) lineage markers, which were detected in circulation as well in the spleen, liver, bone marrow and thymic tissues. The presence of lymphocytes indicates broad engraftment of donor cells in the recipient SCID pigs. Unlike unreconstituted SCID pigs, the engrafted animals thrived and reached puberty under standard housing conditions. This study demonstrates a novel method to extend the survival of SCID pigs, which may improve the availability and use of SCID pigs as a biomedical animal model.

CRISPR-Cas technology has transformed our ability to introduce targeted modifications, allowing unconventional animal models such as pigs to model human diseases and improve its value for food production. The main concern with using the technology is the possibility of introducing unwanted modifications in the genome. In this study, we illustrate a pipeline to comprehensively identify off-targeting events on a global scale in the genome of three different gene-edited pig models. Whole genome sequencing paired with an off-targeting prediction software tool filtered off-targeting events amongst natural variations present in gene-edited pigs. This pipeline confirmed two known off-targeting events in IGH knockout pigs, AR and RBFOX1, and identified other presumably off-targeted loci. Independent validation of the off-targeting events using other gene-edited DNA confirmed two novel off-targeting events in RAG2/IL2RG knockout pig models. This unique strategy offers a novel tool to detect off-targeting events in genetically heterogeneous species after genome editing.

Background:Presenilin 1 (PSEN1) is one of the genes linked to the prevalence of early onset Alzheimer's disease. In mice, inactivation of Psen1 leads to developmental defects, including vertebral malformation and neural development. However, little is known about the role of PSEN1 during the development in other species. Objective:To investigate the role of PSEN1 in vertebral development and the pathogenic mechanism of neurodegeneration using a pig model. Methods:CRISPR/Cas9 system was used to generate pigs with different mutations flanking exon 9 of PSEN1, including those with a deleted exon 9 (Δexon9). Vertebral malformations in PSEN1 mutant pigs were examined by X-ray, micro-CT and micro-MRI. Neuronal cells from the brains of PSEN1 mutant pigs were analyzed by immunoflourescence, followed by image analysis including morphometric evaluation via image J and 3D reconstruction. Results:Pigs with a PSEN1 null mutation (Δexon9-12) died shortly after birth and had significant axial skeletal defects, whereas pigs carrying at least one Δexon9 allele developed normally and remained healthy. Effects of the null mutation on abnormal skeletal development were also observed in fetuses at day 40 of gestation. Abnormal distribution of astrocytes and microglia in the brain was detected in two PSEN1 mutant pigs examined compared to age-matched control pigs. The founder pigs were bred to establish and age PSEN1ΔE9/+ pigs to study their relevance to clinical Alzheimer's diseases. Conclusions:PSEN1 has a critical role for normal vertebral development and PSEN1 mutant pigs serves as novel resources to study Alzheimer's disease.

The composition of the culture medium affects the viability and developmental competency of porcine embryos produced in vitro. Previous transcriptional profiling has identified areas of improvement in the formulation of these media. Xenobiotic metabolism-related genes were upregulated in in vitro-cultured porcine embryos compared to their in vivo-derived counterparts, and the antibiotic gentamicin is a component of porcine embryo culture media. While effective against a broad spectrum of bacteria, gentamicin has been observed to be toxic to rat embryos and may induce changes in gene expression in cell culture. The objective of this study was to determine if gentamicin has an adverse effect on the development of porcine embryos. After in vitro fertilization, presumptive zygotes were placed in either MU4 medium containing gentamicin or MU4 medium without gentamicin. No difference was detected in blastocyst development, total cell number, apoptotic index, or expression of 3 selected genes between embryos cultured with or without gentamicin (p > 0.05). Therefore, porcine embryos are able to tolerate the presence of 10 mu g/mL gentamicin without significant impacts on blastocyst development rate, total cell number, or apoptosis.

Abstract Growth differentiation factor 9 (GDF9) is a secreted protein belonging to the transforming growth factor beta superfamily and has been well characterized for its role during folliculogenesis in the ovary. Although previous studies in mice and sheep have shown that mutations in GDF9 disrupt follicular progression, the exact role of GDF9 in pigs has yet to be elucidated. The objective of this study was to understand the role of GDF9 in ovarian function by rapidly generating GDF9 knockout (GDF9–/–) pigs by using the CRISPR/Cas9 system. Three single-guide RNAs designed to disrupt porcine GDF9 were injected with Cas9 mRNA into zygotes, and blastocyst-stage embryos were transferred into surrogates. One pregnancy was sacrificed on day 100 of gestation to investigate the role of GDF9 during oogenesis. Four female fetuses were recovered with one predicted to be GDF9–/– and the others with in-frame mutations. All four had fully formed oocytes within primordial follicles, confirming that knockout of GDF9 does not disrupt oogenesis. Four GDF9 mutant gilts were generated and were grown past puberty. One gilt was predicted to completely lack functional GDF9 (GDF9–/–), and the gilt never demonstrated standing estrus and had a severely underdeveloped reproductive tract with large ovarian cysts. Further examination revealed that the follicles from the GDF9–/– gilt did not progress past preantral stages, and the uterine vasculature was less extensive than the control pigs. By using the CRISPR/Cas9 system, we demonstrated that GDF9 is a critical growth factor for proper ovarian development and function in pigs. Summary Sentence Inactivation of growth differentiation factor 9 revealed its critical role for proper ovarian development and function in pigs.

In brief Aromatase catalyzes the synthesis of estrogens and has been shown to have an important role during the establishment of pregnancy in the pig. This study confirmed the differential expression of the three aromatase isoforms. Abstract Although three porcine aromatase isoforms have been identified, their gene expression profiles in reproduction are still poorly understood. Here, we identified by Sanger sequencing unique nucleotide signatures for the three paralogous copies of Cyp19 and analyzed by RT-PCR the occurrence of the Cyp19 and Cyp17a1 transcripts at different tissues and stages of conceptus and fetal–placental development. Cyp19a1 and Cyp19a3 expressions were detected in conceptuses and gonads, respectively. Cyp19a2 transcripts were identified on both the conceptuses and the placenta samples. Transcripts for Cyp17a1 were detected predominantly in conceptus and gonads. In the endometrium of day 21 pregnant females, as well as days 12 and 17 pseudopregnant females, we did not detect the expression of Cyp19a1 , Cyp19a2, or Cyp19a3. In our study, we have demonstrated distinct transcriptional regulation for the three functional Cyp19 paralogs and a potential role for Cyp17a1 in controlling the secretion of estrogen from the conceptus and the placenta.

Generating porcine embryos in vitro is a critical process for creating genetically modified pigs as agricultural and biomedical models; however, these embryo technologies have been scarcely applied by the swine industry. Currently, the primary issue with in vitro -produced porcine embryos is low pregnancy rate after transfer and small litter size, which may be exasperated by micromanipulation procedures. Thus, in this review, we discuss improvements that have been made to the in vitro porcine embryo production system to increase the number of live piglets per pregnancy as well as abnormalities in the embryos and piglets that may arise from in vitro culture and manipulation techniques. Furthermore, we examine areas related to embryo production and transfer where improvements are warranted that will have direct applications for increasing pregnancy rate after transfer and the number of live born piglets per litter.

Oocyte activation occurs at the time of fertilization and is a series of cellular events initiated by intracellular Ca2+ increases. Consequently, oocytes are alleviated from their arrested state in meiotic metaphase II (MII), allowing for the completion of meiosis. Oocyte activation is also an essential step for somatic cell nuclear transfer and an important tool to overcome clinical infertility. Traditional artificial activation methods aim to mimic the intracellular Ca2+ changes which occur during fertilization. Recent studies emphasize the importance of cytoplasmic Zn2+ on oocyte maturation and the completion of meiosis, thus suggesting artificial oocyte activation approaches that are centered around the concentration of available Zn(2+)in oocytes. Depletion of intracellular Zn2+ in oocytes with heavy metal chelators leads to successful oocyte activation in the absence of cellular Ca2+ changes, indicating that successful oocyte activation does not always depends on intracellular Ca2+ increases. Current findings lead to new approaches to artificially activate mammalian oocytes by reducing available Zn2+ contents, and the approaches improve the outcome of oocyte activation when combined with existing Ca2+-based oocyte activation methods. Here, we review the important role of Ca2+ and Zn2+ in mammalian oocyte activation and development of novel oocyte activation approaches based on Zn2+ availability. Summary Sentence Artificial oocyte activation is essential for the application of Assisted Reproductive Technology and the ability to control the level of cytoplasmic zinc can facilitate oocyte activation process.

Senecavirus A (SVA) is a cause of vesicular disease in pigs, and infection rates are rising within the swine industry. Recently, anthrax toxin receptor 1 (ANTXR1) was revealed as the receptor for SVA in human cells. Herein, the role of ANTXR1 as a receptor for SVA in pigs was investigated by CRISPR/Cas9 genome editing. Strikingly, ANTXR1 knockout (KO) pigs exhibited features consistent with the rare disease, GAPO syndrome, in humans. Fibroblasts from wild type (WT) pigs supported replication of SVA; whereas, fibroblasts from KO pigs were resistant to infection. During an SVA challenge, clinical symptoms, including vesicular lesions, and circulating viremia were present in infected WT pigs but were absent in KO pigs. Additional ANTXR1-edited piglets were generated that were homozygous for an in-frame (IF) mutation. While IF pigs presented a GAPO phenotype similar to the KO pigs, fibroblasts showed mild infection, and circulating SVA nucleic acid was decreased in IF compared to WT pigs. Thus, this new ANTXR1 mutation resulted in decreased permissiveness of SVA in pigs. Overall, genetic disruption of ANTXR1 in pigs provides a unique model for GAPO syndrome and prevents circulating SVA infection and clinical symptoms, confirming that ANTXR1 acts as a receptor for the virus.

Establishment and maintenance of pregnancy in the pig is a complex process that relies on conceptus regulation of the maternal proinflammatory response to endometrial attachment. Following elongation, pig conceptuses secrete interferon gamma (IFNG) during attachment to the endometrial luminal epithelium. The objective here was to determine if conceptus production of IFNG is important for early development and establishment of pregnancy. CRISPR/Cas9 gene editing and somatic cell nuclear transfer technologies were used to create an IFNG loss-of-function study in pigs. Wild-type (IFNG+/+) and null (IFNG-/-) fibroblast cells were used to create embryos through somatic cell nuclear transfer. IFNG expression was not detected in IFNG-/- conceptuses on either day 15 or day 17 of pregnancy. Ablation of conceptus IFNG production resulted in the reduction of stromal CD3+ and mast cells, which localized to the site of conceptus attachment on day 15. The uteri of recipients with IFNG-/- conceptuses were inflamed, hyperemic and there was an abundance of erythrocytes in the uterine lumen associated with the degenerating conceptuses. The endometrial stromal extracellular matrix was altered in the IFNG-/- embryo pregnancies and there was an increased endometrial mRNA levels for collagen XVII (COL17A1), matrilin 1 (MATN1), secreted phosphoprotein 1 (SPP1), and cysteine-rich secretory protein 3 (CRISP3), which are involved with repair and remodeling of the extracellular matrix. These results indicate conceptus IFNG production is essential in modulating the endometrial proinflammatory response for conceptus attachment and survival in pigs.

Invitro-produced (IVP) porcine embryos are developmentally delayed compared with those derived invivo. Thus, efforts have been made to modify the current medium for IVP of porcine embryos in an attempt to shift the abundance of target transcripts towards the invivo level. The objective of the current study was to identify differences in mRNA abundance that may account for reduced developmental competence in IVP embryos and to determine whether alterations to maturation and culture media directed the transcriptional profiles of IVP embryos towards an invivo state. Following AI of gilts, an oviduct and tip of the uterine horn were flushed on Day 2 to recover 4-cell stage embryos; these were cultured for 4 days in MU3, generating IVM and invitro-cultured (IVC) blastocyst-stage embryos. On Day 6, the gilts were killed, and the contralateral horns were flushed to obtain invivo-derived (IVV) blastocyst-stage embryos. The third group of blastocyst-stage embryos, referred to as invitro-matured and cultured (IVMC), were created by aspirating cumulus–oocyte complexes from slaughterhouse-derived ovaries, maturing and fertilizing invitro, and culturing for 6 days in MU3. Total RNA was extracted from pools of 10 blastocyst-stage embryos with 3 replicates per group. First- and second-strand cDNA was synthesised and sequenced by using the Illumina platform (Illumina Inc.). After removal of adapters and reads that mapped to porcine rRNA genes and the PhiX genome, reads were mapped to the Sus scrofa genome by using STAR (version 2.7.1a) with default options. Pairwise comparisons were performed to test for differential expression of genes by using the Bioconductor package DESEqn 2. Transcripts were differentially abundant (false discovery rate <0.05) between IVV and IVC embryos (2,450), between IVV and IVMC embryos (3,045), and between IVC and IVMC embryos (262). Pathways related to cell cycle were downregulated in IVC and IVMC compared with IVV embryos, and pathways related to amino acid transport in metabolism were upregulated in IVC and IVMC compared with IVV embryos. Of particular interest, message for cytochrome P450 family 1 subfamily A member 1 (CYP1A1) was only present in the IVC (956 reads) and IVMC (381 reads) embryos but not in the IVV embryos (0 reads). The aryl hydrocarbon receptor (AHR) promotes transcription of CYP1A1, which encodes a monooxygenase involved in xenobiotic metabolism. Moreover, abundance of 12 other AHR targets was increased in IVC and IVMC embryos (false discovery rate <0.05) compared with IVV embryos. Thus, production of porcine embryos invitro may activate AHR, resulting in altered transcriptional profiles and reduced competence. This research was funded by USDA-NIFA (2019-67011-29543).

Genetically modified pigs have become valuable tools for generating advances in animal agriculture and human medicine. Importantly, in vitro production and manipulation of embryos is an essential step in the process of creating porcine models. As the in vitro environment is still suboptimal, it is imperative to examine the porcine embryo culture system from several angles to identify methods for improvement. Understanding metabolic characteristics of porcine embryos and considering comparisons with other mammalian species is useful for optimizing culture media formulations. Furthermore, stressors arising from the environment and maternal or paternal factors must be taken into consideration to produce healthy embryos in vitro. In this review, we progress stepwise through in vitro oocyte maturation, fertilization, and embryo culture in pigs to assess the status of current culture systems and address points where improvements can be made.

Elongation of pig conceptuses is a dynamic process, requiring adequate nutrient provisions. Glutamine is used as an energy substrate and is involved in the activation of mechanistic target of rapamycin complex 1 (mTORC1) during porcine preimplantation development. However, the roles of glutamine have not been extensively studied past the blastocyst stage. Therefore, the objective of the current study was to determine if glutaminase (GLS), which is the rate-limiting enzyme in glutamine metabolism, was necessary for conceptus elongation to proceed and was involved in mTORC1 activation. The CRISPR/Cas9 system was used to induce loss-of-function mutations in the GLS gene of porcine fetal fibroblasts. Wild type (GLS(+/+)) and knockout (GLS(-/-)) fibroblasts were used as donor cells for somatic cell nuclear transfer, and GLS(+/+) and GLS(-/-) blastocyst-stage embryos were transferred into surrogates. On day 14 of gestation, GLS(+/+) conceptuses primarily demonstrated filamentous morphologies, and GLS(-/-) conceptuses exhibited spherical, ovoid, tubular, and filamentous morphologies. Thus, GLS(-/-) embryos were able to elongate despite the absence of GLS protein and minimal enzyme activity. Furthermore, spherical GLS(-/-) conceptuses had increased abundance of transcripts related to glutamine and glutamate metabolism and transport compared to filamentous conceptuses of either genotype. Differences in phosphorylation of mTORC1 components and targets were not detected regarding conceptus genotype or morphology, but abundance of two transcriptional targets of mTORC1, cyclin D1, and peroxisome proliferator-activated receptor gamma coactivator 1-alpha was increased in spherical conceptuses. Therefore, porcine GLS is not essential for conceptus elongation and is not required for mTORC1 activation at this developmental timepoint. Summary sentence Knocking out glutaminase in porcine embryos does not prevent development to the blastocyst stage nor elongation of the conceptus to a filamentous morphology.

Pigs have become an important model for agricultural and biomedical purposes. The advent of genomic engineering tools, such as the CRISPR/Cas9 system, has facilitated the production of livestock models with desired modifications. However, precise site-specific modifications in pigs through the homology-directed repair (HDR) pathway remains a challenge. In mammalian embryos, the use of small molecules to inhibit non-homologous end joining (NHEJ) or to improve HDR have been tested, but little is known about their toxicity. The compound RS-1 stimulates the activity of the RAD51 protein, which plays a key role in the HDR mechanism, demonstrating enhancement of HDR events in rabbit and bovine zygotes. Thus, in this study, we evaluated the dosage and temporal effects of RS-1 on porcine embryo development and viability. Additionally, we assessed the effects of its vehicle, DMSO, during embryo in vitro culture. Transient exposure to 7.5 μM of RS-1 did not adversely affect early embryo development and was compatible with subsequent development to term. Additionally, low concentrations of its vehicle, DMSO, did not show any toxicity to in vitro produced embryos. The transient use of RS-1 at 7.5 μM during in vitro culture seems to be the best protocol of choice to reduce the potentially toxic effects of RS-1 while attempting to improve HDR in the pig. Direct injection of the CRISPR/Cas9 system, combined with strategies to increase the frequency of targeted modifications via HDR, have become an important tool to simplify and accelerate the production of genetically modified livestock models.

Glutamine supplementation to porcine embryo culture medium improves development, increases leucine consumption, and enhances mitochondrial activity. In cancer cells, glutamine has been implicated in the activation of mechanistic target of rapamycin complex 1 (mTORC1) to support rapid proliferation. The objective of this study was to determine if glutamine metabolism, known as glutaminolysis, was involved in mTORC1 activation in porcine embryos. Culture with 3.75 mM GlutaMAX improved development to the blastocyst stage compared to culture with 1 mM GlutaMAX, and culture with 0 mM GlutaMAX decreased development compared to all groups with GlutaMAX. Ratios of phosphorylated to total MTOR were increased when embryos were cultured with 3.75 or 10 mM GlutaMAX, which was enhanced by the absence of leucine, but ratios for RPS6K were unchanged. As another indicator of mTORC1 activation, colocalization of MTOR and a lysosomal marker was increased in embryos cultured with 3.75 or 10 mM GlutaMAX in the absence of leucine. Culturing embryos with glutaminase inhibitors decreased development and the ratio of phosphorylated to total MTOR, indicating reduced activation of the complex. Therefore, glutaminolysis is involved in the activation of mTORC1 in porcine embryos, but further studies are needed to characterize downstream effects on development.

To improve efficiency of somatic cell nuclear transfer (SCNT), it is necessary to modify differentiated donor cells to become more amendable for reprogramming by the oocyte cytoplasm. A key feature that distinguishes somatic/differentiated cells from embryonic/undifferentiated cells is cellular metabolism, with somatic cells using oxidative phosphorylation (OXPHOS) while embryonic cells utilize glycolysis. Inducing metabolic reprogramming in donor cells could improve SCNT efficiency by priming cells to become more embryonic in nature before SCNT hypoxia inducible factor 1-α (HIF1-α), a transcription factor that allows for cell survival in low oxygen, promotes a metabolic switch from OXPHOS to glycolysis. We hypothesized that chemically stabilizing HIF1-α in donor cells by use of the hypoxia mimetic, cobalt chloride (CoCl2 ), would promote this metabolic switch in donor cells and subsequently improve the development of SCNT embryos. Donor cell treatment with 100 µM CoCl2 for 24 hr preceding SCNT upregulated messenfer RNA abundance of glycolytic enzymes, improved SCNT development to the blastocyst stage and quality, and affected gene expression in the blastocysts. After transferring blastocysts created from CoCl2 -treated donor cells to surrogates, healthy cloned piglets were produced. Therefore, shifting metabolism toward glycolysis in donor cells by CoCl2 treatment is a simple, economical way of improving the in vitro efficiency of SCNT and is capable of producing live animals.