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Zhou W.,ViaGen Inc. | Gosch G.,Sioux Center | Guerra T.,ViaGen Inc. | Broek D.,Sioux Center | And 4 more authors.
Theriogenology | Year: 2014

Somatic cell nuclear transfer (SCNT), or cloning, is one of the assisted reproductive technologies currently used in agriculture. Commercial applications of SCNT are presently limited to the production of animals of high genetic merit or the production of the most elite show cattle owing to its relatively low efficiency. In current practice, 20% to 40% of SCNT pregnancies do not result in viable offspring. In an effort to better understand some of the anomalies associated with SCNT pregnancies, we investigated amino acid compositions of first trimester amniotic fluid. In this retrospective study, amniotic fluids were collected from SCNT and control IVF pregnancies at Day 75 of gestation and grouped according to the pregnancy results: control IVF (IVF), viable SCNT pregnancies that resulted in live healthy calves (SCNT-HL), nonviable SCNT pregnancies that were aborted before Day 150 (SCNT-ED), and nonviable SCNT pregnancies that were aborted after Day 150 or produced deceased calves (SCNT-LD). High-performance liquid chromatography (HPLC) was used to analyze the concentrations of 22 amino acids (AAs) in the amniotic fluid samples. There were no differences in average AA concentrations between IVF and SCNT-HL groups, whereas SCNT-LD and SCNT-ED had higher levels of total AA concentrations. Concentrations of asparagine, citruline, arginine, and valine were significantly higher in the SCNT-LD group. Both SCNT-LD and SCNT-ED groups had relatively large intragroup variances in AA concentrations. Urea concentration was also measured in the SCNT amniotic fluid samples. No correlations between urea concentrations and arginine concentrations or pregnancy outcomes were found. The findings in this study not only deepen the understanding onSCNT pregnancy anomalies, but also provide a potentially useful screening tool for assessing viable and nonviable SCNT pregnancies. © 2014 Elsevier Inc.


Gregg K.,ViaGen Inc. | Gosch G.,Sioux Center | Guerra T.,ViaGen Inc. | Chen S.H.,ViaGen Inc. | And 4 more authors.
Theriogenology | Year: 2010

The objective was to use the bovine viral diarrhea virus (BVDV) as a model to assess the risk of infectious disease transmission in the system of in vitro embryo production and transfer via somatic cell nuclear transfer (SCNT) technology. The risks of BVDV transmission in the SCNT embryo production were previously evaluated [1]. In that in vitro study, following standard operating procedures (SOP), including pre-nuclear transfer donor cell testing, oocyte decontamination and virus-free cell and embryo culture conditions, SCNT embryos produced were free of detectable viral RNA. The current study focused on the evaluation of the potential risk of disease transmission from SCNT embryos to recipients, and the risk of producing persistently infected animals via SCNT embryo transfer. Blood samples were collected from 553 recipients of SCNT embryos and 438 cloned calves and tested for the presence of BVDV viral RNA via a sensitive real time PCR method. All samples tested were negative. These results, in conjunction with the previous in vitro study, confirmed that the established SCNT embryo production and transfer system is safe and presents no detectable risk of disease transmission. © 2010 Elsevier Inc.


Polejaeva I.A.,Utah State University | Broek D.M.,ViaGen Inc. | Walker S.C.,ViaGen Inc. | Zhou W.,ViaGen Inc. | And 3 more authors.
PLoS ONE | Year: 2013

The objective of this study was to determine whether or not reproductive performance in cattle produced by somatic cell nuclear transfer (SCNT) is significantly different from that of their genetic donors. To address this question, we directed two longitudinal studies using different embryo production procedures: (1) superovulation followed by artificial insemination (AI) and embryo collection and (2) ultrasound-guided ovum pick-up followed by in vitro fertilization (OPU-IVF). Collectively, these two studies represent the largest data set available for any species on the reproductive performance of female clones and their genetic donors as measured by their embryo production outcomes in commercial embryo production program. The large-scale study described herein was conducted over a six-year period of time and provides a unique comparison of 96 clones to the 40 corresponding genetic donors. To our knowledge, this is the first longitudinal study on the reproductive performance of cattle clones using OPU-IVF. With nearly 2,000 reproductive procedures performed and more than 9,200 transferable embryos produced, our observations show that the reproductive performance of cattle produced by SCNT is not different compared to their genetic donors for the production of transferable embryos after either AI followed by embryo collection ( P = 0.77) or OPU-IVF (P = 0.97). These data are in agreement with previous reports showing that the reproductive capabilities of cloned cattle are equal to that of conventionally produced cattle. In conclusion, results of this longitudinal study once again demonstrate that cloning technology, in combination with superovulation, AI and embryo collection or OPU-IVF, provides a valuable tool for faster dissemination of superior maternal genetics. © 2013 Polejaeva et al.


Patent
ViaGen Inc. and Foundation University | Date: 2012-05-29

Genetic material is derived from animals post-mortem, and used in nuclear transfer processes to produce cloned embryos and live cloned animals having genetic make-ups identical to the post mortem animals. The method has particular applicability to the management and breeding of livestock, to the production of animals having desired genetic traits, and to the integration of those genetic traits into selective breeding operations.


PubMed | Recombinetics, Inc., ViaGen Inc., Utah State University and Sioux Center
Type: Journal Article | Journal: PloS one | Year: 2014

The objective of this study was to determine whether or not reproductive performance in cattle produced by somatic cell nuclear transfer (SCNT) is significantly different from that of their genetic donors. To address this question, we directed two longitudinal studies using different embryo production procedures: (1) superovulation followed by artificial insemination (AI) and embryo collection and (2) ultrasound-guided ovum pick-up followed by in vitro fertilization (OPU-IVF). Collectively, these two studies represent the largest data set available for any species on the reproductive performance of female clones and their genetic donors as measured by their embryo production outcomes in commercial embryo production program. The large-scale study described herein was conducted over a six-year period of time and provides a unique comparison of 96 clones to the 40 corresponding genetic donors. To our knowledge, this is the first longitudinal study on the reproductive performance of cattle clones using OPU-IVF. With nearly 2,000 reproductive procedures performed and more than 9,200 transferable embryos produced, our observations show that the reproductive performance of cattle produced by SCNT is not different compared to their genetic donors for the production of transferable embryos after either AI followed by embryo collection (P = 0.77) or OPU-IVF (P = 0.97). These data are in agreement with previous reports showing that the reproductive capabilities of cloned cattle are equal to that of conventionally produced cattle. In conclusion, results of this longitudinal study once again demonstrate that cloning technology, in combination with superovulation, AI and embryo collection or OPU-IVF, provides a valuable tool for faster dissemination of superior maternal genetics.


Gregg K.,ViaGen Inc. | Riddell K.P.,Auburn University | Chen S.H.,ViaGen Inc. | Galik P.K.,Auburn University | And 5 more authors.
Theriogenology | Year: 2010

The objective was to assess the risk of transmission of bovine viral diarrhea virus (BVDV) through embryo production via somatic cell nuclear transfer (SCNT), with oocytes obtained from persistently infected (PI) donors. Using ultrasound-guided follicular aspiration following superstimulation, oocytes were obtained from five female beef cattle, including three that were PI and two that were negative for BVDV. In the three PI cattle, seven aspirations yielded 32 oocytes (PI-1: three aspirations yielding six oocytes; PI-2: two aspirations yielding 14 oocytes; and PI-3: two aspirations yielding 12 oocytes). The oocyte recovery rate was better in negative control cattle, with 32 oocytes obtained from the two cattle in a single superstimulation and aspiration session. Oocytes were processed individually for SCNT, evaluated, and tested for BVDV. Nearly all (31/32) oocytes from the three PI donors were positive for BVDV by PCR, with detected viral RNA copy number ranging from 1 to 1.1 × 105. The proportion of oocytes acceptable for SCNT embryo production (based on oocyte quality and maturation status) was only 16 to 35% from PI donors, but was 81% from control donors. Therefore, routine testing of unacceptable (discarded) oocytes could be an effective approach to identify batches that might contain infected oocytes from PI donors. Identification and removal of high-risk batches of oocytes would minimize the risk of BVDV transmission through SCNT embryo production. © 2010 Elsevier Inc.


Grant
Agency: National Science Foundation | Branch: | Program: SBIR | Phase: Phase I | Award Amount: 149.96K | Year: 2011

This Small Business Innovation Research Phase I project is focused on developing an innovative algorithm for non-invasive bovine embryo viability assessment using near-infrared spectroscopy (NIR). Assisted reproductive technologies have been developed by the livestock industry to obtain large numbers of offspring from genetically superior animals. Embryos produced by in vitro fertilization (IVF) and somatic cell nuclear transfer (SCNT or cloning) could play a central role in cattle production systems by impacting the rates of genetic improvement and efficient dissemination of superior genotypes. Broad-based implementation of these techniques is limited by the inability to select high quality embryos using current subjective visual assessment. The availability of a quantitative embryo selection method able to distinguish the subtle differences that separate a healthy pre-implantation embryo from a poorly performing one would greatly increase the overall efficiency of IVF and SCNT technologies. The research objectives of this project are: identify the differences in NIR spectral characteristics from spent media resulting from embryo culture of viable and non-viable embryos; develop an algorithmic model using the NIR spectral characteristics; and demonstrate that this algorithm can improve embryo selection accuracy, increasing pregnancy rate by at least 50% compared to the current morphological assessment. The broader impact/commercial potential of this project would be greater utilization of in vitro reproductive technologies by cattle breeders, which would have enormous impact on genetic improvement and reproductive management. Currently, a quantitative embryo assessment method in livestock is not available. If successful, the proposed innovative approach will increase pregnancy and calving rates by 50% compared to the present level and reduce the cost of a SCNT calf by $7,500. Reduced cost could substantially increase the technology utilization with a potential financial value of over $350 million. Additionally, the annual economic impact could exceed $100 million in dairy IVF in the U.S. alone. A successful result also will have a significant impact on herd health and help the livestock industry produce more offspring using fewer recipients, which will reduce pressure on the environment, decrease per unit climate emissions and land use, and meet future global nutritional needs. Such efforts will contribute to food security and improve the social and economic well-being of rural communities. The NIR spectral data from the project could lead to discoveries that enhance the scientific understanding of early embryogenesis and drive further progress in reproductive technology.


Trademark
ViaGen Inc. | Date: 2010-06-22

Mammalian embryos; cloned embryos of mammalian animals; live mammalian animals; cloned live mammalian animals. Cloning services in respect of mammalian animals.


Somatic cell nuclear transfer (SCNT) technology has become a powerful tool for reproductive biology to preserve and propagate valuable genetics for livestock. Embryo production through SCNT involves enucleation of the oocyte and insertion of a somatic donor cell into the oocyte. These procedures lead to a few small openings on the zona pellucida that may elevate risk of viral infection for the produced SCNT embryos. The oocytes used for SCNT are mainly obtained from abattoirs where viral contamination is almost inevitable. Therefore, a systematic evaluation of risk of disease transmission through SCNT embryo production is necessary prior large scale implementation of this technology in the livestock industry. The objective of the current study was to evaluate the risk of disease transmission via SCNT embryo production and transfer by testing for the presence of porcine reproductive and respiratory syndrome virus (PRRSV) throughout the process of SCNT embryo production. The presence of PRRSV in each step of SCNT embryo production, from donor cells to pre-implantation SCNT embryo culture, was carefully examined using a real-time PCR assay with a sensitivity of five copies per-reaction. All 114 donor cell lines derived from pig skin tissue over a period of 7 years in our facility tested negative for PRRSV. Out of the 68 pooled follicular fluid samples collected from 736 ovaries, only four (5.9%) were positive indicating a small amount of viral molecule present in the oocyte donor population. All 801 Day 7 SCNT embryos produced in four separate trials and over 11,571 washed oocytes obtained in 67 batches over 10 months tested negative. These oocytes were collected from multiple abattoirs processing animals from areas with high density of pig population and correspond to a donor population of over 5828 individuals. These results indicate that the oocytes from abattoirs were free of PRRSV infection and therefore could be safely used for in vitro embryo production. Additionally, the established SCNT embryo production system, including donor cell testing, oocytes decontamination, and pathogen free embryo reconstruction and culturing, bears no risk of PRRSV transmission.


Grant
Agency: NSF | Branch: Standard Grant | Program: | Phase: | Award Amount: 149.96K | Year: 2011

This Small Business Innovation Research Phase I project is focused on developing an innovative algorithm for non-invasive bovine embryo viability assessment using near-infrared spectroscopy (NIR). Assisted reproductive technologies have been developed by the livestock industry to obtain large numbers of offspring from genetically superior animals. Embryos produced by in vitro fertilization (IVF) and somatic cell nuclear transfer (SCNT or cloning) could play a central role in cattle production systems by impacting the rates of genetic improvement and efficient dissemination of superior genotypes. Broad-based implementation of these techniques is limited by the inability to select high quality embryos using current subjective visual assessment. The availability of a quantitative embryo selection method able to distinguish the subtle differences that separate a healthy pre-implantation embryo from a poorly performing one would greatly increase the overall efficiency of IVF and SCNT technologies. The research objectives of this project are: identify the differences in NIR spectral characteristics from spent media resulting from embryo culture of viable and non-viable embryos; develop an algorithmic model using the NIR spectral characteristics; and demonstrate that this algorithm can improve embryo selection accuracy, increasing pregnancy rate by at least 50% compared to the current morphological assessment.

The broader impact/commercial potential of this project would be greater utilization of in vitro reproductive technologies by cattle breeders, which would have enormous impact on genetic improvement and reproductive management. Currently, a quantitative embryo assessment method in livestock is not available. If successful, the proposed innovative approach will increase pregnancy and calving rates by 50% compared to the present level and reduce the cost of a SCNT calf by $7,500. Reduced cost could substantially increase the technology utilization with a potential financial value of over $350 million. Additionally, the annual economic impact could exceed $100 million in dairy IVF in the U.S. alone. A successful result also will have a significant impact on herd health and help the livestock industry produce more offspring using fewer recipients, which will reduce pressure on the environment, decrease per unit climate emissions and land use, and meet future global nutritional needs. Such efforts will contribute to food security and improve the social and economic well-being of rural communities. The NIR spectral data from the project could lead to discoveries that enhance the scientific understanding of early embryogenesis and drive further progress in reproductive technology.

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