bluuebunny/crossref_metadata_2025 huggingface dataset:
283 GENERATION AND CHARACTERIZATION OF BOVINE INDUCED PLURIPOTENT STEM CELLS
Authors: Koo O. J. , Kwon H. S. , Kwon D. K. , Kang K. S. , Lee B. C. , Jang G.
Published: December 2012

Stem cells in large animals are an excellent model for cell therapy research and fine resources for producing transgenic animals. However, there are only few reports of stem cells in large animals because of technical differences between species. In this report, we successfully generate bovine induced pluripotent stem cells (iPSC) using 4 human reprogramming factors (Oct4, Sox2, Klf4, and c-myc) under control of PiggyBac transposition vector. Fibroblasts derived from bovine fetuses were transfected using FugeneHD agent. After 21 days, colony-shaped structures on the culture plates were mechanically detached and then seeded on a mouse embryonic fibroblast (MEF) feeder layer pretreated with mitomycin C. The culture medium was DMEM/F12 supplemented with 20% serum replacement, 5 ng mL–1 basic fibroblast growth factor (bFGF), 0.1 mM β-mercaptoethanol, 1% NEAA, and 1% penicillin-streptomycin antibiotics. The iPSC colonies showed alkaline phosphatase activity and expressed several pluripotency markers (Oct4, Sox2, SSEA1, and SSEA4). To confirm differentiation potential, the iPSC were cultured as embryoid bodies and then plated again. βIII-tubulin (ectoderm) and GFAP or α-SMA (mesoderm) were well expressed on the attached cells. The results revealed that the bovine fibroblasts were well inducted to iPSC that had potential of multilineage differentiation. We hope this technology contributes to improving transgenic cattle production.
This study was financially supported by IPET (grant # 109023-05-3-CG000, 111078-03-1-CG000) and the BK21 program for Veterinary Science.
285 GENERATION OF CANINE INDUCED PLURIPOTENT STEM CELLS FROM CANINE FETAL FIBROBLAST AND ADULT FIBROBLAST OF CLONED DOG
Authors: Kwon H. S. , Oh H. J. , Lee D. H. , Kim D. E. , Kang S. K. , Lee B. C.
Published: December 2012

Induced pluripotent stem cells (iPSC) derived from a patient’s fibroblasts have been used as fine resources for studying disease mechanisms and therapeutic strategies. The dog is considered invaluable in human disease research because its genetic diseases are strikingly similar to those of human. Therefore, we generated cloned dogs and transgenic cloned dogs via somatic cell nuclear transfer. In this study, we tried to derive canine iPSCs from canine fibroblasts to establish a way to make iPSC from skin fibroblasts of transgenic cloned dogs. We isolated canine fetal fibroblast (FF) from normal beagles and adult skin fibroblast (ASF) from cloned beagles. Both ASF and FF were infected with all-in-one retroviral vector that delivers human reprogramming factors (Oct4, Sox2, Klf4, c-Myc). Ten to twenty-one days after infection, the colony-shaped structure was picked and plated on a mouse embryonic fibroblast (MEF) feeder layer, pretreated with mitomycin C. Then, all cells were cultured with DMEM/F12 supplemented with 20% fetal bovine serum, 5 ng mL–1 basic fibroblast growth factor (bFGF), 5 ng mL–1 LIF, 0.1 mM β-mercaptoethanol, 1% NEAA, and 1% penicillin-streptomycin. Alkaline phosphatase (AP) activity and expression of Oct4, Sox2, SSEA1, and SSEA4, were observed in the cells to characterise the iPS cell colonies. In vitro differentiation of 10th-passage canine iPSC was performed through embryonic body formation. About 50 canine iPS-like colonies were formed on a 100-mm dish. As a result, the canine iPSC from FF (iPSC-FF) and canine iPSC from ASF (iPSC-ASF) showed typical colony morphology, and both stained positively for AP. The expression of pluripotency-associated transcription factors Oct4 and Sox2 was positively displayed in iPSC-FF colonies. The stem cell markers SSEA1 and SSEA4 were negative in canine iPSC-FF. The canine iPS-FF spontaneously differentiated into all 3 germ layers in vitro, showing positive expressions of βIII-tubulin (ectoderm), α-SMA (mesoderm), and GATA6 (endoderm). As for iPS-ASF, characterisation and in vitro differentiation experiment are in progress. These results show that canine iPS-FF are similar to embryonic stem cells in terms of morphology and the ability to differentiate into 3 germ layers. Although we did not demonstrate complete verification of canine iPS-ASF of the cloned dog, their morphology, AP expression, and iPS-FF generation should indicate the possibility of iPSC production in the cloned dog. In conclusion, retroviral transduction of 4 human reprogramming factors can reprogram canine fetal fibroblasts into canine iPSC. The technique of producing canine iPSC will stimulate the utilisation of transgenic cloned dogs and expand the range of human diseases or therapeutic application.
This study was supported by RDA (#PJ0089752012), RNL Bio (#550-20120006), IPET (#311011-05-1-SB010), Research Institute for Veterinary Science, and Nestlé Purina Korea.
287 PORCINE ADIPOSE-DERIVED STEM CELLS IN CO-CULTURE FUSE ACTIVELY WITH MOUSE MYOTUBES AND EXPRESS MYOGENIC MARKERS
Authors: Jain S. , Milner D. J. , Bionaz M. , Cameron J. A. , Wheeler M. B.
Published: December 2012

Mesenchymal stem cells (MSC) have been shown to be useful in regenerative medicine with their capability to give rise to various different cell types, including osteoblasts, adipocytes, chondrocytes, muscle cells, and neurons. Among MSC, bone marrow-derived stem cells (BMSC) are considered the primary source. Recently, many studies have shown the regenerative capabilities of adipose-derived stem cells (ASC). The ASC, with their greater abundance and ease of harvest, provide clear advantages over BMSC. We have previously demonstrated the myogenic differentiation of porcine ASC when co-cultured with differentiating C2C12 myoblasts in a myogenic differentiation-promoting medium. In this study, we sought to examine the myogenic potential of porcine ASC when co-cultured with fully differentiated murine myotubes. For the present study, we used porcine ASC isolated from the back fat of a transgenic gree fluorescent protein (GFP)-expressing pig at passage 3. The ASC were added to mouse C2C12 myotube cultures that had been induced towards myogenesis for 72 h. As controls, we co-cultured, in the same conditions, GFP-expressing endothelial cells (ENDO) from the aorta of the same pig. Additionally, we cultured ASC, ENDO, and C2C12 cells alone in myogenic-differentiation medium. Cultures were harvested at 12, 24, and 48 h after addition of porcine cells to myotube cultures for measurement of mouse- or porcine-specific myogenic markers by quantitative RT-PCR and immunohistochemistry. We were able to observe fusion of ASC GFP-expressing cells with pre-formed mouse myotubes by detection of myotubes expressing GFP. Additionally, immunofluorescent staining of co-cultures with an antibody specific for porcine nuclear Lamin A demonstrated the presence of ASC nuclei incorporated into myotubes. We observed large increases in gene expression of porcine-specific myogenin (MYOG; >900-fold) and desmin (DES; 8-fold). Unexpectedly, ENDO in co-culture with myotubes also had increased expression of DES (4-fold) and MYOG (400-fold), possibly indicating their de-differentiation and adaptation to a myogenic phenotype. In addition, expression of mouse-specific DES and MYOG were boosted in C2C12 myotubes when co-cultured for 48 h compared with C2C12 alone, suggesting enhanced myogenesis or prolonged survival of myotubes in co-culture. Cultures of ASC and ENDO alone did not display increased expression of myogenic markers. These results provide support for the use of ASC for muscle regeneration strategies, as in the case of damaged muscles and muscular dystrophy. In addition, compared with our previous observations, where ASC were co-cultured with undifferentiated C2C12, the ASC co-cultured with myotubes appeared to have an enhanced fusion and expression of myogenic markers. Finally, the capacity of ENDO to fuse and actively expressed muscle-specific genes deserves further investigation.
294 NAÏVE STATE-LIKE PLURIPOTENT STEM CELL LINES DERIVED FROM PORCINE EMBRYONIC FIBROBLASTS
Authors: Park J.-K. , Choi K.-H. , Son D.-C. , Oh J.-I. , Lee C.-K.
Published: December 2012

A recent study has reported that pluripotent stem cells can be categorized according to their pluripotent state. The first is the “naïve” state, which is characterised by small, round or dome-shaped colony morphologies, LIF and BMP4 signalling pathways, and 2 active X chromosomes in females; mouse embryonic stem cells (mESC) represent this type. A second “primed” state has also been described and is possible in mouse epiblast stem cells (mEpiSC) or human embryonic stem cells (hESC). These primed state pluripotent stem cells display flattened monolayer colony morphologies, FGF and nodal/activin signalling pathways, and X chromosome inactivation in females. Meanwhile, a few studies have reported that primed pluripotent stem cell lines could be reverted to a naïve pluripotent state using various exogenous factors including GSK3β and MEK inhibitors (2i), LIF, hypoxic conditions, and upregulation of Oct3 or klf4. Therefore, the purpose of this study was to investigate whether a LIF-dependent naïve pluripotent stem cell line could be derived from porcine embryonic fibroblasts (PEF) via various previously reported factors. We were able to successfully induce PEF into a naïve state-like pluripotent stem cell line by viral infection using FUW-tetO-hOCT4, FUW-tetO-hSOX2, FUW-tetO-hKlf4, FUW-tetO-hMYC, and FUW-M2rtTA obtained from Addgene and addition of 2i and LIF. These naive state-like pluripotent stem cells display mESC-like morphologies, clonogenicity by trypsin, and expression of Oct4, Sox2, Nanog, and SSEA1 using PCR, immunocytochemistry, and fluorescence-activated cell sorting. All cell lines maintained stemness characteristics and stable morphology for more than 30 passages. In addition, naïve state-like pluripotent stem cells could be induced to differentiate to fibroblast-like cells by withdrawal of doxycycline, lif, and 2i. These differentiated cells could be regenerated into naïve state-like pluripotent stem cells by addition of doxycycline, lif, and 2i. We suggest that, as a nonpermissive species, the porcine species undergoes reprogramming into a primed state during the establishment of pluripotent stem cell lines and needs various exogenous factors, including continuous transgene expression, GSK3β and MEK inhibitors (2i), and LIF to be induced into naïve state-like pluripotent stem cells.
This work was supported by the BioGreen 21 Program (PJ0081382011), Rural Development Administration, Republic of Korea.
309 PROTEOMIC ANALYSIS OF THE BLASTOCOEL FLUID AND REMAINING CELLS OF BOVINE BLASTOCYSTS
Authors: Jensen P. L. , Groendahl M. L. , Beck H. C. , Petersen J. , Stroebech L. , Christensen S. T. , Soerensen P. D. , Andersen C. Y.
Published: December 2012

Human embryonic stem cells (hESC) are derived from the human blastocyst and possess the potential to differentiate into any cell type present in the adult human body. Human ESC are considered to have great potential in regenerative medicine for the future treatment of severe diseases and conditions such as Parkinson’s disease, diabetes, and spinal cord injury. One of today’s challenges in regenerative medicine is to define proper culture conditions for hESC. The natural milieu in the blastocyst may provide clues on how to improve culture conditions, and the aim of the present study was to determine the proteome of the blastocoel fluid and the remaining cells of bovine blastocysts. Bovine blastocysts were produced by in vitro fertilization of oocytes retrieved from slaughterhouse ovaries. The blastocoel from 195 blastocysts (1–8 nL per blastocyst) were isolated by micromanipulation and analysed by nano-HPLC tandem mass spectrometry along with the remaining cells of the blastocyst. Searching the mass spectrometry data against a combined bovine database (SwissProt/TrEMBL), we identified 263 proteins in the blastocoel fluid and 1606 proteins in the cellular compartment of the blastocyst. A Venn diagram showed 124 proteins in overlap between the two compartments of the blastocyst. Several heat shock proteins and specific antioxidants were identified in both the blastocoel and cell material. A selection of proteins identified in the blastocoel fluid is to be tested on hESC in cell culture experiments, with proliferation of undifferentiated cells as the primary endpoint. The results from this study provide new knowledge about early mammalian preimplantation development, and the data can be used in the continued pursue of improving culture conditions for hESC, which further facilitates the clinical application of these cells.
318 EFFECT OF BREED AND FOLLICULAR STATUS ON RESPONSE TO SUPEROVULATION IN SOUTH AFRICAN GOATS
Authors: Lehloenya K. C. , Mpebe N. A. , Gonzalez-Bulnes A.
Published: December 2012

This study evaluated the effect of breed and follicular status on quantity and quality of embryos recovered following superovulation in South African goats. Mature, nonlactating Boer (n = 10) and unimproved indigenous (n = 10) goats were used in this trial. Oestrous cycles of does were synchronised with controlled internal drug release (CIDR®; Pfizer Animal Health, New York, NY, USA) dispensers inserted for 9 days and injected with prostaglandin at CIDR insertion. All does were superovulated with pFSH, administered in 7 dosages, at 12 h intervals, starting from 48 h before CIDR removal. Ultrasonographic scans were performed at initiation of the superovulation treatment. Does were cervically inseminated with fresh undiluted semen at 36 and 48 h following CIDR withdrawal. Embryos were surgically collected at Day 6 following AI. The total number of recovered structures (unfertilised ova and embryos) was evaluated microscopically for the stage of development and quality, using morphological criteria. The structures were classified as unfertilised ova (if there was no cleavage), degenerate embryos (embryos at 8-cell stage and earlier stage), or as transferable grade 1, 2, and 3 embryos (morphologically intact compacted morulae, early blastocysts, and expanded blastocysts). All data were analysed using the ANOVA procedures of SAS (SAS Institute Inc., Cary, NC, USA). The time from CIDR removal to onset of oestrus did not differ significantly between breeds. Breed had no effect on the total number of corpora lutea, structures (unfertilised ova and embryos), unfertilised ova, embryos, degenerated embryos, and transferable embryos. The total number of follicles at the onset of pFSH treatment was positively correlated with the number of structures (unfertilised ova plus embryos) recovered. Similarly, the total number of embryos recovered was positively correlated with the number of follicles 2 to 3 mm and 4 to 5 mm, and the total number of follicles. The number of follicles 4 to 5 mm and the total number of follicles at the onset of pFSH treatment were positively correlated with total number of the transferrable embryos at the beginning of a superovulation treatment. The total number of transferable embryos was negatively correlated with the presence of follicles with a diameter >6 mm at the beginning of the superovulation treatment. In conclusion, breed had no effect on response to superovulation. The total number of follicles and follicle diameters 2 to 3 mm and 4 to 5 mm were positively correlated with embryo yield. The presence of follicles >6 mm at the beginning of superovulation negatively affected the quality of embryos. The results of this study indicate that breed has no effect on response to superovulation. However, the number and size of follicles at the beginning of a superovulation treatment affect both embryo yield and quality.
32 CLONING OF ELITE QUARANTINE SNIFFING DOG BY SOMATIC CELL NUCLEAR TRANSFER
Authors: Oh H. J. , Kim M. J. , Kim G. A. , Choi J. , Park E. J. , Jo Y. K. , Park J. E. , Lee J. , Kang G. , Park Y. H. , Lee B. C.
Published: December 2012

Somatic cell nuclear transfer (SCNT) in assisted reproductive technologies has been considered for the conservation of valuable or endangered animals. Dogs that were originally bred for hunting, such as beagles, have an exceptional ability to detect a particular smell from many others. For that reason, the beagles have been used to detect quarantine risk items from a wide range of goods in assorted luggage without scaring or disrupting the passengers. Though very useful and highly in need, elite quarantine sniffing beagles with excellent abilities are rare; much time, effort, and money are required in producing them. Here, we have applied SCNT for propagation of elite quarantine sniffing dogs to save time and economic burden. Ear fibroblasts from a 10-year-old adult male elite quarantine sniffing beagle were isolated and cultured in vitro as donor cells. For SCNT, in vivo-matured oocytes, obtained by flushing the uterine tubes of oocyte donors (mixed breed), were used. The oocytes were enucleated, microinjected with donor cells, fused by electrical stimulation, and activated chemically. Reconstructed oocytes were surgically transferred into the uterine tube of naturally synchronous recipient females. A total of 212 activated cloned embryos were transferred into 12 female recipient dogs and 4 recipients became pregnant. The 4 pregnant recipients delivered 4 pups through caesarean section or natural delivery, but 1 died right after birth and did not show an abnormality. Other live puppies exhibited normal phenotypes; their appearance was similar to that of the donor dog. All cloned pups were genetically identical to the donor dog and their mitochondrial DNA was from their oocyte donor dogs. When the cloned pups were 16 weeks old, we conducted a Volhard test, which is commonly used to describe the following puppy aptitudes: social attraction, following, restraint, social dominance, elevation dominance, retrieving, touch sensitivity, sound sensitivity, and sight sensitivity. Dog behavior data on differences in transcript abundance were analyzed by a general linear mixed model. The 3 cloned pups showed similar behavioral tendencies. The present study demonstrates that NT technique using donor cell derived from 1 elite quarantine sniffing dog is useful to produce a large number of quarantine sniffing dogs.
This study was supported by RDA (no. PJ0089752012), RNL Bio (no. 550-20120006), IPET (no. 311062-04-1-SB010), Research Institute for Veterinary Science, Nestlé Purina Korea, and TS Corporation.
TRANSGENIC ANIMALS IN AGRICULTURE
Authors: Murray James D.
Published: December 2012

The first reports on the effects of human growth hormone in transgenic mice, engendered speculation about how genetic engineering could be applied to improve livestock for use in agriculture. However, 30 years later this promise has yet to be realised as there are no GE animals approved for food consumption anywhere in the world. Initial work mostly either concentrated on the production of animals (sheep and swine) with deregulated growth hormone (GH) genes or represented work to improve our understanding of gene constructs for the control of gene expression. Although a variety of GH transgenic lines of livestock were successfully produced, most had compromised health and were not suitable for further development. However, several lines of transgenic farm animals and fish could be potentially useful genotypes for use in production agriculture, including GH pigs, phytase pigs, lysozyme goats, lactoferrin cows, and lactalbumin pigs. To date, none of these lines have yet been approved for human consumption and thus have not made it into production. Many of these lines have been placed in frozen storage or have been lost and would need to be redeveloped. The reasons for this are varied and include simply retirement of the developer, through to insufficient funds, pressure from anti-biotechnology activists, lack of a regulatory process, public opposition, or lack of a perceived need by industry. The combination of these factors has led to a virtual cessation of research and development of transgenic animals for use in agriculture in North America, Europe, and Australia, early leaders in developing this technology, and the emergence of programs in places such as Brazil and China, where the next generation of GE animals for agriculture will be developed. However, recent advances in the methodology to produce transgenic animals, such as the use of transposons or site-directed nucleases such as TALENs, may lead to renewed efforts to develop targeted GE animals for use in agriculture.
WHY DO WE NEED TRANSGENIC ANIMALS?
Authors: Roberts R. Michael
Published: December 2012

Genetic modification of animals through cross breeding and selection has probably been practiced since wild animals were first domesticated, but became more intensive beginning ~200 years ago with the creation of defined breeds with specialised phenotypes. This generality applies both to agriculturally important species, such as chickens and cattle, and to companion animals, such as cats and dogs. It also applies to plants. In the 20th century, new technologies designed to improve agricultural productivity, perhaps best illustrated by the development of artificial insemination and sperm cryopreservation for the dairy industry, as well as improved nutrition and veterinary care, all fueled by generous government support of agricultural research, accelerated progress towards providing low-cost, safe, and nutritious food for a burgeoning world population. Progress in some cases has been quite remarkable, especially in milk and meat production. The report by Palmiter, Brinster and colleagues in 1982, that mice harboring a transgene for growth hormone under the control of a metallothionein gene promoter grew faster than control siblings, raised the spectre of using relatively simple, one-step gene insertions, rather than cross-breeding and selection, to improve the production of milk, meat, and fibre, and enhance animal health quickly and directly. Although agronomy and horticulture have been able to benefit greatly from applying genetic modification to plants, there has been only very slow application of transgenesis to animal agriculture and aquaculture. The main goal of my talk will be to illustrate some of the difficulties, both scientific and social, that have been encountered in bringing genetic modification and related technologies, such as cloning, into mainstream agriculture. I shall also discuss the competitive advantage held by rodents over alternative animal models in biomedical sciences but argue that the latter still have an important role to play in this regard. A final topic will be to review the potential value of pluripotent stem cells for introducing genetic change in domestic species and for testing the efficacy and safety of grafts derived from pluripotent stem cells before human clinical trials.
Erratum to: 4 INFLUENCE OF A MATERNAL DIABETES MELLITUS TYPE 1 ON LIPID AND CHOLESTEROL METABOLISM IN RABBIT PRE-IMPLANTATION EMBRYOS
Authors: Schindler M. , Pendzialek M. , Plösch T. , Knelangen J. M. , Gürke J. , Hauke E. , Fischer B. , Navarrete Santos A.
Published: December 2012

The incidence of overweight and obesity has reached epidemic levels worldwide. Even more alarming is the increasing prevalence of metabolic diseases in younger children and adolescents. The rate of women with diabetes mellitus in child-bearing age is rising, too. According to the developmental origins of health and disease (DOHaD) paradigm, exposure to a hyperglycaemic environment in utero may programme physiology and metabolism permanently, with long-term consequences for offspring health. Experimental evidence indicates that programming of obesity does occur during early embryo development, a period where many women are unaware of pregnancy. To study effects of maternal diabetes mellitus on early embryo development, we induced a type I diabetes through alloxan treatment of female rabbits. In diabetic rabbits, the triglyceride and cholesterol concentrations were altered in serum and the cholesterol concentration in the uterine secretions was elevated. Lipid content of 6-day-old blastocysts was analysed after Oil Red staining and whole mount histochemistry or with Nile Red by fluorescence-activated cell sorting (FACS). Analysis by FACS revealed an approximately 2-fold increase in lipid droplets in blastocysts grown under diabetic conditions. The expression of genes important for lipid metabolism, such as fatty acid transport protein 4 (FATP4), fatty acid-binding protein 4 (FABP4), carnitine palmitoyltransferase 1 (CPT-1), and lipoprotein lipase (LPL), were determined by real-time PCR and showed distinct differences between diabetic and control blastocysts. Immunohistochemical staining of FABP4 was clearly increased in blastocysts grown under diabetic conditions and showed a cell lineage-specific distribution. Two transcription factors, peroxisome proliferator-activated receptor α (PPARα) and PPARγ, with key functions in lipid metabolism and adipogenic differentiation, were increased in blastocysts from diabetic rabbits. We show that maternal diabetes mellitus leads to alteration in lipid metabolism and to triglyceride accumulation in blastocysts. Its long-lasting consequences (e.g. for adipose cell differentiation) need attention and further investigation.
41 RAPID PRODUCTION OF α-1,3-GALACTOSYLTRANSFERASE-DEFICIENT MINIATURE PIGS BY NUCLEAR TRANSFER FOLLOWING NEONATAL SKIN BIOPSY AND LOSS OF HETEROZYGOSITY
Authors: Kim Y. J. , Kim M. J. , Kim M. J. , Ahn J. S. , Ryu J. H. , Heo S. Y. , Park S. M. , Kang J. H. , Choi Y. J. , Ahn K. S. , Shim H.
Published: December 2012

Production of α-1,3-galactosyltransferase (αGT)-deficient pigs is a fundamental task to overcome hyperacute rejection in pig-to-human xenotransplantation. However, the procedure to produce such pigs requires a great deal of cost, time, and labor. Heterozygote αGT knockout (KO) pigs should be bred at least for 2 generations to ultimately produce homozygote progenies. Here, we show that αGT-deficient miniature pigs could be produced in much reduced time using mitotic recombination in neonatal ear skin fibroblasts. Miniature pig fetal fibroblasts were transfected with αGT-targeting vector and treated with neomycin. Eighty-seven colonies were screened by PCR, and 2 among them were identified as homologous recombinants with monoallelic disruption of αGT (targeting efficiency = 2.3%). One of the two colonies was chosen and used for subsequent NT. In total, 559 nuclear transfer embryos reconstructed with αGT gene-targeted fetal fibroblasts were transferred to 5 surrogate gilts. Three gilts became pregnant and developed to term with 3 live piglets delivered. Being analyzed by PCR, all piglets born were found to be αGT gene targeted. Fibroblasts were isolated from ear skin biopsies of a 10-day-old αGT gene-targeted miniature pig. To increase the number of mitotic recombination that occurs spontaneously during cell proliferation, fibroblasts were cultured for 6 to 8 passages. Then, these cells were treated with IB4-conjugated Dynabeads; IB4 lectin binds to galactose-α-1,3-galactose (αGal), the antigen formed by αGT, and is responsible for hyperacute rejection (HAR). Using a magnetic column, fibroblasts bound to IB4 lectin were removed. Remaining cells lacking αGal were cultured further until colony formation. A total of 22 fibroblast colonies were screened by PCR, and 15 among them were confirmed as loss of heterozygosity (LOH) with biallelic disruption of αGT by mitotic recombination (LOH efficiency = 68%). One of these clonal cultures of αGT-deficient fibroblasts was chosen and used for subsequent NT. In total, 729 nuclear transfer embryos reconstructed with αGT-deficient neonatal ear skin fibroblasts were transferred to 4 surrogate gilts. Two gilts became pregnant, and 3 live piglets were produced. Based on PCR, both αGT alleles were targeted in all piglets born. Only 8 months were required from the production of heterozygote to homozygote KO in contrast to approximately 2 years consumed for conventional breeding of heterozygotes. The present study demonstrates that the time required for the production of αGT-deficient miniature pigs could be reduced significantly by neonatal skin biopsies and subsequent selection of mitotic recombinants. Such a procedure may be beneficial for the production of other homozygote KO animals, especially in species that require substantial periods of gestation such as pigs.
43 PATTERN OF NUCLEOLI FORMATION IN RACCOON-PORCINE INTERSPECIES SOMATIC CELL NUCLEAR TRANSFER EMBRYOS
Authors: Nam Y. H. , Jeon Y. , Cheong S. A. , Kwak S. S. , Hyun S. H.
Published: December 2012

Recently, great focus has been on the rescue of endangered animals through somatic cell nuclear transfer (SCNT). Because it is difficult to obtain the oocytes of endangered species, interspecies SCNT (iSCNT) methods have been attempted. Numerous iSCNT embryos have shown unsuccessful development due to aberrations in expression of housekeeping genes and genes dependent on the major embryonic genome activation (EGA). In particular, aberrant EGA may cause the arrest of nucleoli formation and developmental block in embryos. According to this concept, we performed raccoon iSCNT using porcine oocytes and analyzed iSCNT embryo development pattern and formation of nucleoli. Enucleated porcine oocytes were fused with raccoon fibroblasts by electrofusion. Cleavage and blastocyst formation were evaluated under a stereomicroscope at 48 and 168 h post-activation (hpa), respectively. To confirm the formation of nucleoli, which can be detected by C23 antibody labeling in many mammalian species, C23 immunocytochemistry was performed at 48 and 72 hpa. A total of 158 iSCNT embryos were cultured; 68.5% of the raccoon iSCNT embryos were cleaved at 48 hpa (1-cell stage: 9.7%; 2-cell stage: 14.4%; 4-cell stage: 34.1%; 6-cell stage: 12.7%; 8-cell stage: 7.3%; fragmented: 21.8%). But, the embryos seen as 5- to 8-cell stage did not have the same number of nuclei as their blastomere number. When raccoon iSCNT embryos were stained by Hoechst 33342, 5- to 8-blastomere raccoon iSCNT embryos had only 4 nuclei. The raccoon iSCNT embryos did not develop past the 4-cell stage and failed to form blastocysts. In the control group, 65.2% of pig SCNT embryos were cleaved at 48 hpa (1-cell stage: 8.0%; 2-cell stage: 4.2%; 4-cell stage: 23.6%; 6-cell stage: 13.6%; 8-cell stage: 23.8%; fragmented: 26.8%), and 10.0% of pig SCNT embryos developed to blastocysts. In raccoon iSCNT embryos, raccoon nuclei failed to form nucleoli at 48 and 72 hpa. By contrast, pig SCNT embryos showed 18.8 and 87.9% nucleoli formation at 48 and 72 hpa. Our results demonstrate that 4-cell-stage embryos of raccoon-porcine hybrid embryos may be produced by SCNT methods. The pig oocytes partly supported the remodeling and reprogramming of the raccoon somatic cell nuclei, but they were unable to support nucleoli formation. Moreover, aberrant nucleoli formation caused the unsuccessful development of raccoon SCNT embryos to the blastocyst stage.
This work was supported by a grant from the Next Generation BioGreen 21 program (no. PJ008121012011), Rural Development Administration, Republic of Korea.
Erratum to: 56 SINGLE LAYER CENTRIFUGATION THROUGH PURESPERM® 80 IMPROVES QUALITY OF CRYOPRESERVED DOG SPERMATOZOA
Authors: Alcaráz L. , Hidalgo M. , Galvez M. J. , Acha D. , Ortiz I. , Demyda-Peyrás S. , Gonzales C. , Portero J. , Quesada F. , Ramirez L. , Dorado J.
Published: December 2012

Density gradient centrifugation with PureSperm® (PureSperm® 40 + PureSperm® 80; Nidacon International, Mölndal, Sweden) has been satisfactorily used to enhance the quality of dog semen samples; however, no studies have been performed on the effect of single layer centrifugation (SLC) with PureSperm® on frozen–thawed dog semen. The aim of this study was to investigate if SLC with PureSperm® 80 can improve the post-thaw semen quality of dog. Semen from 5 dogs was collected by digital manipulation. Two ejaculates from each dog were centrifuged with Tris-based extender, supernatant was removed, and sperm pellet was suspended to a final concentration of 300–400 × 106 sperm mL–1 with CaniPROTM Freeze A plus 20% egg yolk at 22°C. Extended semen was cooled to 5°C within an hour and then diluted to a final concentration of 150–200 × 106 sperm mL–1 in CaniPROTM Freeze B plus 20% egg yolk at 5°C, loaded in 0.5-mL plastic straws and frozen horizontally in ranks placed 4 cm above the surface of liquid nitrogen vapors for 10 min, after which they were directly placed in liquid nitrogen. After 24 to 48 h of storage, straws were thawed in a water bath at 37°C for 30 s. After thawing, semen samples were divided in 2 aliquots: one of them was used as control and the other one was processed by SLC PureSperm® 80. Assessment of sperm motility (assessed by computerized-assisted semen analysis), morphology (Diff-Quick staining), and viability [triple fluorescent stain of propidium iodine/isothiocyanate-labeled peanut (Arachis hypogaea) agglutinin/Rhodamine 123] were evaluated in control and treated semen samples. Data were studied by ANOVA. Results are expressed as mean ± SEM. Significant (P < 0.001) differences were found between SLC-treated and control semen for sperm motility (percentage of total motile spermatozoa: 93.65 ± 0.05 v. 83.79 ± 0.13; percentage of progressive motile spermatozoa: 79.38 ± 6.66 v. 54.61 ± 16.11), morphology (86.45 ± 0.01 v. 83.51 ± 0.01), and viability (percentage of viable sperm with an intact acrosome: 58.32 ± 0.04 v. 36.50 ± 0.17; percentage of viable sperm with an acrosome reaction: 2.81 ± 0.01 v. 9.74 ± 0.21). Based on our results, we can conclude that SLC with PureSperm® 80 is an alternative and successful method for improving the quality of frozen–thawed dog spermatozoa, selecting good-quality spermatozoa (motile, morphologically normal, viable, and acrosome intact spermatozoa) from the rest of the semen sample.
59 EFFECT OF STORAGE DURATION ON POST-THAW PARAMETERS OF BULL SEMEN
Authors: Carwell D. B. , Scott B. R. , Len J. , Blackburn H. , Bondioli K. R. , Gentry G. T. , Godke R. A.
Published: December 2012

It has been proposed that once good-quality sperm are collected, extended, and cryopreserved that the post-thaw quality will remain high regardless of the duration of storage. A previous study has suggested that there is no effect on post-thaw sperm motility of bovine semen stored in liquid nitrogen up to 13 years. However, no studies have reported the effect of extended storage of bovine semen in LN2. In this study, cryopreserved semen from Angus bulls (n = 25) was utilized. Time frames were assigned to each bull based on the year of collection and cryopreservation: time frame 1 (1960–1975), time frame 2 (1976–1991), and time frame 3 (1992–2006). Due to differences in packaging methods across time periods (glass ampules and plastic straws), thawing methods utilized were different and based on the packaging type. Semen packaged in glass ampules (n = 7) were thawed in a 37°C water bath for 80 s, while those in plastic straws (n = 18) were thawed in a 37°C water bath for 30 s. Once thawed all packages were wiped dry and either scribed (glass ampule) or the end cut (plastic straw) and the semen expelled into a 1.5-mL microcentrifuge tube and maintained at 37°C for evaluation. For sperm motility, a 20-µL sample was placed onto a prewarmed microscope slide for analysis. Sperm motility parameters (total motility and progressive motility) were evaluated by 2 experienced technicians and averaged. A hemocytometer was used to determine sperm concentration in a final dilution of 1 : 100. An eosin/nigrosin stain was used to determine sperm morphology. Parameters were analyzed using one-way ANOVA. There was no difference in sperm concentration between time frames 1 and 2 (53 × 106 mL–1 and 59 × 106 mL–1, respectively) or time frames 1 and 3 (53 × 106 mL–1 and 37 × 106 mL–1, respectively). However, time frame 2 exhibited a higher (P < 0.05) sperm concentration compared with time frame 3 (59 × 106 mL–1 v. 37 × 106 mL–1, respectively). There were no differences among time frames 1, 2, or 3 for total (42, 51, and 55%, respectively) and progressive sperm motility (29, 38, and 41%, respectively). Similarly, there were no differences among time frames 1, 2, and 3 for percent normal sperm (80 ± 3.6, 76 ± 5.0, and 71 ± 4.0%, respectively) and abnormal sperm (19 ± 3.6, 23 ± 5.0, and 28 ± 4.0%, respectively). Furthermore, time frames 1, 2, and 3 did not exhibit a difference in primary (9 ± 2.5, 7 ± 2.6, and 6 ± 1.0%, respectively), secondary (2 ± 0.8, 5 ± 1.4, 6 ± 2.0%, respectively), or tertiary abnormalities (7 ± 2.4, 10 ± 2.7, and 16 ± 2.7%, respectively). These data suggest that once good-quality bovine sperm is cryopreserved, the duration of storage (up to 43 years) has no effect on Angus bull post-thaw semen parameters.
62 LONG-TERM STORAGE OF BOVINE EMBRYOS: BIRTH OF A CALF AFTER 22 YEARS OF CRYOPRESERVATION
Authors: Detterer J. , Gehring M. , Maimer E. , Meinecke-Tillmann S.
Published: December 2012

After the pioneering work of Rostand (1946 CR Acad. Sci. III 222, 1524–1525) and Polge et al. (1949 Nature 164, 666–669) on semen cryopreservation and the reports on the first live young born after storage of mouse embryos for up to 8 days (Whittingham et al. 1972 Science 178, 411–414) or 8 months (Whittingham and Whitten 1974 J. Reprod. Fertil. 36, 433–435) in liquid nitrogen, the world’s first calf was obtained in Cambridge nearly 40 years ago, after freezing and thawing of a bovine embryo (Wilmut and Rowson 1973 Vet. Rec. 92, 686–690). Since then, especially in humans (case reports), the birth of healthy offspring after long-term storage of semen, zygotes, and embryos has been repeatedly reported (e.g. Dowling-Lacey et al. 2011 Fertil. Steril. 95, 1120.e1–3). But practical experiences in different other species such as mice or cattle show contradictory results on the fertility after transfer of long-term-stored gametes or embryos. Here, we report the birth of normal Holstein calves after long-term embryo preservation. After storage for 15 to 22 years, 4 embryos were thawed (air-thaw for 10 s and 25°C water bath for 20 s), which had been originally frozen in 10% glycerol (3 embryos) or in 1.5 M ethylenglycol (1 embryo) following a slow freezing protocol. Removal of glycerol was accomplished with a 4-step dilution procedure and 10.3% sucrose (EMCARETM Thawing System, ICPbio Reproduction, Glenfield, New Zealand), whereas ethylenglycol was removed with a washing step in EMCARETM (Holding Solution, ICPbio Reproduction). The embryos were transferred into the uterine horn ipsilateral to the corpus luteum of recipient cows at 7 days post-oestrus. One embryo was classified as expanded blastocyst grade 1 (7-1), 2 embryos were classified as morulae grade 1 (4-1), and the embryo frozen with ethyleneglycol was classified as a morula grade 2 (4-2) (IETS classification; 40× magnification). Three pregnancies were established (1 × 7-1; 1 × 4-1, and 1 × 4-2), and 3 healthy bull calves were born (18.1.2011, stored 15 years; 6.3.2012, stored 22 years; and 29.3.2012, stored 15 years). These case reports document that long-term-stored bovine embryos maintained developmental competence for up to 22 years of cryopreservation in liquid nitrogen. To our knowledge these cases represent the “oldest” cryopreserved bovine embryos resulting in healthy calves in Germany.
68 WHOLE-OVARY CRYOPRESERVATION: A DIRECT COMPARISON OF CONVENTIONAL AND DIRECTIONAL FREEZING
Authors: Maffei S. , Silva J. R. V. , Hanenberg M. , Pennarossa G. , Brevini T. A. L. , Arav A. , Gandolfi F.
Published: December 2012

Whole-ovary cryopreservation followed by retransplantation with vascular anastomosis is a promising method to restore cancer-patient fertility. However, protocols need to be improved to minimize the damages related to an inadequate cooling rate caused by the large volume of whole organs. The aim of this study was to compare conventional slow-freezing (SF) versus directional-freezing (DF) techniques. Of 30 sheep ovaries collected at the local abattoir, 6 were used as fresh controls and 24 were perfused for 5 min with cryoprotectant solution made of Leibovitz L-15 medium, 10% FCS, and 1.5-M dimethyl sulfoxide. Samples were inserted in glass freezing tubes filled with the same solution. Twelve ovaries were frozen with a Kryo 560M apparatus (Planer, UK) using a cooling rate of 0.5°C min–1 from 4 to –40°C and 5°C min–1 from –40 to –100°C followed by direct plunging in liquid nitrogen. The other 12 ovaries were frozen using an MTG 1315 apparatus (Core Dynamics, Ness Ziona, Israel). Tubes were pushed along the thermal gradient (4 to –70°C) at 0.01 mm s–1 resulting in a cooling rate of 0.3°C min–1 and then plunged in liquid nitrogen. After 2 weeks, samples were thawed at 37°C for 2 min with gentle shaking. Morphological analysis showed that the rate of intact follicles was 94% in control samples, 87% in DF samples, and 58.3% in SF samples (P < 0.05). No significant differences were found in stromal cell density between fresh and DF ovaries (17 833 and 17 041 nuclei mm–2, respectively), whereas SF ovaries showed a lower density (10 875 nuclei mm–2; P < 0.001). To assess vessel integrity, thawed ovaries were perfused with 25% Indian ink/167 UI of heparin mL–1 saline solution and embedded in paraffin. Fresh and DF ovaries displayed a similar rate of perfused vessels (82.7 and 80.2%, respectively), whereas SF ovaries significantly lower number of perfused vessels (52.3%), indicating the occurrence of vascular injury. Follicle viability after freezing was evaluated by culturing individual cortical fragments (2 × 2 × 1 mm) for 7 days. A high rate of morphologically normal follicles was found in both fresh (85%) and DF ovaries (78%). The number of intermediate and primary follicles was significantly increased in both groups when compared to Day 0 (P < 0.001). By contrast, cortical slices obtained from SF ovaries showed a higher rate of degenerated follicles, oocytes with pyknotic nuclei, and a significant decrease of follicle number. Furthermore, qPCR and immunohistochemical assay for Ki-67 showed a similar cell proliferation in fresh and DF ovary but a significantly lower one in SF samples. We conclude that directional freezing allows a better preservation of both cortical and stromal tissue, as well as the vasculature, thus overcoming 2 of the main restrictions associated with whole-organ freezing.
Supported by AIRC IG 10376 and by Carraresi Foundation.
74 GERM CELLS AND TESTICULAR SOMATIC CELLS HAVE DIFFERENT SENSITIVITY TO CRYOPRESERVATION
Authors: Robbins H. , Dores C. , Coyle K. , Dobrinski I.
Published: December 2012

Spermatogonial stem cells (SSC) are the foundation of spermatogenesis. Undifferentiated spermatogonia, containing SSC, represent only 2 to 5% of cells recovered from immature mammalian testis. Cryopreservation in liquid nitrogen allows for long-term storage of cells. Preservation of germ cells can serve as a means of genetic preservation from immature males when sperm storage is not an option. Studies have investigated the effects of cryopreservation on the spermatogenic potential of SSC and the efficiency of various cryopreservation protocols. Preliminary observations indicated that germ cells may survive cryopreservation better than testicular somatic cells, resulting in a post-thaw cell population enriched in germ cells. However, this has not been critically evaluated. The objective of this study was to test the hypothesis that germ cells are less susceptible to cryo-damage than testicular somatic cells. Cells were harvested from the testes of 1-wk-old piglets by 2-step enzymatic digestion. The initial cell suspension was subjected to differential adhesion to enrich the cell population for germ cells. Cells were plated in DMEM + 5% fetal bovine serum and incubated at 37°C in 5% CO2 in air. After 18 h, cells in suspension and cells slightly attached were recovered by trypsinization (1 : 10 trypsin-ethylenediaminetetraacetic acid) for 30 s and replated. This was repeated 24 and 36 h after initial plating. The enriched population was placed into cryovials at a concentration of 30 × 106 cells in freezing media (70% DMEM + 20% fetal bovine serum + 10% dimethyl sulfoxide), kept for 24 h at –80°C in a cryogenic freezing container and transferred to liquid nitrogen for 1 week. Aliquots of cells before freezing and after thawing at 37°C followed by incubation at 37°C in 5% CO2 in air for 1 h were analyzed for viability by propidium iodide (PI) exclusion and immunofluorescence for the germ cell marker VASA to identify viable germ cells (VASA+/PI–), nonviable germ cells (VASA+/PI+), viable somatic cells (VASA–/PI–), and nonviable somatic cells (VASA–/PI+). The percentage of viable germ cells after freezing and thawing was compared to the percentage of viable somatic cells by ANOVA. After enrichment by differential plating, the cell population had 95.6 ± 0.9% viability and contained 27.1 ± 7.4% germ cells (n = 3 replicates). After cryopreservation, the overall cell viability was 77.5 ± 1.6%, and 25.8 ± 8.0% were germ cells. The overall viability after cryopreservation could potentially have benefited from the 1-h incubation prior to analysis. The viability of the germ cell population after freezing and thawing was higher (92.1 ± 3.1%) than somatic cell viability (72.3 ± 1.7%; P < 0.01). These results indicate that porcine germ cells survive cryopreservation better than do testicular somatic cells. Therefore, cryostorage of germ cells can be an efficient means for preservation of male genetic material.
Supported by NIH ORIP/DCM RR17359.
76 THE EFFECT OF GLYCEROL CONCENTRATIONS IN EGG-YOLK CITRATE EXTENDER ON THE QUALITY OF CRYOPRESERVED NGUNI BULL SEMEN
Authors: Seshoka M. M. , Mphaphathi M. L. , Ramukhithi F. V. , Netshirovha T. R. , Hlungwani C. , Nedambale T. L.
Published: December 2012

There are bull shortages in South African poor rural areas. Artificial-insemination technology could play a significant role on breeding emerging farmer’s cattle. The objective of this study was to compare glycerol concentrations (0, 4, 8, or 12%) during freezing of Nguni bull semen to conduct AI in different villages. Semen was collected by electro ejaculator from 2 Nguni bulls of known and proven fertility. Collected semen samples were kept in a thermo flask (37°C) and transported to the laboratories within 10 min after collections. Semen samples were pooled and evaluated by Sperm Class Analyser® and allocated randomly per treatment. Semen was then diluted (1 : 2 v:v) with egg-yolk citrate extender supplemented with either 0% (negative control), 4, 8, and 12% of glycerol concentration or AndroMed® (positive control). Semen samples were equilibrated for 4 h at 5°C. After equilibration period, samples were loaded into 0.5-mL straws and placed horizontally into the controlled rate (–5, –8, –10, –12, –15, –25, –35°C min–1) from 5°C until target temperature of –80°C is reached. The frozen semen straws were stored in a liquid nitrogen tank (–196°C) until thawing. Treatment means were separated using Fisher’s protected t-test least, and data are presented as mean ± SD. There was a significant differences (P < 0.05) between raw total sperm motility (83.3 ± 19.3) and frozen–thawed sperm with either 0% (0.0 ± 0.0), 4% (30.2 ± 13.4), 8% (47.9 ± 12.5), or 12% (61.5 ± 4.7) of glycerol and on AndroMed® (27.7 ± 17.8) group. Regardless of the glycerol concentrations used, the freezing-thawing process reduced (P < 0.05) the Nguni total sperm motility rate compared to uncryopreserved sperm (83.3 ± 19.3). In conclusion, egg-yolk citrate extender supplemented with 12% glycerol yielded a better (P < 0.05) total sperm motility rate (61.5 ± 4.7) as compared with the 0% (0.0 ± 0.0), 4% (30.2 ± 13.4), 8% (47.9 ± 12.5), and AndroMed® (27.7 ± 17.8) group. Further studies are required to test other levels of glycerol concentrations (>12%) on freezing Nguni semen and conducting AI.