bluuebunny/crossref_metadata_2025 huggingface dataset:
288 DEVELOPMENT OF AN EXPERIMENTAL MODEL FOR ANOSMIA FOR CELL THERAPY PURPOSES
Authors: Carvalho R. C. , Rodrigues M. N. , Fonseca E. T. , Abreu-Silva A. L. , Miglino M. A.
Published: December 2012

The olfactory sense is most important for social and environmental interaction, contributing to the quality of life. Its dysfunction, called anosmia, is followed by severe social problems. Anatomically, this is associated with changes in the nasal epithelium and the central and peripheral nervous system. Anosmia can be caused by infection, trauma, and cancer. Along with other olfactory disorders, anosmia occurs in ~50% of the population between 65 and 80 years of age and 75% of the population above 80 years of age. Because of the dramatic effect of such diseases, we aim to develop an experimental model of anosmia in rats as well as to culture stem cells that may be used for future cell therapy purposes. We used 15 adult Wistar rats (60 days). As a first step of our project, the animals were submitted to an experimental surgery to ablate the olfactory bulb (OB). The ocular bulb was used as reference to localizate transfrontal craniotomy. The surgical time for this procedure ranged from 40 to 50 min. The olfactory bulb has an elliptical shape and consists of soft tissue, almost gelatinous, which breaks up easily when manipulated or restrained by surgical instruments. Therefore, we chose to aspirate the tissue, using a urethral probe coupled to an aspirator. Removal of the OB resulted in bilateral cavities, elongated, situated between the cribriform plate of the ethmoid bone and brain. The postoperative period passed without major incidents. The skin suture was removed between 7 and 10 days after surgery. The animals were clinically evaluated each day for a period of 60 days. No animal showed signs of pain or discomfort, apathy, loss of appetite, or hypersensitivity in the surgical wound. However, the consumption of food and water was increased. Some animals lost their feeding selectivity, eating pieces of paper, shavings, and surgical drapes used to line the cages. In addition, we observed behavioural changes such as excitability and aggressiveness. Tests of revulsion at the smell of cats and perception of pungent odor demonstrated the lack of odor perception, consequently regarded as signs of anosmia. We conclude that the surgical technique used was effective and easy to perform in order to establish an experimental model for anosmia in rats. As a next step, potential stem cells derived from the olfactory epithelium were successfully isolated. A characterisation of these cells is currently under way, as well as preclinical tests to reveal the recovery of olfactory functions after cell therapy.
290 EFFECT OF EXOGENOUS Oct4 PROTEIN ON DIRECT CONVERSION OF HUMAN FIBROBLASTS INTO NEURAL STEM CELLS
Authors: Kang J. H. , Park S. M. , Heo S. Y. , Shim H.
Published: December 2012

The generation of neural stem cells (NSC) from somatic cells may provide unlimited source of neuronal cells for autologous transplantation to patients of neurological disorders. Recently, direct conversion of fibroblasts into NSC by epigenetic reprogramming has been reported (Han et al. 2012 Cell Stem Cell 10, 465–472; Thier et al. 2012 Cell Stem Cell 10, 473–479; Ring et al. 2012 Cell Stem Cell 11, 100–109). These reprogrammed cells are referred to as induced neural stem cells (iNSC) and they share the characteristics of NSC in their morphology, molecular marker expressions, and capacity to differentiate into neurons, astrocytes, and oligodendrocytes. One of the procedures to convert fibroblasts into iNSC is restriction of Oct4 activity to the initial phase of reprogramming, while Sox2, Klf4, and c-Myc are constitutively expressed. In the present study, we examined the effect of Oct4 in reprogramming of human fibroblasts into iNSC. Oct4 protein was modified by the addition of poly-arginine protein transduction domain to easily penetrate into the cell membrane. We transduced Oct4 protein, in contrast to the previous reports where the Oct4 gene was virally introduced. First, human fibroblasts were transfected by retroviral vectors carrying the genes encoding Sox2, Klf4, and c-Myc. Then, transfected cells were cultured in ReNcell NSC maintenance medium containing Oct4 protein. After 4 days, Oct4 protein was removed from the medium. With Oct4 protein transduction, 21 flat colonies were formed from 4 × 105 fibroblasts. These colonies were picked and passaged for subculture and later became iNSC. However, in the absence of Oct4 protein, no colonies were obtained from the same number of fibroblasts that were initially plated. Approximately 40 days after transduction of reprogramming factors, cluster of iNSC were obtained. These cells expressed molecular markers of human NSC, including Nestin, Sox2, Pax6, and Blbp. Moreover, these iNSC could differentiate into neurons, astrocytes, and oligodendrocytes in vitro. Results of the present study demonstrate that transduction of exogenous Oct4 protein may be essential to the direct conversion of human fibroblasts into iNSC using a combination of reprogramming factors Sox2, Klf4, and c-Myc.
292 COMPARISON OF DIFFERENTIAL PLATING METHODS TO OBTAIN ENRICHED POPULATIONS OF BOVINE SPERMATOGONIAL STEM CELLS
Authors: Giassetti M. I. , Barros F. R. O. , Worst R. A. , Saurin G. C. P. , Bruno A. H. , Moreira P. V. , Hamilton T. R. , Nichi M. , D'Ávila Assumpção M. E. O. , Visintin J. A.
Published: December 2012

Spermatogonial stem cells (SSC) represent only 0.1% of all germ cells found in adult animal testicles. The main goal of differential plating treatments is to isolate SSC and enrich the SSC population in a cell subculture. The aim of this study was to determine which differential plating treatment – laminin, lectin from Datura stramonium (DSA), Matrigel (BD Biosciences, San Jose, CA, USA), bovine serum albumin (BSA), or PBS/control – was the most efficient for isolating bovine SSC. Thus, 3 g of testicular parenchyma of adult Nelore bulls was minced and consecutively digested in two steps: collagenase (1 mg mL–1) and trypsin (2.5 mg mL–1). Cells from each testicle were plated (3 × 106 viable cells) in a culture dish coated with 1 mL of each of the following plating treatments: laminin (20 µg mL–1), DSA (20 µg mL–1), Matrigel (5%), BSA (0.5 mg mL–1), or PBS. Cells were cultured for 18 h in a high-humidity atmosphere containing 5% CO2 at 37°C. Supernatants were centrifuged and cell concentration and viability were assessed by Trypan Blue exclusion method. Then, levels of thiobarbituric acid reactive substances (TBARS) were evaluated in a 20 mM ascorbic acid solution and in 25µL of 4 mM ferrous sulfate solution immediately after incubation with 2 × 106 viable cells (n = 30) from each testicle (n = 6) for all treatments. The supernatant was collected and the same volume of 0.1% thiobarbituric acid was added. The TBARS level was quantified by spectrophotometry (532 nm). Approximately 1 × 106 viable cells (n = 55) from each testis (n = 11) for all treatments were fixed with 70% ethanol and incubated with an anti-α six integrin (A6Int) antibody, followed by Alexa Fluor 488 (BioLegends®, San Diego, CA, USA) and propidium iodide labelling. The percentage of marked cells with the anti-A6Int was determined by flow cytometry (Guava Technologies, Hayward, CA, USA). Kruskal-Wallis ANOVA and post hoc Bonferroni tests were performed; P < 0.05 was considered significantly different (Stata version 12.0, Stata Corp., College Station, TX, USA). No treatment side effects were observed on percentage of A6Int-positive cells recovered, TBARS level, or rate of viable cells after and before differential plating. A difference in cell viability was observed between the Matrigel and laminin treatments (P = 0.04). In conclusion, our results showed no quantitative difference between enriched SSC populations purified by differential plating treatments, as was expected. Thus, this experiment needs to be complemented with better characterisation of purified bovine SSC for their molecular markers expression using real time RT-PCR and flow cytometry. Only then will we be able to reach a conclusion on the effectiveness of these treatments.
Financial support: FAPESP (2011/20115-1).
298 STIMULATION OF ENDOGENOUS OCT4 IN BOVINE FETAL FIBROBLASTS WITH SYNTHETIC mRNA TRANSFECTION
Authors: Adams T. L. , Farmer S. E. , Godke R. A. , Bondioli K. R.
Published: December 2012

Induced pluripotent cells have been produced with viral vectors and other transgenic approaches that modify the genome of the host cell. Transfection with mRNA encoding transcription factors eliminates genomic modification and has been successful with human cells. The objective of this study was to deliver synthetic mRNA to bovine fetal fibroblasts in a manner consistent with induced pluripotency. Plasmids encoding green fluorescent protein (GFP) and human OCT4 were used as templates for in vitro transcription of mRNA (ivtRNA) and used to transfect bovine fetal fibroblasts. Expression of GFP was measured by flow cytometry and cell viability by seeding a constant cell number and determining final cell count. Differences between treatments were tested by ANOVA at P ≤ 0.05. In the first experiment, ivtRNA concentrations of 140, 280, and 560 ng/well of a 24-well plate were used and cell number and fluorescence determined after 24 h. Transfected treatments had significantly fewer cells and greater fluorescence than controls. Cells transfected with 280 ng/well ivtRNA had the highest fluorescence. Cells were then transfected with 280 ng/well of ivtRNA and total cells and fluorescence determined after 12, 24, 36, 48, and 60 h. Cell number was significantly lower than controls for all time points but similar between time points. Fluorescence was greater than controls for all time points and peaked at 24 to 36 h. There was no difference in fluorescence between 24 and 36 h, but these time points were significantly greater than all other time points. From these data, we determined that sustained expression could be maintained with transfection every other day. Cells were transfected every other day with ivtRNA encoding GFP and fluorescence measured every 3 days. Fluorescence was greater than controls for all time points but did not increase with repeated transfection. After 6 transfections, there were no viable cells left. In the final experiment, cells were transfected every other day for 12 days with 140, 280, or 560 ng/well ivtRNA encoding human Oct4. After 12 days, RNA was isolated and bovine OCT4 transcripts measured by RT-PCR. Primers and PCR conditions were designed so that bovine OCT4 transcripts were amplified but human OCT4 ivtRNA was not. The bovine poly-adenylate polymerase (PAP) transcript was also amplified. After gel electrophoresis of RT-PCR products, band intensities were determined and a ratio of OCT4/PAP calculated. This ratio was 0.7 for controls and 1.2 for the 140 ng/well ivtRNA treatment. No bovine OCT4 expression was detected in the higher ivtRNA treatments because of poor cell viability. From these experiments, we conclude that ivtRNA can be transfected into and expressed by bovine fetal fibroblasts. Repeated transfections with ivtRNA encoding human OCT4 induced expression of the endogenous bovine OCT4 but cell viability was reduced. Further experiments utilising ivtRNA with modified bases are planned to reduce the cell toxicity of ivtRNA.
304 THE DEVELOPMENTAL TOXICITY EVALUATION OF 5-FLUOROURACIL AND INDOMETHACIN IN HUMAN EMBRYONIC STEM CELLS
Authors: Jung E. M. , Jeung E. B.
Published: December 2012

Embryonic stem cells have pluripotency and differentiate into and constitute the cells and tissues of our body. In this study, using human embryonic stem cells (hESC), we evaluated novel methods for screening toxicological chemicals during developmental process. We elucidated developmental toxicity of two well-known chemicals, 5-fluorouracil (5-FU) and indomethacin (Indo) in hESC. The undifferentiated hESC were treated with the chemicals (10–4 to 104 µM of 5-FU and Indo) in a dose-dependent manner during 1 to 3 days. Surface markers (SSEA-4, TRA-1-60, and TRA-1-81) expressed only in undifferentiated hESC were monitored by immunocytochemistry to ensure the characterisation of undifferentiated hESC. Moreover, expression of embryonic stem cell-specific genes was assessed with real-time PCR after treatment of 5-FU and Indo (10–2, 100, and102 µM of 5-FU and Indo). The expression of surface markers was not significantly affected by treatment of 5-FU and Indo. The expression of transcription factors (Oct-4, Sox-2, Nanog, and hTERT) was significantly decreased by high concentrations of 5-FU and Indo (102 µM). However, no difference was observed in treatment of low concentration of 5-FU and Indo (10–2 µM). Taken together, these results suggest that 5-FU and Indo have cytotoxic effects, and modulate the expression of transcription factors that have pivotal roles in undifferentiated hESC. Therefore, we suggest that hESC may have potential to test toxicity of chemicals during embryonic developmental stage.
310 DOES PROGESTERONE OR EQUINE CHORIONIC GONADOTROPIN IMPROVE THE SUPEROVULATORY RESPONSE AND EMBRYO QUALITY IN WOOD BISON DURING THE ANOVULATORY SEASON?
Authors: Palomino J. M. , Mapletoft R. J. , Anzar M. , Woodbury M. R. , Cervantes M. P. , Adams G. P.
Published: December 2012

Superovulation protocols are being developed in wood bison (Bison bison athabascae), a threatened Canadian species. In initial studies, 2 doses of FSH diluted in hyaluronan given 48 h apart were successful for inducing ovarian superstimulation in wood bison, and ovulation rate was improved by final treatment with hCG instead of LH (Palomino et al. 2012 Reprod. Fertil. Dev. 24, 226). In that study, exogenous progesterone had no effect on the number of ovulations, but embryo quality could not be evaluated because of the low number of embryos collected. In beef cattle, replacement of the final doses of FSH with eCG has resulted in the recovery of a greater number of ova/embryos. The objective of the present study was to evaluate the effect of exogenous progesterone (PRID) on embryo quality and to determine if the addition of eCG increases the ovulation rate in superstimulated wood bison. Follicular ablation was done to synchronize follicular wave emergence in 26 wood bison cows during the anovulatory season (May). Cows were assigned randomly to 3 groups: PRID/no eCG (n = 8), PRID/eCG (n = 9), and no PRID/eCG (n = 9). A PRID was inserted on the day of follicular ablation (Day –1) in respective groups. In all bison, FSH diluted in hyaluronan (5 mg mL–1, MAP-5, Bioniche Animal Health Inc., Belleville, Ontario, Canada) was given intramuscularly on Day 0 (300 mg) and Day 2 (100 mg). On Day 3, a single dose of 450 IU of eCG (Pregnecol, Bioniche Animal Health Inc.) was given intramuscularly and the PRID were removed in the corresponding groups. On Day 5, all bison were given 2500 IU of hCG (Chorulon, Merck, Whitehouse Station, NJ, USA) intramuscularly to induce ovulation. Bison were inseminated with chilled semen 12 and 24 h later. Nonsurgical embryo collection was performed on Day 14. Transrectal ultrasonography was done to record the ovarian response, ovulation rate, and number of corpora lutea (CL). Results were compared by ANOVA and Chi-squared test (Table 1). The number of ovulatory-sized follicles (≥9 mm) on Day 5 did not differ among groups. Ovulation rate was lowest in bison treated with both a PRID and eCG (P < 0.05). There were no differences among groups in the number of CL on Day 14, the number of ova/embryos collected, or the number of transferable embryos. The superovulatory response and embryo collection rate in the present study were higher than in any previous reports in bison. The ovulation rate was not improved by the addition of eCG treatment, and exogenous progesterone had no effect on embryo quality.

Table 1.Response of superstimulated wood bison (mean ± SEM) to treatment with exogenous progesterone (PRID) and/or eCG
333 ESTABLISHMENT OF NOVEL METHOD FOR REPEATED CONSTRUCTION OF ENGINEERED ZINC FINGER NUCLEASE
Authors: Fujii W. , Kano K. , Sugiura K. , Naito K.
Published: December 2012

Zinc finger nucleases (ZFN), which are artificial restriction enzymes consisting of an engineered zinc-finger domain (ZF) and an endonuclease domain, can be used for the induction of site-directed mutation and the efficient generation of gene knockout animals. However, the repeated construction of various ZFN sequences is both expensive and time consuming. In this study, we attempted to establish a novel method for inexpensive and rapid ZFN construction. First, we constructed ZFN against mouse Rosa26 and original mouse Gli3 gene loci using short PCR primer sets (>30 bp), which contained 21 bp of the ZF recognition helix for a specific DNA triplet. We prepared 18 sets of such primers and PCR was performed using one of these primer sets and the partial ZF sequence as a template, which was obtained from the first to second DNA recognition helix of mouse Zif268. The PCR products were joined by overlap-PCR and nested PCR, and then inserted into a vector coding the endonuclease domain of FokI nuclease. By these steps, we successfully synthesised intended ZFN vectors containing 4 to 6 fingers. Next, we evaluated the functions of constructed ZFN. The mRNA of constructed ZFN were transcribed in vitro and injected into the cytoplasm of C57BL/6N zygotes. After 24 h of culture, 2-cell stage embryos were subjected to genomic PCR of the target locus, and the PCR products were directly sequenced. When ZFN mRNA for mouse Rosa26 was injected, 3- to 146-bp deletions were detected in 92.8% of injected embryos. This result was almost the same as previously reported for ZFN, indicating that our novel construction method can synthesise functional ZFN, which work as a site-directed nuclease, and that efficiency was comparable with those constructed by conventional PCR methods using long oligonucleotide sets (60 bp).
337 NONMEIOTIC INTROGRESSION OF QUANTITATIVE TRAIT NUCLEOTIDES AND CORRECTION OF CONGENITAL MUTATIONS IN LIVESTOCK WITH TRANSCRIPTION ACTIVATOR-LIKE EFFECTOR NUCLEASES
Authors: Fahrenkrug S. C. , Tan W. , Lillico S. G. , Stverakova D. , Proudfoot C. , Williamson G. , Long C. R. , Whitelaw C. B. A. , Carlson D. F.
Published: December 2012

Genetic enhancement of livestock productivity and welfare are major goals of breeding and genetics programs. However, the introgression of desirable alleles across breeds is slow and inaccurate. The development of gene editing technologies would provide the opportunity to accelerate the genetic improvement of a diversity of livestock breeds. Transcription activator-like effector nucleases (TALEN) are programmable nucleases that join the modular DNA binding domain of transcription activator-like (TAL) effectors with FokI endonuclease. We found that TALEN could be easily manufactured and that 64% displayed activity in swine and cattle primary fibroblasts, with cleavage of 1.5 to 45% of chromosomes in cell populations, as measured by Surveyor assay. Clonal isolation and sequencing revealed that up to 84% of cells contained at least one modified allele, with up to 24% of cells containing biallelic or homozygous chromosomal modification. Co-transfection of a customized TALEN pair with a template containing a specific allele was effective at the nonmeiotic introgression of quantitative trait into naïve cattle breeds. We will also describe the repair of 2 recently described embryonic lethal mutations that are segregating in important dairy cattle breeds (JH1 and HH1). Injection of TALEN mRNA into the cytoplasm of pig and cattle zygotes was capable of inducing gene knockout (KO) in 27 to 75% of embryos analysed (n = 4–59), nearly half of which (8/19) harbored biallelic modification. We will present data describing efforts towards gene conversion by direct injection of livestock embryos. Finally, we will present alternative strategies for the incorporation of gene editing in livestock production systems by cloning or embryo treatment.
GENETICALLY ENGINEERED PIG MODELS FOR TRANSLATIONAL DIABETES RESEARCH
Authors: Wolf Eckhard
Published: December 2012

Animal models play crucial roles for understanding disease mechanisms and for the development and evaluation of therapeutic strategies. In biomedicine, classical rodent models are most widely used for several reasons, including standardization of genetics and environment, cost efficiency, and the possibility to introduce targeted genetic modifications for the generation of tailored disease models. However, due to differences in anatomical and physiological characteristics, rodent models do not always reflect the situation of human patients sufficiently well to be predictive in terms of efficacy and safety of new therapies. In this respect, the pig has been discussed as a missing link between mouse models and human patients. As a monogastric omnivore, the pig shares many anatomical and physiological similarities with humans. Importantly, the techniques for genetic modification of pigs have been refined to a level allowing almost the same spectrum of alterations as in mouse models (Aigner et al. 2010 J. Mol. Med. (Berl.) 88, 653–664). These include inducible transgene expression systems (Klymiuk et al. 2012 FASEB J. 26, 1086–1099) as well as the introduction of targeted genetic modifications (Klymiuk et al. 2012 J. Mol. Med. (Berl.) 90, 597–608). A major focus of our laboratory is the generation, characterisation, and implementation of pig models for translational diabetes research. Transgenic pigs expressing a dominant negative receptor for the incretin hormone glucose-dependent insulinotropic polypeptide (GIP) demonstrated a crucial role of the GIP system for the physiological age-related expansion of pancreatic β-cell mass. Moreover, this animal model shares important characteristics of type 2 diabetes mellitus: impaired incretin effect, reduced glucose tolerance and insulin secretion, and a progressive reduction of β-cell mass (Renner et al. 2010 Diabetes 59, 1228–1238). More recently, we used this model to search for metabolic biomarkers which are associated with progression in the pre-diabetic period and identified specific amino acid and lipid signatures as candidate biomarkers (Renner et al. 2012 Diabetes 61, 2166–2175). Further, we created the first pig model for permanent neonatal diabetes by expression C94Y mutant insulin in the β-cells of transgenic pigs. In addition to their use as biomedical models, pigs may also serve as organ and tissue donors for xenotransplantation. Transplantation of encapsulated porcine pancreatic islets to type 1 diabetic patients with severe unaware hypoglycemia has already entered clinical studies, but encapsulation may shorten the lifespan of the islets. Therefore, in order to overcome the rejection of pig islets by human T-cells, we generated transgenic pigs expressing the optimized CTLA-4Ig variant LEA29Y in the pancreatic β-cells. Islets from LEA29Y transgenic pigs rescued diabetes and were protected against rejection in a humanized mouse model (Klymiuk et al. 2012 Diabetes 61, 1527–1532).
PORCINE MODELS FOR HUMAN CANCER
Authors: Flisikowska Tatiana , Leuchs Simon , Saalfrank Anja , Eser Stefan , Kind Alexander , Wolf Eckhard , Saur Dieter , Schnieke Angelika
Published: December 2012

Cancers are a leading cause of death worldwide and a major priority for biomedical research. Most animal models of solid cancers are in rodents, particularly genetically engineered mice. However, mice differ significantly from humans in size, lifespan, physiology, anatomy, and diet, limiting their usefulness for some studies. Pigs are increasingly recognised as a valuable adjunct to pre-clinical research. Our aim is to provide a series of genetically defined pigs that model serious and common human cancers. These will allow new diagnostic and therapeutic strategies to be investigated at human scale, and longitudinal studies under conditions that mimic the human patient. We are thus engaged in a program of gene targeting to replicate in pigs a series of genetic lesions known to underlie human cancers. Here, we describe results from two key tumour suppressor genes: adenomatous polyposis coli (APC) and p53 (TP53). Somatic mutations resulting in inactivation or altered p53 function are present in most human cancers, and germline TP53 mutations are responsible for Li-Fraumeni multiple cancer syndrome. TP53R175H is the most frequent missense mutation in many sporadic human cancers. We have created gene-targeted knockout pigs and pigs carrying a latent TP53R167H mutant allele orthologous to human mutant TP53R175H that can be activated by Cre recombination to model the occurrence of oncogenic mutant p53 in chosen tissues (Leuchs et al. 2012 PLoS One, in press). In vitro studies indicate that porcine TP53R167H resembles human TP53R175H in altered function, and homozygous knockout of porcine TP53 results in transformation of porcine MSCs. APC plays a vital initiating role in both sporadic colorectal cancer (CRC) and the inherited predisposition to colorectal cancer, familial adenomatous polyposis (FAP). We generated gene-targeted cloned pigs carrying two different nonsense mutations in APC (APC1061 and APC1311) at sites orthologous to human germline mutations responsible for FAP. At 1 year of age, the APC1311 mutation resulted in >100 lesions, including ~60 polyps, exclusively in the large intestine. Importantly, this accords with the location and onset of human FAP in early adulthood, and contrasts with equivalent mutations in mice where polyps develop in the small intestine. Histological and molecular analysis showed that the porcine model recapitulates all major features of early stage human FAP (Flisikowska et al. 2012 Gastroenterology, in press). Tumorigenesis involves multiple genetic alterations over time. It will now be possible to mimic this progression in pigs by combining these and other mutations. We are confident that pig models will make a significant contribution to human oncology.
35 EFFECT OF CULTURE AT LOW OR ATMOSPHERIC OXYGEN TENSION IN SOMATIC DONOR CELLS FOR HORSE NUCLEAR TRANSFER
Authors: Gambini A. , Jarazo J. , De Stefano A. , Karlaninan F. , Salamone D.
Published: December 2012

Somatic donor cells play a major role during the NT procedure. In vitro culture conditions may affect the capability of these cells to be reprogrammed and to allow embryo development. The aim of this study was to evaluate the effect of in vitro culture at low (5%) or atmospheric (20%) oxygen tension in somatic donor cells for cloned equine embryo production. Adult fibroblasts were obtained through culture of minced tissue from neck biopsies of one horse skin. They were cultured in DMEM supplemented with 10% fetal bovine serum (FBS) and 1% antibiotics in 2 groups: (1) 5% CO2 and (2) 5% CO2 and 5% O2, both groups in humidified air at 39°C. Quiescence of donor cells was induced by growth to confluency for 3 to 5 days prior to NT. Oocyte collection, maturation, cloning, and activation procedures were performed as described by Gambini et al. (2012 Biol. Reprod. 87, 1–9.). After activation, reconstructed embryos (RE) were cultured in DMEM/F12 supplemented with 5% FBS in the well of the well system as 3 reconstructed embryos per well. Cleavage and blastocyst formation (7–8 days) of the experimental groups were assessed. In vitro development, on a per-well and RE basis, was compared using the chi-square test. No statistical differences were observed in cleavage [(1): 48/84, 57%; (2): 54/87, 62%). No difference was observed in blastocyst rates on a per-well basis [(1): 5/28, 18%; (2): 4/29, 14%] or on a per-RE basis [(1): 5/84, 6%; (2): 4/87, 5%]. This work suggests that the oxygen tension during the in vitro culture of somatic donor cells does not affect the quantity of the cloned equine blastocyst produced. Further studies are required to determine if these conditions would affect in vivo embryo development.
39 FACTORS INFLUENCING THE EFFICIENCY OF GENERATING GENETICALLY ENGINEERED PIGS BY NUCLEAR TRANSFER: MULTI-FACTORIAL ANALYSIS OF A LARGE DATA SET
Authors: Kurome M. , Geistlinger L. , Kessler B. , Zakhartchenko V. , Klymiuk N. , Wuensch A. , Flisikowski K. , Flisikowska T. , Merkl C. , Nagashima H. , Schnieke A. , Zimmer R. , Wolf E.
Published: December 2012

Somatic cell nuclear transfer (SCNT) using genetically engineered donor cells is currently the most widely used strategy to generate genetically tailored pig models for biomedical research. Although this approach facilitates a similar spectrum of genetic modifications as in rodent models, including inducible transgene expression and gene targeting, the outcome in terms of live cloned piglets is quite variable. We used a large data set from 274 SCNT experiments (in total, 18 649 reconstructed embryos transferred into 193 recipients), performed over a period of 3 years, and evaluated it by multivariate analysis for combined assessment of multiple factors and their relative contribution to the efficiency of generation of genetically modified cloned pigs. Specifically, we addressed the effects of season, type of genetic modification (additive gene transfer v. gene targeting), donor cell source (mesenchymal stem cells, postnatal fibroblasts, fetal fibroblasts, and kidney cells), serial NT, and pre-selection of SCNT embryos for early development. Target parameters were pregnancy and delivery rates, as well as the numbers of born, live, and healthy offspring. Cloning efficiency was calculated as the number of cloned piglets relative to the number of transferred SCNT embryos. We used robust linear models adjusted to the underlying empirical distribution of the cloning outcome, as a straightforward approach to determine the statistically significant part of the network of factors affecting pig cloning. The SCNT experiments performed during the winter using fetal fibroblasts or kidney cells after additive gene transfer resulted in the highest number of live and healthy offspring, whereas sequential NT and NT experiments performed during the summer decreased it. Although the effects of individual factors may be different between various laboratories, the statistical approach described in this study may help to identify and optimize the most critical specific factors to cloning success in programs aimed at the generation of genetically engineered pig models.
Supported by the DFG (FOR535, FOR793), the Bayerische Forschungsstiftung, and Mukoviszidose e.V.
52 IMPLANTATION OF TRANSGENIC BOVINE CLONED EMBRYOS DERIVED FROM TRANSFECTED CELLS BY PiggyBac TRANSPOSITION
Authors: Choi W. J. , Lee S. J. , Lee W. W. , Kim S. J. , Saadeldin I. M. , Cho J. K. , Lee B. C. , Jang G.
Published: December 2012

A gene-delivery system, PiggyBac (PB) transposition, has been applied to transgene expression in mammalian cells or animals. In this study, to produce transgenic cattle, we used PB in bovine fibroblasts and then the transfected cells were microinjected into enucleated bovine oocytes to produce embryos and offspring. For this study, 2 different fluorescence genes (GFP, transcribed by constitutive promoter and RFP, transcribed by tetracycline-dependent promoter), which were flanked by PB sequences, were transfected into the bovine fetal fibroblasts by the FuGENE transfection protocol. The developmental rate of blastocysts among the cleaved embryos derived from GFP cells and doxycycline-induced RFP cells was developed at 23.1% (31/134) and 40.9% (442/1082), respectively. After transferring the GFP- or RFP-expressing blastocysts into recipient cows, pregnancies were detected by ultrasonography from both recipients of GFP or RFP. To know gene expression in fetal stage, embryonic sacs were collected surgically. The primary cells were successfully isolated from both embryonic sacs. Every cell from the GFP embryonic sac expressed GFP. When the cells from the RFP embryonic sac were treated with doxycycline, RFP was homogenously expressed. In conclusion, this study demonstrated that PB transposition could be applied to deliver genes into bovine somatic cells. Furthermore, transgenic embryos from transfected cells using the PB system were developed into blastocysts, implanted, and were able to form embryonic sacs. The PB system will be a useful method to produce transgenic cattle.
This study was financially supported by IPET (grant no. 109023-05-3-CG000, 111078-03-1-CG000) and the BK21 Program for Veterinary Science.
54 EXPRESSION PATTERN OF DNMT1 AND DNMT3a GENES DURING INTERGENERIC SOMATIC CELL NUCLEAR TRANSFER EMBRYO DEVELOPMENT
Authors: Morovic M. , Strejcek F. , Ostrup O. , Lucas-Hahn A. , Petersen B. , Niemann H. , Hyttel P. , Laurincik J.
Published: December 2012

One of the most-discussed reasons for developmental incompetence of embryos constructed by the cloning procedure is inadequate reprogramming of the transferred nucleus to a state equivalent to that of an early embryonic nucleus. Previous studies have shown species-dependent expression patterns of DNA methyltransferase (DNMT) genes in mammalian oocytes and preimplantation embryos, and also a correlation between incomplete DNA methylation and the lack of NT success in mammals. In the present study, the expression pattern of DNMT1 and DNTM3a genes at the 2-cell and 4-cell stages of bovine versus porcine intergeneric nuclear transfer (iSCNT) embryos was observed by reverse-transcriptase (RT) PCR. All pools were done in triplicate and contained 10 iSCNT embryos. The species-specific primers for DNMT1 and DNMT3a genes were designed for determination of de novo synthesis of epigenetic enzymes. As positive controls, porcine and bovine parthenogenetic embryos were used. Gene transcription for bovine DNMT1 (bDNMT1) and DNMT3a (bDNMT3a) was not observed in 2- and 4-cell stage embryos generated by bovine fibroblast transfer into the porcine ooplasm; however, using primers for pig DNMT1 (pDNMT1) and DNMT3a (pDNMT3a), positive results were obtained. In the 2- and 4-cell-stage embryos constructed using porcine fibroblast and bovine ooplasm, only the bovine-specific primers showed positive signals. Based on the different timing of major genome activation during embryonic development in bovine and porcine embryos, the strong influence of ooplasm on introduced fibroblast was expected. Despite the mRNA presence of DNMT1 and DNMT3a enzymes of oocyte origin, de novo transcription of somatic DNMT1 and DNMT3a genes was not detected and iSCNT embryos did not develop beyond the 4-cell stage. These results strongly suggest species-specific and maternally driven regulation of epigenetic reprogramming during early embryogenesis.
This work was supported by VEGA 1/0077/11.
57 TREHALOSE SUPPLEMENTED EXTENDER PRESERVE ACROSOMAL INTEGRITY IN POST-THAW BOAR SPERM
Authors: Athurupana R. , Funahashi H.
Published: December 2012

Cryopreservation of boar semen is still considered suboptimal due to lower fertility when compared to fresh semen. The use of glycerol for boar semen cryopreservation may be a reason of low fertility results. Trehalose is a nonreducing disaccharide known to stabilize proteins and biologic membranes during processes such as cryopreservation. The aim of this study was to evaluate the effects of trehalose on boar sperm cryosurvival in an egg-yolk-based extender. The semen samples collected from different individual Berkshires were diluted in egg-yolk-based freezing extender containing glycerol (final concentration 68.5 and 274 mM) or trehalose (50 and 100 mM). Then the samples were cryopreserved using the straw freezing procedure. Frozen sperms were thawed at 39°C in water. Post-thawed sperm were analyzed for motility (under microscope by a conventional method), viability, and acrosome integrity (under fluorescence microscope following LIVE/DEAD or CTC staining, respectively). Statistical analyses of results from 5 replicated trials were performed by ANOVA with a Bonferroni/Dunn post hoc test (significance; P < 0.05). The extender supplemented with 100 mM trehalose exhibited significantly higher acrosomal integrity (41.6%) compared with other extenders (P < 0.05, n = 5; Table 1). The trend of motility and viability was higher in 274 mM glycerol (28.0 and 52.9%) and 100 mM trehalose extenders (23.6 and 53.9%), but those were not significant. These results demonstrate that the presence of trehalose at 100 mM during cryopreservation improves the acrosome integrity of boar sperm, without any reduction in viability and motility, after thawing.

Table 1.Effect of glycerol and trehalose on post-thaw boar sperm
65 THE EFFECTS OF CUMULUS CELLS ON DEVELOPMENTAL RATES OF VITRIFIED MOUSE OOCYTES IN C57BL/6J STRAIN
Authors: Kashiwazaki N. , Kohaya N. , Fujiwara K. , Furui K. , Ito J.
Published: December 2012

Unfertilized oocytes are one of the most desired germ-cell stages for cryopreservation because these cryopreserved oocytes can be used for assisted reproductive technologies, including IVF and intracytoplasmic sperm injection. However, in general, the fertility and developmental ability of cryopreserved oocytes are still low. We have recently reported that, in the presence of surrounding cumulus cells, matured mouse oocytes vitrified using calcium-free media and ethylene glycol retain their developmental competence (Kohaya et al. 2011 J. Reprod. Dev. 57, 675–680). Since the previous study was carried out using ICR mice (closed colony), we examined whether our protocol can be applied for C57BL/6J mice (inbred strain), which are commonly used for production of transgenic and knockout mice. The effect of cumulus cells on the ability of C57BL/6J mouse oocytes to be fertilized and develop in vitro was examined. Cumulus oocyte complexes (COC) derived from female mice with super ovulation were collected by flushing. Cumulus cells were removed for a portion of the oocytes (DO) using hyarulonidase. Oocytes from both treatment groups (COC and DO) were then vitrified according to the protocol we previously reported (Kohaya et al. 2011). After warming, vitrified COC and DO were used for IVF. All percentage data were subjected to arcsine transformation before statistical analysis. Data were analyzed by one-way ANOVA and Tukey’s test. Significance was considered at P < 0.05. The pronuclear formation rate of vitrified DO after IVF (20/58, 33.3%) was reduced compared with vitrified COC (55/90, 62.1%). Vitrified COC showed significantly (P < 0.05) higher developmental ability to develop into the 2-cell (50/90, 57.0%) and blastocyst stages (42/90, 45.9%) compared with vitrified DO [24.8% (16/58) and 18.4% (11/58), respectively]. The vitrified COC developed to term at a high success rate (51/90, 56.7%) being equivalent to the rate obtained with IVF using fresh COC (52/90, 57.8%). Taken together, the current results clearly demonstrate that, in the presence of surrounding cumulus cells, matured mouse oocytes vitrified using calcium-free media and ethylene glycol retain their developmental competence. These findings will contribute to improve oocytes vitrification in not only experimental animals but also in clinical application in human infertility.
66 BUFFALO-BULL SEMEN-FERTILITY EVALUATION IN RELATION TO MOTILITY AND INTEGRITY OF ACROSOME, PLASMA MEMBRANE, AND SPERM DNA
Authors: Kumar D. , Kumar P. , Singh P. , Yadav S. P. , Yadav P. S.
Published: December 2012

The breeding-soundness examination is conducted to identify and select bulls with an acceptable reproductive efficiency. In buffalo, there is meagre information regarding fertility index in relation to sperm attributes so that a future breeding bull could be selected at the age of maturity. So, to predict the fertility of buffalo breeding bulls, the present study was conducted to assess the motility parameters and integrity of acrosome, plasma membrane, and DNA of cryopreserved semen of high- and low-fertile bulls. The fertility of bulls was classified on the basis of conception rates, where buffalo bulls with conception rate <35% were considered as low-fertility bulls and those with conception rate >55% were considered as high-fertility bulls. A computer-assisted semen analyser was used for motility and viability studies, whereas integrity of acrosome, plasma membrane, and DNA were assessed by Pisum sativum agglutinin–fluorescein isothiocyanate, Annexin-V/PI, and TUNEL assay kit, respectively, under the florescence microscope. At least 200 spermatozoa were evaluated from each group, and results were analysed by ANOVA. The level of significance was observed at P < 0.05. The mean (±SE) values for various motility parameters of sperm for high-fertile bulls were total motility (56.8 ± 3.2%), average path velocity (87.22 ± 1.6 µm s–1), straight linear velocity (68.93 ± 1.9 µm s–1), and curvilinear velocity (156.52 ± 4.3 µm s–1). These values were significantly higher than those of low-fertile bulls (43.8 ± 1.7%, 79.02 ± 2.4 µm s–1, 63.42 ± 1.2 µm s–1, and 142.37 ± 2.8 µm s–1, respectively). The amplitude of lateral head displacement (6.8 ± 0.07 v. 6.5 ± 0.1 µm), beat cross frequency (33.9 ± 0.4 v. 33.43 ± 0.5 Hz), straightness (79.2 ± 0.7 v. 78.7 ± 0.6%), linearity (45.5 ± 0.4 v. 45.5 ± 0.7%), and viability (71.2 ± 0.8 v. 68.9 ± 0.8%) did not differ in both groups. The average percentage of intactness of sperm acrosome of high-fertile bulls was significantly higher (81.82 ± 0.87%) than that of low-fertility bulls (76.86 ± 0.87%). Furthermore, to assess the functionality of plasma membrane of sperm, we analyzed different stages of apoptotic-like events. The percentage of apoptotic sperm differed significantly between high-fertility (15.59 ± 0.75%) and low-fertility (25.94 ± 0.5%) bulls. The percentages of early necrotic, necrotic, and viable sperm did not differ in 2 groups. The DNA integrity in high-fertility (90.24 ± 0.94%) and low-fertility (88.37 ± 0.91%) bulls was not significantly different. In conclusion, the various parameters such as average path velocity, curvilinear velocity, straight linear velocity, and total motility; acrosomal integrity; and percentage of apoptotic sperm are useful for evaluating the semen quality of a bull to reduce the risk of using poor-fertility bulls in an AI program.
73 EFFECT OF THE BUFFER SYSTEM, CRYOPROTECTANT, AND ANTIOXIDANT ON SPERM FROM ENDANGERED CATALONIAN SHEEP XISQUETA AND ARANESA AND GOAT BLANCA DE RASQUERA BREEDS DURING PRESERVATION
Authors: Palomo M.-J. , Tabarez A. , Garcia W. , Terre M. , Ferrando A. , Jordana J.
Published: December 2012

In an attempt to increase the possibilities of a better sperm cryopreservation from these endangered small ruminant catalonian breeds, we are studying different strategies. One of them is to study the effect of the buffer system Tes-Tris (TEST) compared with the Tris and citric acid (TRIS) buffer system, testing simultaneously both systems in a 1% (w/v) soybean lecithin or in a 15% (v/v) powdered egg yolk–based media supplemented both with 5% glycerol and to assess also the inclusion of 5 mM of butylated hydroxytoluene (BHT) as an antioxidant on the cryopreservation media. However, the main objective of the present study was to test first the effect of the different media when the sperm were cultured 4 h at 5°C. Briefly, fresh ejaculates from 6 young bucks of Blanca de Rasquera breed (1 year old) were collected by an artificial vagina in favourable reproductive period and immediately mixed in equal quantities. Pooled ejaculates were split into 2 equal aliquots and washed by centrifugation (twice for 10 min at 600g) in TRIS- or TEST-based media without cryoprotectants and antioxidant. Afterward each pellet was split into 4 equal aliquots, re-suspended in TRIS or TEST, depending on the experimental group, soybean lecithin, or powdered egg yolk-based media, and supplemented or not with BHT and kept for 4 h at 5°C. Likewise, fresh ejaculates from 8 young rams (4 rams of Xisqueta and 4 rams of Aranesa breed, 1 year old) were collected and processed as buck semen samples. Sperm survival before cryopreservation was determined by eosine-nigrosine stain, and sperm motion parameters were analysed by a computer-assisted sperm analysis system (ISAS®). Six replications were performed in both species, and General Lineal Model (SAS®, Cary, NC, USA) was used for the statistical analysis. The highest sperm viability percentage (mean ± SE) on goat sperm cultured 4 h at 5°C was observed in the extender with TRIS buffer system in powdered egg yolk-based media supplemented with BHT (81.1 ± 2.8), not showing significant differences with the others extenders, except with the viability of the samples in the extenders with TEST buffer system in soybean lecithin-based media supplemented (56.5 ± 2.9; P < 0.001) or not with BHT (60.1 ± 5.1; P < 0.001). On the other hand, no significant differences on sperm viability were observed on ram sperm between treatments. Nevertheless, the sperm quality motion characteristics (data not shown) were quite different between all the treatments in both species. Considering that the present results are still preliminary, we suggest that more analysis should be made to explain how the different composition of the extenders affect sperm quality during the cryopreservation process.
Supported by INIA (RZ2009-00008-00-00), Generalitat de Catalunya (2009SGR0621 and CUR-DIUE), and FSE and Fundacion Carolina.
77 ADDITION OF CHOLESTEROL IN FRESH GOAT SPERM IMPROVES CRYOSURVIVAL RATES
Authors: Silva B. G. , Moraes E. A. , Oliveira C. S. , Ferrari Junior W. D. , Matos W. C. G. , Graham J. K. , Lima L. R.
Published: December 2012

Cryopreservation causes irreversible damage to goat sperm membranes, measured by a loss of motile and functional normal cells, compared with fresh sperm. The objective of this study was to determine if the addition of cholesterol-loaded cyclodextrin (CLC) to goat semen improved sperm cryosurvival. The CLC was prepared as described by Purdy and Graham (2004 Cryobiology 48, 36–45) with some modifications: 200 mg of cholesterol were dissolved in 1 mL of chloroform and 1 g of methyl-beta-cyclodextrin was dissolved in 2 mL of methanol. A 0.45-mL aliquot of the cholesterol solution was added to the cyclodextrin solution, after which the mixture was poured into a glass Petri dish and the solvents allowed to evaporate on a warm plate for 24 h. The resulting crystals were removed from the dish and stored at 22°C. A working solution of the CLC was prepared by adding 50 mg of CLC to 1 mL TALP at 37°C. Thirty ejaculates from 5 bucks were collected, diluted 1 : 1 in Tris diluent, divided into 7 equal aliquots, and centrifuged at 800g for 10 min. The sperm pellets were resuspended in Tris diluent, to which 0, 0.75, 1.5, 3.0, 4.5, 6.0, or 7.5 mg of CLC/120 million sperm were added. All treatments were incubated for 15 min at room temperature and then cooled to 4°C over 2 h. The samples were then diluted with Tris-egg-yolk diluent containing 2% glycerol, and the sperm were packaged into 0.5-mL straws, frozen in static liquid-nitrogen vapour for 20 min, and plunged into liquid nitrogen. Straws were thawed in 37°C water for 30 s, extended in Tris, and analyzed using optic microscopy. To test thermal resistance, after thawing, 0.5 mL of semen from each treatment were placed in 1.5-mL Eppendorf tubes in a water bath at 37°C for 3 h. At 0, 60, 120, and 180 min, subsamples were evaluated for sperm progressive motility. A hyposmotic test was also conducted by adding 10 µL of sperm to 2 mL of each solution and incubating them for 1 h/37°C. Sequentially, 20 µL of sperm was diluted in hypoosmotic solution (150 mOsm), and the samples were evaluated using phase-contrast microscopy. A total of 100 spermatozoa were counted in at least 5 different fields, and sperm tails were classified as either noncoiled or coiled. Data were analyzed using ANOVA, and treatment means were separated, using the SNK test at 5% probability. The sperm motility (50.4, 33.8, and 22.5%) was significantly higher for sperm treated with 0.75 mg of cholesterol after 0, 60, and 120 min of incubation after thawing, when compared with other treatments. No treatment differences in the hypoosmotic swelling test were observed. The addition of 0.75 mg of cholesterol to fresh goat semen improved sperm motility after cryopreservation for up to 3 h.
Supported by FACEPE and CAPES.