bluuebunny/crossref_metadata_2025 huggingface dataset:
417 REPORTER GENE BIOASSAY OF FOLLICLE-STIMULATING HORMONE ACTIVITY IN MARE SERUM
Authors: Sahmi F. , Sayasith K. , Portela V. , Price C. A.
Published: August 2010

Equine chorionic gonadotropin is secreted by the mare placenta and possesses both LH and FSH bioactivities in nonequine species. In ruminants, eCG is used commercially to induce superovulation. The production of commercial eCG is hampered by the variation in FSH and LH bioactivity of eCG between mares, and potentially results in batch-to-batch variation in eCG bioactivity. The objective of this study was to establish a cell-line-based bioassay of FSH activity in serum for use at eCG production facilities. Several cell lines were used for this study: HEK293 (kidney cells), KGN (a human granulosa cell line), and a new bovine granulosa cell line. The HEK293 and bovine granulosa cell lines did not express the FSH receptor (FSHR); therefore, the strategy was to cotransfect those cells with a FSHR expression plasmid and a cAMP reporter gene (β-galactosidase; β-Gal). The KGN cells transfected with β-Gal failed to respond to FSH and were not used further. The HEK293 and bovine cell lines responded to FSH in a dose-dependent manner, with a visible increase in β-Gal activity measured by colorimetric assay. The cells responded to eCG but not to LH, IGF1, or estradiol, demonstrating specificity for FSH activity. The minimum time of incubation required for clear bioactivity was 4 h. Activity was detected in serum from pregnant but not estrous mares. Attempts to create stable cell lines expressing both FSHR and β-Gal plasmids were not productive. We therefore attempted to create frozen batches of transiently transfected HEK293 cells. Several incubation conditions were tested and we succeeded in detecting β-Gal activity in response to eCG in thawed cells. The choice of serum during transfection had a major effect on the ability of the cells to respond to eCG after thawing, and the time interval between transfection and freezing significantly altered the magnitude of the response to eCG. The cells responded visibly to eCG treatment after 4-h incubation. In summary, we have developed a reasonably fast, colorimetric bioassay for FSH activity that can be used for serum in an on-farm setting.

Supported by NSERC, AAFC, and Bioniche Animal Health.
426 TRANSGENIC PIGS EXPRESSING HIGH LEVELS OF HUMAN THROMBOMODULIN ON VASCULAR ENDOTHELIUM
Authors: Klymiuk N. , Kessler B. , Kurome M. , Wuensch A. , Faber C. , Nagashima H. , Wolf E.
Published: August 2010

The development of a1,3-galactosyltransferase deficient pigs along with overexpression of complement regulatory proteins was an important step to overcome hyperacute rejection of pig organs transplanted into primates. However, the lack of galactose-α1,3-galactose (αGal) epitopes does not protect against subsequent rejection mechanisms, such as acute vascular rejection, which plays a crucial role to achieve prolonged graft survival in pig-to-human xenotransplantation. Acute vascular rejection involves incompatibilities between human blood and porcine endothelial cells, leading to coagulation disorders. Indeed, thrombotic microangiopathy is one of the key characteristics of rejected pig-to-primate grafts from aGal-deficient donor animals. One of the strategies to overcome this coagulation problem by genetic modification of donor pigs is expression of human thrombomodulin (TM) on the porcine endothelium. Although porcine TM is able to bind circulating human thrombin, the resulting heterodimer is unable to cleave human protein C, which promotes the coagulation cascade. We addressed this problem by the development of a vector placing the single-exon open reading frame of human TM under the control of a 6-kb promoter fragment obtained from the porcine TM gene. In addition, we replaced the 3′-untranslated region (UTR) by a polyadenylation box from the bovine growth hormone gene to avoid interferon-y-dependent destabilization of thrombomodulin transcripts. This vector was linked to a floxed neomycin cassette and transfected into porcine fetal fibroblasts. Positive cell clones were selected with G418, mixed, and then used for nuclear transfer. Donor fibroblasts were electrofused with enucleated oocytes resulting in 358 reconstructed embryos (91.1% fusion rate). One or two days later, 294 embryos were transferred to 3 synchronized gilts. In total, 8 cloned transgenic piglets with 1 or 2 independent integration sites of the transgene were born. Heart, kidney, lung, and liver of 5 founder animals were analyzed by immunohistochemistry They were found to express thrombomodulin strongly and strictly limited to the vascular endothelium of at least heart and kidney. The staining intensity was estimated qualitatively, by grading from weak to strong immunoreactivity (brown color, using DAB as chromogen). Cells from founder pigs expressing TM strongly have been used for recloning. The effect of human TM to normalize blood coagulation will be tested in experiments involving ex vivo perfusion of transgenic hearts and kidneys with human blood.

Supported by the Deutsche Forschungsgemeinschaft (FOR 535).
432 INHERITANCE OF LENTIVIRAL PHOSPHOGLYCERATE KINASE-ENHANCED GREEN FLUORESCENT PROTEIN (PGK-eGFP) INTEGRANTS OF TRANSGENIC CATTLE
Authors: Reichenbach M. , Habermann F. A. , Reichenbach H. D. , Guengoer T. , Weber F. , Zerbe H. , Sinowatz F. , Pfeifer A. , Wolf E.
Published: August 2010

An alternative approach to classic techniques for the generation of transgenic livestock is the use of viral vectors. Using lentiviral vectors (LV) we previously generated transgenic founder cattle with integrants carrying phosphoglycerate kinase (PGK) promoter-enhanced green fluorescent protein (eGFP) expression cassettes (Hofmann et al. 2004 Biol. Reprod. 71, 405-409). The aim of this work was to investigate the transmission of LV-PGK-eGFP integrants through the female and male germ line of transgenic founder cattle in resulting embryos, fetuses, and offspring. The female founder animal was superovulated and artificially inseminated with a nontransgenic bull. Six of the 16 embryos obtained were transferred to synchronized recipient heifers, resulting in 2 pregnancies and birth of 1 healthy male transgenic calf, expressing eGFP as detected by in vivo imaging and real-time PCR. Cryopreserved semen of the founder bull and matured COC of nontransgenic cows were used for in vitro embryo production as previously described by Hiendleder et al. (2004 Biol. Reprod. 71, 217-223). The rates of cleavage and development to blastocysts in vitro corresponded to 52.3 ± 3.8% and 23.5 ± 4.6%, respectively. In vivo expression of eGFP was observed at blastocyst stage (Day 7 after IVF) and was seen in 93.8% (198/211) of all blastocysts. Twenty-four eGFP-positive embryos were transferred to 9 synchronized recipients. Analysis of 2 embryos flushed on Day 15, 2 fetuses recovered on Day 45, and a healthy male transgenic calf revealed consistent high-level expression of eGFP in all tissues investigated. These observations show for the first time transmission of lentiviral integrants through the germ line of female and male transgenic founder cattle. Although eGFP transgenic cattle have been produced before by nuclear transfer from transfected cells, lentiviral transgenesis has the advantage that only one copy of the provirus is integrated at a particular chromosomal integration site. High-fidelity expression of eGFP in embryos, fetuses, and offspring of founders provides an interesting tool for developmental studies in cattle, including interactions of gametes, embryos, and fetuses with their maternal environment.
435 REDUCTION OF CELL CYCLE LENGTH AND INCREASE IN S-PHASE BY GROWTH FACTORS IN PIG FETAL FIBROBLASTS
Authors: Waghmare S. , Mir B.
Published: August 2010

Gene targeting in primary somatic cells is inefficient compared with embryonic stem cells. This is because of a slow rate of cell proliferation, fewer cells in S-phase at a given time point under normal culture conditions, and low rate of homologous recombination. Homologous recombination occurs mainly in late S-phase and increase in gene targeting efficiency has been reported in S-phase synchronized cells in bovine and rhesus macaque fetal fibroblasts. In this study we tested several growth factors: platelet-derived growth factor (PDGF), tumor necrosis factor a (TNFα), epidermal growth factor (EGF), fibroblast growth factor (FGF), transforming growth factor β1 (TGFβ1), insulin-like growth factor 1 (ILGF-1) and insulin-like growth factor II (ILGF-II) individually and in various combinations to see the effect on cell proliferation rate. Each experimental set consisted of 3 replicates. TGFβ1-, ILGF1-, ILGFII-, and FGF-treated cells grew very slowly compared with untreated cells. However, a combination of 3 growth factors: PDGF (15 ng mL-1), EGF (50 ng mL-1) and TNFa (100 pg mL-1), herein referred to as the cocktail, accelerated cell proliferation rate and reduced cell cycle length on average from 24.5 ± 0.2 to 20.4 ± 0.5 h with no significant change in number of cells in S-phase. Further, cells grown in the presence of the cocktail showed changes in morphology. The cells became spindle-shaped and occupied less surface area per cell compared with untreated cells. Importantly, cocktail-treated cells maintained a normal karyotype without any chromosomal abnormality. Thymidine has been used successfully to block various cell types in S-phase but it failed to synchronize these cells in S-phase in the concentration range of 2 to 10 mM for 24 to 48 h. However, serum starvation (0.2% fetal bovine serum) for 48 h blocked the cell proliferation rate effectively and synchronized cells in G0 phase (80-82% cells). After releasing from the block, cells were grown in the absence or presence of cocktail and cell cycle analysis was done at different time points by flow cytometry. Each time point was repeated 3 times. We observed the maximum number of cells in S-phase at 22 to 23 h (61.33% ± 7.77 in cocktail-treated cells v. 41.7% ± 3.28 in untreated cells). In summary, the cocktail-treated cells showed changes in cell morphology, higher proliferation rate, reduction in cell cycle length by 16.7%, and maximum percentage of cells in S-phase following serum starvation but maintained normal karyotypes. This high proliferation rate, reduction in cell cycle length, and maximum number of cells in S-phase should be very helpful in increasing the efficiency of gene-targeting in pig fetal fibroblasts.
438 REPEATED ULTRASOUND-GUIDED FOLLICULAR ASPIRATION IN PREGNANT SUCKLED BEEF COWS PRETREATED WITH OR WITHOUT eCG
Authors: Aller J. F. , Mucci N. , Kaiser G. , Ríos G. , Callejas B. , Alberio R. H.
Published: August 2010

In vivo oocyte recovery during the first trimester of pregnancy may be an alternative method of obtaining gametes for IVM/IVF/IVC from genetically valuable females. This study was conducted to evaluate i) the influence of the day of gestation on the follicular growth and number and quality of oocytes recovered by ovum pick-up (OPU) in pregnant suckled beef cows with or without eCG pre-stimulation, and ii) the possible effects on pregnancy outcome. Twelve pregnant suckled Angus cows (40 days after FTAI) were randomly assigned to 2 groups (n = 6/group). Group 1 treatment included: Day 0 = dominant follicle ablation (DFA) to synchronize the emergence of a new follicular wave, Day 1 = eCG treatment (2,000 IU, i.m. Syntex, Argentina) and Day 5 = OPU (follicles >3 mm were aspirated). Group 2: as for Group 1 with no eCG treatment. In both groups, OPU was repeated 5 times (Days 45, 59, 73, 87, and 101 of gestation) and ovarian follicles were classified into 3classes according to diameter (class I, <5 mm; class II, 5-9 mm; and class III, ≥10 mm). The collected COCs were categorized in 4 categories (I to IV) on the basis of both cumulus cells and appearance of oocyte cytoplasm. Categories I + II were considered as viable. Data were analyzed by PROC MIXED (SAS Institute, Cary, NC, USA). There was a significant treatment by follicular class interaction (P < 0.001). The numbers (mean ± SEM) of class II (4.3 ± 0.9) and III (2.5 ± 0.4) follicles/cow/session observed in eCG-treated cows were greater than for nontreated cows (0.9 ± 0.1 and 0.9 ± 0.1, respectively). In contrast, the number (mean ± SEM) of class I follicles/cow/session was lower for cows with eCG treatment (2.8 ± 0.4) than for nontreated cows (5.7 ± 0.5). A significant treatment by day of gestation interaction for aspirated follicles was observed (P = 0.03). The numbers (mean ± SEM) of recovered oocytes and viable oocytes/cow/session were greater (P < 0.05) for eCG-treated cows (2.2 ± 0.2 and 1.6 ± 0.4 respectively) than for nontreated cows (1.0 ± 0.2 and 0.9 ± 0.2, respectively). No donor pregnancies were lost either during or following OPU procedure. The overall efficiency of OPU procedure is shown in Table 1. We can conclude that (1) eCG-treated pregnant suckled cows can be a source of oocytes for IVF to at least 100 days of gestation and (2) repeated DFA/eCG treatment/OPU procedures did not affect the pregnancy outcome.

Table 1.Overall efficiency of repeated DFA/eCG treatment/OPU procedures in pregnant suckled cows
440 EVALUATION OF FOLLICULAR GROWTH AFTER EXOGENOUS STIMULATION IN MICE USING ULTRASOUND BIOMICROSCOPY
Authors: Campos-Junior P. H. A. , Silva C. S. , Grázia J. G. V. , Batista R. I. T. P. , Maffili V. V. , Santos R. R. , Viana J. H. M.
Published: August 2010

Real-time follicular dynamics studies have often been restricted to large animals because of the resolution of ultrasound equipment available. The recent advances in image technology, including significant gains in spatial resolution, allowed these evaluations to be performed in small rodents, which are important models to understand folliculogenesis. The aim of this study was to evaluate exogenous stimulated follicular growth in mice using a high resolution ultrasound. Female mice (n = 15) received a 5 IU i.p. injection of eCG (Vetecor®, Calier) and 48 h later a 5 IU injection of hCG (Novormon®, Schering-Plough), and were mated thereafter. In experiment 1, animals (n = 7) were anesthetized with ketamin/xilazin solution and evaluated at 3, 9, 15, 21, 27, 33, 45, and 51 h after eCG injection. The ovaries were identified with ultrasound biomicroscopy (UBM, Vevo 660®, Visual Sonics, Toronto, ON, Canada) coupled with real-time micro-visualization probe (RMV 707b), and the follicular population was measured, quantified, and classified into small follicles (≤449 μm) and large follicles (≥450 μm). The number of small follicles decreased (P < 0.05) from 30.44 ± 15.91 to 14.79 ± 7.23, and the number of large follicles (≥450 μm) increased from 0.36 ± 0.74 to 4.21 ± 4.25. For both size classes, however, statistical differences only occurred at 45 h after eCG; that is, close to the moment of hCG injection. In experiment 2, animals (n = 8) were evaluated every 2 h beginning 4.5 h after hCG to check for ovulations. The largest follicles achieved a mean maximum size of 596.7 ± 106.0 μm, 5.8 ± 2.3 h after hCG application. In 5 animals, large follicles were observed at 2 distinct moments, 5.0 ± 1.2 h and 10.4 ± 1.0 h after hCG, respectively. Results suggest that the later stages of follicular growth in mice are only achieved a few hours before ovulation, and that ovulations induced by hCG are not synchronized.

Financial suport: Fapemig, CBTC São Rafael, CNPq.
444 FOLLICLE ASPIRATION/ABLATION AS A TECHNIQUE TO AVOID OVULATION AND INDUCE DIESTRUS CONCENTRATIONS OF PROGESTERONE DURING THE ESTROUS CYCLE IN MARES
Authors: Ignacio F. S. , Montechiesi D. F. , Bergfelt D. R. , Puoli Filho J. N. P. , Carvalho L. R. , Meira C.
Published: August 2010

The present study aimed to demonstrate that ultrasound-guided follicle aspiration/ablation could circumvent ovulation and induce progesterone production in potential recipient mares sufficient to reach diestrus concentrations and potentially support pregnancy following embryo transfer. Ovulation (Day 0) was detected and follicular development was monitored using transrectal ultrasonography in 29 horse mares during the estrous cycle. On Day 5, all mares were given PGF2α (7.5 mg of Dinoprost trometamine, Lutalyse®, Pfizer, Paulinia, Brazil) and randomly assigned to a control and follicle ablation groups. Mares in the control group (n = 5) were not subject to follicle ablation and were allowed to ovulate. Mares in the ablation groups were distributed according to the diameter of the largest follicle to be ablatedusing transvaginal ultrasonography as follows: F25-29 mm (n = 6); F30-34 mm (n = 6); F35-40 mm (n = 6); F ≥ 40 (n = 6). Blood samples were collected at 24-h intervals from the day before ovulation or follicle ablation until detection of the next ovulation. Plasma progesterone concentrations were measured using a commercial RIA kit with intra- and interassay CV of 7.6% and 8.3%, respectively. Concentrations of progesterone ≥2 ng mL-1 were considered representative of diestrus in the ablation groups. Groups were compared using ANOVA and mean differences among groups were located with Tukey’s test. There was an effect of group (P < 0.05) and the percentage of mares and mean (± SEM) days post-ablation or ovulation that progesterone was ≥2 ng mL-1 for each follicle group were as follows: F25-29, 83% and 6.2 ± 1.1ab; F30-34, 66% and 7.3 ± 0.5a; F35-40, 83% and 5.4 ± 0.6ab; F ≥ 40, 100% and 4.0 ± 0.4bc; and control, 100% and 1.6 ± 0.4c. Values with different superscripts are different (P < 0.05). There was no difference (P > 0.05) in maximum concentrations of progesterone among ablation groups. For 7 days after follicle ablation, concentrations of progesterone (≥2 ng mL-1) were not different (P > 0.05) between ablated and control groups. Correspondingly, the time to spontaneous luteal regression (i.e. progesterone <2 ng mL-1) post-ablation or ovulation was not different (P > 0.05) among groups. In conclusion, aspiration/ablation of follicles ≥25 mm following luteal regression during the estrous cycle resulted in the production of progesterone such that concentrations attained diestrus values that could potentially sustain pregnancy following embryo transfer.

The authors wish to acknowledge FUNDUNESP for funding the project.
446 BOVINE POSTPARTUM DISEASES IN DIFFERENT SYSTEMS OF MILK PRODUCTION
Authors: da Rosa D. C. , Pessoa G. A. , Leonardi C. E. P. , Vasconcelos F. F. , Silva C. A. M. , Rubin M. I. B.
Published: August 2010

Reproductive data from 304 cows were analyzed in milk herds of the western central region of the Rio Grande do Sul (RS), Brazil, to evaluate the effect of diseases in the postpartum period and their effect on the reproductive issues related to specialist (n = 180), semi-specialist (n = 84), and non-specialist (n = 40) systems of milk production. For statistical analysis ANOVA and correlation at 5% significance were used. The results revealed that 42% of females had postpartum disorders (129/304) and 58% were healthy (175/304). At 60 days after AI, 51% (114/225) of the cows were pregnant. Sick postpartum animals had a low rate of pregnancy (19%). In the specialized and non-specialized systems, a correlation (P = 0.00227; 0.0310) was detected between healthy animals and pregnant, among patients and non-pregnant (P = 0.0289; 0.0160), respectively. The incidence of uterine infection detected by ultrasound after parturition (28%) was higher than the incidence in animals with mastitis (16%). None of the production systems has proved to be an effective model to prevent postpartum disease. The incidence of disease postpartum, as well as the incidence of uterine infections, was similar between the systems of production. The neosporose, uterine infections and mastitis were the reproductive health problems most prevalent in the study.

This study was support by CNPq graduate funding to the first and second authors.
45 PROLONGING THE FIRST CELL CYCLE IN NUCLEAR TRANSFER BOVINE EMBRYOS DOES NOT INCREASE CLONING EFFICIENCY
Authors: Blaza R. G. , Oliver J. E. , Oback B. , Wells D. N.
Published: August 2010

We hypothesized that reprogramming a somatic cell following NT is a time-dependent process that can be improved by artificially prolonging the first cell cycle of the cloned embryo. Eleven candidate drugs were initially screened for their ability to reversibly delay the onset of the first cleavage in bovine parthenotes without affecting subsequent in vitro embryo development. After identifying the cyclin-dependent kinase inhibitor butyrolactone-1 (BLT1; BIOMOL International, Plymouth Meeting, PA, USA) as a suitable candidate, we determined its optimal concentration and exposure time. We then performed zona-free bovine NT with serum-starved male skin fibroblasts. Commencing 10 h after the start of IVC, reconstructed 1-cell embryos were treated with either 200 μM BLT1 or 0.4% DMSO in SOF culture medium for 8 to 11 h. After thorough washing, cleavage rates were recorded and culture continued until Day 7. Labeling with 5-bromo-2-deoxyuridine (BrdU) was used to determine DNA replication during the first cell cycle. Some embryos were also transferred singularly to recipient cows. Embryo development was analyzed by a generalized linear model with binomial variation and pregnancy rates by Fisher’s exact test. At 0, 2, 4, 6, and 10 h after the start of IVC, 0% (0/28), 8% (5/61), 67% (39/58), 90% (54/60), and 100% (16/16), respectively, of NT reconstructs had incorporated BrdU, indicating that all 1-cell NT embryos were in S-phase at the start of treatment. After 8 to 11 h of incubation in BLT1, only 28% (119/429) of NT embryos had cleaved, compared with 93% of DMSO-treated controls (297/319). After removing BLT1 in those embryos arrested at the 1-cell-stage, there was no BrdU incorporation over the subsequent 1 h (0/17), embryos entered mitosis and by 4 h, 90% had cleaved (86/96). Thus, BLT1-arrested embryos were at a post-replicative stage prior to M-phase. Rates of in vitro embryo development on Day 7, from late morula to expanded blastocyst stages, of either grade 1-3 or grade 1-2 quality, in the BLT1 treatment were not different compared with controls (129/275 = 47% v. 151/309 = 49% and 33% v. 33%, respectively). Nuclei counts in expanded blastocysts from the BLT1 treatment were not significantly different than controls (109 v. 121, n = 31). Embryo survival on Day 35 of pregnancy and for calves to 1 month ofage was also not different between BLT1 and control treatments (13/31 = 42% v. 12/29 = 41% and 6% v. 10%, respectively). In conclusion, treating 1-cell NT embryos in S-phase for 8 to 11 h with 200 μM BLT1 arrested embryos in G2 and delayed cleavage by approximately 6 h. Cell cycle arrest was fully reversible after drug withdrawal, with rates of cleavage and in vitro development comparable to that of controls. The prolongation of the first cell cycle in bovine NT embryos using this method did not, however, increase cloning efficiency. Arrest for longer periods, at other stages of the cell cycle, and using alternative reagents may be beneficial.
48 MUSCLE CHARACTERISTICS OF BOVINE CLONE OFFSPRING F1 COMPARED WITH CLONES
Authors: Cassar-Malek I. , Heyman Y. , Picard B. , Richard C. , Chavatte-Palmer P. , Jurie C.
Published: August 2010

Information on clone offspring F1 is limited, especially in species with a long inter-generation interval such as cattle. As cloned cattle exhibit a slight delay in muscle maturation until puberty (Jurie et al. 2009 Animal 3, 244-250), the present study aimed to investigate the contractile and metabolic muscle characteristics of F1 at 8, 12, and 18 months of age. Repeated biopsies of the semitendinosus muscle were collected on 10 F1 heifers born after AI of cloned cows at the experimental farm of INRA. Muscle characteristics of these offspring were compared with those of 9 female clones and 8 AI control heifers previously biopsied at the same ages. All animals (clones, F1, and controls) were female Holstein, born and raised under the same conditions in the same farm. Biopsy samples were stored frozen at -80°C until analysis for contractile and metabolic characteristics. The type of contractile fibers was determined from the proportion of the different myosin heavy chain (MyHC) isoforms separated by electrophoresis. Oxidative metabolism was assessed by isocitrate dehydrogenase (ICDH) and cytochrome-c oxidase (COX) activities (μmol min-1 per gram of muscle). Data were analyzed separately for each time of biopsy using the GLM procedure of SAS (SAS Institute, Cary, NC, USA). The statistical model contained the group as fixed effect. When a significant effect was detected, differences between least squares means were further separated by the PDIFF option of SAS. Comparison of contractile characteristics from the 3 groups of animals is presented in Table 1. The proportion of MyHC I (slow oxidative isoform) and MyHC IIx (fast glycolytic isoform) in the muscles of F1 was not significantly different from those of controls at 8 and 12 months of age. F1 had different muscle contractile properties compared with clones at 12 months of age. At 8 months of age, F1 had greater ICDH activity than controls (1.39 ± 0.22 v. 0.54 ± 0.007; P ≤ 0.002) and greater COX activities (11.4 ± 1.6 v. 4.2 ± 0.9; P ≤ 0.003), but this was not observed later on. Altogether, these data indicate that the muscles of F1 were more oxidative than those of controls. This was not related to a higher percentage of type I fibers but rather to a higher percentage of type IIA fibers. A delay in muscle maturation was only partially found in F1.

Table 1.Contractile characteristics (mean ± SE) of the semitendinosus muscle from controls (n = 8), clones (n = 9) and clone offspring (F1, n = 10) heifers
53 EFFECT OF THE TIME INTERVAL BETWEEN OVARY COLLECTION AND OOCYTE IN VITRO MATURATION ON EQUINE CLONED EMBRYO DEVELOPMENT
Authors: Gambini A. , Jarazo J. , Olivera R. , Salamone D.
Published: August 2010

The availability of viable equine oocytes is a limiting factor on in vitro embryo production; therefore, it is necessary to assess some of the variables that affect oocyte viability. The aim of our study was to evaluate one of those variables: the effect of time between the collection of the ovary and oocyte in vitro maturation. Ovaries of slaughtered mares were collected during the breeding season (Argentine, Southern hemisphere). They were separated in bags every half hour and treated separately after arriving at the laboratory. COCs were recovered by a combination of scraping and washing of all visible follicles with a syringe filled with DMEM supplemented with 1 mM sodium pyruvate and 15 IU mL-1 heparin. COCs were matured for 24 to 26 h in 3 groups, according to time interval: 4 to 7 (group I), 7 to 10 (II), and 10 to 12 (III) hours. The medium for maturation was TCM-199 supplemented with 10% fetal bovine serum (FBS), 1 μL mL-1 insulin-transferrin-selenium, 1 mM sodium pyruvate, 100 mM cysteamine, and 0.1 mg mL-1 of FSH at 39°C in a humidified atmosphere of 5% CO2 in air. The cumulus was removed by a trypsin treatment and vortexing in hyaluronidase (1 mg mL-1). Cloning and fusion procedures were performed following the zona-free technique described by Lagutina et al. (2007 Theriogenology 67, 90-98). Two experiments were carried out by using different activation protocols. In experiment 1, the activation process was 22 mM ionomycin in H-TALP for 4 min followed by 3h culture in 1.9 mM 6-DMAP in SOF, whereas in experiment 2, we used 8.7 mM ionomycin in H-TALP for 4 min followed by 4 h culture in 1 mM 6-DMAP and 10 mg mL-1 cycloheximide in SOF. Embryos were cultured in wells of well (WOW) system. Half of the medium was renewed on Day 3 with fresh SOF and on Day 5 with DMEM/F12 with 10% FBS. Cleavage was assessed 48 h after activation; the rate of blastocyst formation was recorded at Days 8 and 9. Results were compared using chi-square test (P < 0.05). In experiment 1, maturation rates were significantly different between group I (n = 135, 54.1%) and III (n = 94, 40.4%), group II did not differ from them (n = 138, 53%). Cleavage rates differed statistically between II (n = 44, 75%) and III (n = 27, 40.7%), but not with group I (n = 53, 98%). No significant differences were found in blastocyst development; however, we observed a certain tendency towards an increase in the blastocyst rate as the time interval was lower (I: 3/53, 5.7%; II: 1/44, 2.3%; III: 0/27, 0%). In experiment 2, there were no significant differences between group I and II in rates of maturation (n = 56, 59% v. n = 111, 44.5%), cleavage (n = 22, 91% v. n = 34, 82%) or blastocyst rates (1/22, 4.5% v. 7/34, 20.6%). We conclude that cloned equine embryo development, using the two activation protocols tested, is not affected when the time interval between ovary collection and oocyte IVM is within 4 to 10 h.
55 EFFECT OF FUSION-ACTIVATION INTERVAL AND EMBRYO AGGREGATION ON IN VITRO AND IN VIVO DEVELOPMENT OF CLONED BOVINE EMBRYOS PRODUCED BY HANDMADE CLONING
Authors: Gerger R. P. C. , Forell F. , Mezzalira J. C. , Zago F. , Vieira F. K. , Machado Jr J. , Olwheiler L. U. , Rauber L. P. , Rodrigues J. L. , Ambrósio C. E. , Miglino M. A. , Mezzalira A. , Bertolini M.
Published: August 2010

Despite the apparent success of cloning by somatic cell nuclear transfer (SCNT), the efficiency in development to term remains low, with a high rate of losses occurring throughout pregnancy due to faulty reprogramming and conceptus abnormalities. As the ideal fusion-activation interval for optimal nuclear reprogramming after cloning is still ill-defined, the aim of this study was to determine the effect of 2 distinct fusion-activation intervals and embryo aggregation on in vitro development of cloned bovine embryos. Bovine COCs from slaughterhouse ovaries were used after IVM for the production of cloned embryos by handmade cloning, according to our established procedures (Ribeiro et al. 2009 Cloning Stem Cells, in press). Following cumulus and zona removal, oocytes were manually bisected, with hemi-cytoplasts selected by DNA staining. Two hemi-cytoplasts and an adult skin somatic cell were attached and fused with a 15V AC pre-pulse for 5 s, followed by a double 1.2 kV cm-1 DC pulse for 20 μs. Reconstructed embryos were activated in ionomycin exactly at 2 or 4 h post-fusion (2 hpf or 4 hpf), followed by an incubation in 6-DMAP for 4 h. Cloned embryos from both fusion-activation intervals were in vitro-cultured in the well of the well (WOW) system for 7 days, allocating one (1 × 100%) or two (2 × 100%) cloned embryos per WOW. Grade 1 Day-7 blastocysts were transferred to synchronous recipients. Cleavage (Day 2) and blastocyst (Day 7) rates, on a per WOW basis, and pregnancy (Days 30 and 150) rates were compared using the chi-square or the Fisher test, with results from 9 replications summarized in Table 1. Increasing the fusion-activation interval to 4 h decreased cleavage but not blastocyst rates in 1 × 100% embryos. Also, blastocyst rates were lower in 1 × 100% embryos activated 2 h post-fusion. In general, cleavage and blastocysts rates for 2 × 100% embryos (91.5 and 46.0%) were higher than for 1 × 100% embryo counterparts (74.4 and 31.3%), respectively, regardless of the activation time. In addition, blastocyst rates for 4 hpf-activated embryos (50.3%), based on cleavage, were higher than for 2 hpf-activated embryos (38.3%), irrespective of the aggregation scheme. Nonetheless, despite differences in in vitro development, pregnancy rates and conceptus development in the first half of pregnancy were similar between groups. A longer fusion-activation interval (4 hpf) or embryo aggregation (2 × 100%) increased blastocyst yield but did not improve in vivo development and pregnancy maintenance following the transfer to female recipients in cattle.

Table 1.In vitro and in vivo development of cloned bovine embryos

This study was supported by FAPESP and CAPES, Brazil.
60 EFFECTS OF REPETITIVE IONOMYCIN TREATMENT ON BOVINE SOMATIC CELL NUCLEAR TRANSFER (SCNT) EMBRYOS
Authors: Jeong S. H. , Kim S. , Choi M. K. , Na J. M. , Choi J. , Jeon Y. P. , Shin T. Y. , Hyun S. H. , Hwang W. S.
Published: August 2010

In bovine somatic cell nuclear transfer (SCNT), oocyte activation is an essential element for the successful development of cloned embryos. Chemical treatment with ionomycin induces transient levels of Ca2+, and this has been used in the activation of reconstructed embryos. In vitro-fertilized oocytes are naturally activated by sperm following several Ca2+ transients known collectively as Ca2+ oscillations. We aimed to improve the developmental efficiency of embryos produced by SCNT by mimicking this Ca2+ oscillation artificially. SCNT was performed as follows; bovine calf skin fibroblast cells were transferred into the perivitelline space of IVM oocytes, and then these NT couplets were treated with electrical fusion (2 pulses, 1.75 kV cm-1, 15 μs). Reconstructed embryos were subsequently cultured in SOF medium (5% CO2, 5% O2, and 38°C). To mimic Ca2+ oscillation, we carried out 3 different repetitive ionomycin (10 μM) treatments at 1-h intervals. There were 3 groups: group 1 (4 min, 1 time), group 2 (30 s, 4 times), and group 3 (1 min, 4 times). The difference in embryo development amongst these experimental groups was then analyzed using the one-way ANOVA test after arcsine transformation to maintain homogeneity of variance. All analyses were performed using SAS (version 8.1, SAS Institute, Cary, NC, USA). Significant differences among the treatments were determined when the P-value was <0.05. In experiment 1, to assess developmental efficiency, the cleavage rate was investigated on Day 2 and the formation rate of blastocysts (BL) was examined on Day 7. In group 3, a significant increase in BL formation was observed [47/263 (17.8%), 50/259 (19.3%), and 67/258 (26.0%), respectively]. In experiment 2, culturing each group of embryos with different ionomycin treatments caused no significant differences among the groups in terms of the total number of BL (164.3, 158.5, and 145.1, respectively). In experiment 3, expression of apoptosis-related genes in each group was evaluated by real-time PCR and the TUNEL assay. The 3 BL within each group were analyzed for the expression of apotosis-related genes. Expression of the anti-apoptotic Bcl-2 gene was increased in group 3, whereas the expression of the pro-apoptotic Bax was decreased. A decrease in the number of apoptotic nuclei was also observed in group 3. In conclusion, the present study demonstrated that repetitive ionomycin treatment is an improved activation method that can increase the developmental competence of SCNT embryos by decreasing the incidence of apoptosis.
61 MITOCHONDRIA DISTRIBUTION IN FERTILIZED, PARTHENOGENETIC, AND CLONED BOVINE EMBRYOS
Authors: Kaiser G. G. , Ross P. J. , Wang K. , Cibelli J. B.
Published: August 2010

In this study we evaluated mitochondrial distribution of individual bovine embryos after IVF, parthenogenetic activation (PG), and somatic cell nuclear transfer (SCNT). COCs obtained from slaughterhouse ovaries were matured in vitro for at least 18 h in TCM-199 supplemented with hormones, and then divided into 3 groups. SCNT and PG oocytes were stripped by vortexing in HEPES-HECM (hamster embryo culture medium) medium (HH) containing hyaluronidase and metaphase II (MII) oocytes selected by visualization of a polar body. The PG group oocytes were exposed 24h post-maturation to 5 μM ionomycin in HH for 4 min, then rinsed 3 times in HH and allocated to a 4-h culture in 2 mM DMAP in KSOM for activation. The SCNT group oocytes were included in a nuclear transfer procedure performed as previously described (Ross et al. 2006 Biotechniques 41, 741-750). Activation was performed as described for the PG group. The IVF group COCs were co-incubated for 20 h with 106 spermatozoa/mL in IVF-TALP supplemented with heparin. To label mitochondria, 1 mM MitoTracker CMXRos Red (Molecular Probes, Eugene, OR, USA) was added to HH at a final concentration of 0.3 μM. Samples were cultured for 15 min, washed in HH, placed in a glass-bottomed 35-mm Petri dish, and then observed and live photographed by using a spinning disk confocal microscope (Nikon Eclipse TE2000-E + CARV Confocal) equipped with a Cascade 512 B camera (Roper Scientific, Tucson, AZ, USA) using a Nikon 40×, 1.3 NA oil objective lens. Z series images were taken acquiring 15 focal planes at 10-μm intervals. Analysis was performed using Metamorph software. Samples were taken at pronuclear, 4 cell, and morula stages. Each sample was classified for its mitochondrial localization in pericytoplasm, cytoplasm, and perinuclear. Data was analyzed by proc glimmix (SAS, Cary, NC, USA). Significance was set at P < 0.05. A similar pericytoplasmic distribution of mitochondria for all treatments up to the 4-cell stage was observed. At the pronucelar stage, mitochondria distribution was mostly pericytoplasmic, changing to cytoplasmic at the 4-cell stage. At the morula stage there was a significantly higher number of embryos with perinuclear distribution in IVF than in PG and SCNT embryos (Table 1). Our findings demonstrate that mitochondrial reorganization differs in fertilized more-developed embryos compared with their activated counterparts. This may have implications for further embryo development, mainly after SCNT.

Table 1.Mitochondria distribution in fertilized, parthenogenetic, and cloned bovine embryos
65 BLASTOCYSTS DERIVED FROM ADULT FIBROBLASTS OF RHESUS MONKEY (MACACA MULATTA) USING INTERSPECIES SOMATIC CELL NUCLEAR TRANSFER
Authors: Kwon D. K. , Kang J. T. , Park S. J. , Gomez M. N. L. , Kim S. J. , Atikuzzaman M. , Koo O. J. , Jang G. , Lee B. C.
Published: August 2010

Interspecies somatic cell nuclear transfer (iSCNT) has alternatively chosen in primate SCNT because of the difficulty in collecting enough oocytes for research. The purpose of this experiment is to produce iSCNT-derived blastocysts using enucleated cow (Bos taurus) metaphase II oocytes and adult rhesus monkey (Macaca mulatta) fibroblasts. Ear skin tissueofrhesus monkey (male, 6 years old) was collected by biopsy and fibroblasts were isolated. Immature COCs from cow ovaries were collected and matured in vitro in TCM-199. Squish enucleation was done in the presence of bisbenzimide and cytochalasin B. After enucleation, a single rhesus monkey somatic cell was injected into the perivitelline space of an enucleated oocyte through the slit in the zona pellucida made during enucleation. Subsequently, the rhesus monkey somatic cell and cow oocyte membranes were electrically fused. The nonactivated interspecies cloned couplets were cultured for 2 h to allow reprogramming to occur. Then, couplets were activated using a 2-step protocol consisting of treatment with 5 μM ionomycin for 4 to 5 min and subsequently with 2mM 6-DMAP for 4 h. Activated iSCNT embryos were cultured for 10 days inmodified SOF with various conditions (at 37 to39°C, 5 to 5.5% CO2 and 5 to 20% O2) to examine the effects ofIVC conditions. As a results, most embryos were arrested at the 8- to 16-cell stage and only 3 blastocysts were derived from rhesus monkey iSCNT. The blastocyst developmental rate was 0.26% generated from the total IVC activated interspecies embryos (n = 1153). Among the 3 blastocysts, 2 of them were used for counting nuclear number using bisbenzimide staining. The nuclear number of the 2 iSCNT-derived blastocysts was 51 and 24, respectively. The other iSCNT-derived blastocyst was used for analyzing mitochondrial (mt)DNAto confirm that it contained both cow and rhesus monkey mtDNA. As a result, mtDNA from both rhesus monkey and cow were detected inPCR analysis. The band intensity was more dominant for cow mtDNA than for rhesus monkey mtDNA. Although the blastocyst developmental rate is extremely low, it is confirmed that two phylogenetically distant species including primate could develop in vitro until the blastocyst stage by iSCNT. The in vitro developmental system of this rhesus monkey iSCNT-derived blastocysts provides a platform for further improvement of developmental rate and quality of rhesus monkey iSCNT-derived blastocysts. It also provides an opportunity to establish rhesus monkey iSCNT-derived embryonic stem cell lines for study of rhesus monkey nucleus and cow mitochondria interaction mechanisms during early developmental stages.

This study was financially supported by the Korean MEST, through the BK21 program for Veterinary Science, and SNU foundation (Benefactor; RNL Bio).
66 ISOLATION, CHARACTERIZATION, AND NUCLEAR REPROGRAMMING OF STEM CELLS DERIVED FROM ADULT PORCINE LIVER AND FAT
Authors: Li P. , Estrada J. , Zhang F. , Waghmare S. , Mir B.
Published: August 2010

Genetic manipulation of the porcine genome to produce genetically modified pigs for various biomedical and agriculture applications has been hampered by the lack of an ideal cell type that can grow in culture for a long period of time and is amenable to various genetic manipulations with high efficiency. The cell type currently used for various genetic manipulations is fetal fibroblast. These cells have very limited life span in culture and the efficiency of gene targeting is very low. There is no report of isolation of functional embryonic stem cells (ESC) from pig that would have been used to produce transgenic pigs with high efficiency as has been possible in mice. Recently, porcine induced pluripotent stem cells (iPSC) have been reported by 3 groups. However, they have yet to be tested for genetic manipulations and production of transgenic pigs. In this study, we developed a simple but novel strategy to recover stem cells from adult porcine liver and adipose tissues. Small colonies with few cells became visible as early as 2 to 3 days under reduced oxygen conditions on collagen-coated plates, and a full-grown colony with a fibroblast-like morphology took 10 to 14 days to form. Ten single colonies per tissue were isolated, subcultured, and monitored for growth and gene expression. Both of these cell types maintained steady growth through 70 population doublings (at the time of writing) and are still growing without any change in their morphology. Reverse transcription PCR was used to monitor gene expression. Both cell types show strong expression of c-Myc and KLF4. Moreover, low expression of Oct-4 and Lin28, 2 important genes related to pluripotency, was also detected in cells derived from adipose tissues, indicating that these cells could be easily reprogrammed to pluripotent state. These cells maintained a normal karyotype after long-term culture. Cell lines with stable genetic modifications and extended expression of transgene were obtained when these cells were transfected with a plasmid containing the neomycin resistance gene and selected under G418. Further, these 2 cell types, liver stem cells (LSC) and fat stem cells (FSC), and fetal fibroblasts (FF) as a control were used as nuclear donors to produce somatic cell nuclear transfer (SCNT) embryos. The average fusion rates were 87, 81, and 89% for LSC, FSC, and FF, respectively. Of 2 recipients receiving nuclear transfer embryos produced with each cell type, one established pregnancy at Day 30 (50%). Efficiencies were 5% (11 fetuses/223 embryos transferred), 1.8% (4 fetuses/228 embryos transferred),and 5% (11 fetuses/219 embryos transferred) for LSC, FSC, and FF, respectively. Thus, these adult liver and fat stem cells are attractive cell types for cloning valuable adult animals with high efficiency and for SCNT transgenesis.
69 EFFECTS OF CELL TYPE, PRE-ACTIVATION PROTOCOL, AND CULTURE CONDITIONS ON DEVELOPMENT OF PORCINE HANDMADE CLONED EMBRYOS
Authors: Mezzalira J. C. , Ohlweiler L. U. , Massie A. , Monaco E. , Silva E. P. , Yuan Y. , Mezzalira A. , Bertolini M. , Krisher R. L. , Wheeler M. B.
Published: August 2010

Despite the rather successful and widespread use of cloning in various species, distinct cell types from the same species and even the same genotype display differences in blastocyst yield. Moreover, variations in the protocol for embryo production can influence development to the blastocyst stage and subsequent fetal development. The aim of this study was to evaluate the effect of 2 cell types and 2 embryo pre-activation protocols with or without the presence of FCS in the in vitro culture medium on development of handmade pig cloned embryos to the blastocyst stage. Cumulus-oocyte complexes recovered from sow ovaries were in vitro-matured for 38 to 40 h. Denuded matured oocytes selected by the presence of a polar body had the zona pellucida removed in a 0.2% protease HEPES-buffered solution +25% FCS, followed by manual bisection and UV screening of enucleated halves using Hoechst stain. Clone embryo reconstruction was performed using a phytohemoagglutinin solution to adhere 2 cytoplasts and a somatic cell. Adipocyte-derived mesenchymal stem cells (ADMSC) from a Yorkshire pig or granulosa cells (GC) from an Ossabaw pig were used as nuclear donors. Following electrical fusion, couplets were pretreated with a brief exposure to cytochalasin B (CB) or cytochalasin B + cycloheximide (CB+CX) in the presence of serum before the electrical activation (Naruse et al. 2007 Theriogenology 68, 709-716; Du et al. 2009 Reprod. Fertil. Dev. 21, 114). Activated embryos were in vitro-cultured in the well of the well (WOW) system, with 2 embryos per microwell, for 7 days in PZM-3 medium +0.3% BSA in the presence (FBS+) or absence (FBS-) of 10% FCS. Cleavage (Day 2, chi-square test) and blastocyst (Day 7, Fisher test) rates, on a per WOW basis, were compared for a level of significance of 5%. Our preliminary data indicate that the presence of serum in the IVC affected cleavage and blastocyst yield in a cell-type-dependent manner. The presence of serum enhanced the blastocyst yield for ADMSC, whereas for GC, only the absence of serum allowed any blastocyst development. The cell type and the pre-activation protocol did not appear to affect cleavage and embryo development to the blastocyst stage. Despite the low number of replications, our results reinforce the importance of optimizing the embryo production system taking into consideration the individual requirements for distinct cell types, procedures, and culture conditions.

Table 1.Effects of cell type, pre-activation process and in vitro culture (IVC) medium on development of handmade pig cloned embryos