bluuebunny/crossref_metadata_2025 huggingface dataset:
258 IMPACT OF MILRINONE AND FORSKOLIN ON THE EFFICIENCY AND QUALITY OF BOVINE OOCYTE IN VITRO MATURATION
Authors: Stachowiak E. , Papis K. , Karasiewicz J. , Modlinski J. A.
Published: December 2012

The efficiency of in vitro maturation (IVM) of bovine oocytes remains inferior compared with maturation in vivo. Recently, some modifications of in vitro maturation (IVM) procedures have been proposed, such as simulated physiological maturation (Gilchrist 2011 Reprod. Fertil. Dev. 23, 23–31). In our experiment, a comparison of the traditional IVM efficiency with maturation after oocyte meiosis inhibition using roscovitine or with a modified two-step maturation using forskolin (cyclic adenosine monophosphate stimulator) and milrinone (type-3 phosphodiesterase inhibitor) was performed. Control oocytes obtained from slaughterhouse-derived ovaries were subjected to the traditional 24-h maturation in TCM-199 medium supplemented with sodium pyruvate, l-glutamine, gentamicin, 10% FCS, and hormones (pregnant mare’s serum gonadotropin and hCG, PG 600, Intervet, Kenilworth, NJ, USA). The roscovitine (50 µM, 24 h) inhibitory treatment was accomplished in the same medium (without hormones) and subsequently, traditional 24-h IVM was performed. The same TCM-199 medium (with hormones) supplemented with forskolin (100 µM) and milrinone (50 µM) was used for the first step (17 h) of the two-step maturation, whereas the second step (7 h) was performed in the same TCM-199 medium devoid of forskolin and milrinone. Fertilization with frozen sperm processed using TALP media was performed in TALP supplemented with heparin, penicillamine, hypotaurine, epinephrine, and BSA. In vitro culture of presumptive zygotes was performed in CR1aa medium. Portions of oocytes from all treatments after maturation and after fertilization procedures were stained and subjected to microscopic analysis. There were no differences in terms of maturation and fertilization rates between treatments. However, roscovitine-mediated inhibition of maturation performed in our experimental conditions was efficient and reversible, but harmful for subsequent embryo development. On the other hand, two-step maturation was equally as efficient as (but not better than) traditional IVM in all aspects examined in the present study (Table 1). In conclusion, the forskolin and milrinone combined treatment during the IVM procedure gives hope for fully efficient IVM. However, to achieve this goal, more research is necessary.

Table 1.Development of embryos after different oocyte maturation procedures1
268 CYCLIC GUANOSINE MONOPHOSPHATE INHIBITS PHOSPHODIESTERASE 3 ACTIVITY IN PORCINE OOCYTES
Authors: Zaffalon F. G. , Guimmelette C. , Leal C. L. V. , Richard F. J.
Published: December 2012

The level of cyclic adenosine monophosphate (cAMP) within oocytes has been shown to play a critical role in maintaining meiotic arrest. High levels of intracellular cAMP prevent spontaneous oocyte maturation in vitro, whereas a decrease in oocyte cAMP is associated with the resumption of meiosis. Another cyclic nucleotide that also was recently proposed as being involved in meiotic resumption is cyclic guanosine monophosphate (cGMP), which could be regulating phosphodiesterase (PDE) 3 activity. The aim of this study was to determine whether cGMP inhibits cAMP-PDE activity in porcine oocytes. With the method described previously by Sasseville et al. (2006 BMC Dev. Biol. 6, 47), PDE activity was measured in groups of 10 oocytes cultured in the absence (control) or presence of different concentrations of cGMP (1, 3, 10, 30, 100, 300, 1000, and 3000 nM) or with the PDE3 inhibitor cilostamide (10 µM). Before assaying PDE activity, the cumulus–oocyte complexes (COC) were matured in vitro for 24 h in the presence of pregnant mare serum gonadotropin (5 IU) and hCG (5 IU) at 38.5°C in 5% CO2. The COC were homogenised in a hypotonic buffer. Data were analysed using one-way ANOVA followed by Duncan’s post-hoc test. Differences with P < 0.05 were considered significant. Results showed that 300, 1000, and 3000 nM cGMP inhibited PDE3 activity (7.9, 5.1, and 4.1 fmol min–1 per COC; P < 0.05) at levels below the controls (13.2 fmol min–1 per COC) and were similar to the activity observed in the presence of (2.4 fmol min–1 per COC; P > 0.05). The other concentrations tested were similar to activity levels seen in the control (1 to 100 nM; 12.2, 11.3, 10.8, 11.5, and 10.4; P > 0.05). In conclusion, the results support the concept that increasing concentrations of cGMP inhibit PDE activity, suggesting the inhibition of the predominant form of cAMP-PDE present in porcine oocytes, PDE3. These results support the hypothesis that cGMP inhibits PDE activity in porcine oocytes. Further work is needed to determine the role this plays in maintaining high cAMP levels and inhibiting oocyte nuclear maturation.
Financial support from FGZ FAPESP 2010/20188-6 and 2010/18023-9 is acknowledged.
276 ACTIVATION OF ROUND SPERMATID-INJECTED OOCYTES USING PHOSPHOLIPASE C ZETA IN PIGS
Authors: Ito J. , Yuhara E. , Nakamura A. , Kashiwazaki N.
Published: December 2012

In several mammalian species, the generation of offspring by round spermatid injection has been reported. However, in domestic species, including pigs, no one has reported success to date. One of the reasons is that round spermatid-injected oocytes require artificial stimuli for oocyte activation, but the developmental ability of the oocytes is low in pigs, suggesting that a more optimal activation protocol is needed. During fertilization, a sperm-derived factor induces repetitive increases in intracellular calcium, known as calcium oscillations. It is now acknowledged that phospholipase C zeta (PLCζ) has an essential role in inducing calcium oscillations, not only in mammals, but also in several other vertebrates. Therefore, if PLCζ is used as a stimulus for oocyte activation, the efficiency of oocyte activation can be improved. Recently, we found that equine PLCζ (ePLCζ) has higher activity than those of other mammalian species to be studied. In the present study, we examined whether injection of ePLCζ complementary RNA (cRNA) improves the activation of round spermatid-injected oocytes in pigs. First, we examined whether ePLCζ is expressed in round spermatids. Porcine round spermatids were isolated from adult testes, and immunostaining using anti-PLCζ antibody was carried out. The PLCζ was localised at the head and tail in mature sperm, and a part of the round spermatid was also stained. Next, we evaluated the developmental ability of round spermatid-injected oocytes activated by different protocols (electrical pulses v. injection of ePLCζ cRNA). The cytoplasts were then injected with round spermatids. One hour later, the oocytes were divided into two groups. In group 1, the oocytes were activated by a direct current pulse (150 V mm–1 and 60 µs). In group 2, the oocytes were injected with ePLCζ cRNA as follows: the reagent (0.1 µg µL–1) was diluted in injection buffer [100 mM KCl and 10 mM HEPES (pH = 7.0)], loaded into glass micropipettes by aspiration, and delivered to the ooplasm by pneumatic pressure (Narishige, Tokyo, Japan). Each oocyte received 3 to 10 pL (1 to 3% of the total volume of the oocyte). After the stimulations, oocytes were cultured in PZM-5 under 38.5°C in a humidified incubator (95% air, 5% CO2). In the ePLCζ-injected group, rates of pronuclear formation (n = 22/32, 68.8%) and blastocysts (n = 2/43, 4.7%) were higher than those in the electrical pulse-treated group (n = 9/41, 22%; and n = 0/51, 0%, respectively; P < 0.05). In conclusion, our data suggest that injection of PLCζ is effective for activation of round spermatid-injected oocytes in pigs.
289 STEM CELL CULTURE AND DIFFERENTIATION IN MICROFLUIDIC DEVICES
Authors: Khetan E. , Maki A. J. , Wheeler M. B.
Published: December 2012

Advancements in micro and nanotechnology have allowed scientists a powerful platform to study biological systems. Microfluidics is one area of advancement with great promise. Microfluidics deals with the behaviour, specific control, and manipulation of microliter and nanoliter volumes of fluid. The small-scale design of these microfluidic devices permits laminar flow, characterised as parallel streams flowing without disruption between currents. With the introduction of micro-technology and microfluidic platforms for cell culture, stem cell research can be put into a new context. Inside microfluidics, microenvironments can be more precisely controlled and they provide a more in vivo-like environment for the cells to grow and hence can serve as a better way of culturing the cells. In the current study, we examined the influence of microfluidic devices on the development of stem cells. Adipose-derived stem cells (ADSC) were isolated from pigs and seeded in a microfluidic device to differentiate toward adipogenic, osteogenic, and chondrogenic lineages using specific differentiation-promoting media (Monaco et al. 2009 Open Tissue Eng. Regen. Med. J. 2, 20–33). Five thousand cells were seeded per channel at a density of 5 000 000 cells mL–1. The microchannel dimensions were 5 mm long, 1 mm wid, and 200 µm deep. Cells were maintained for 14 days and then stained with respective staining dyes: Oil Red O for adipogenesis, Alizarin Red for osteogenesis, and Toluidine Blue for chondrogenesis. Cells differentiated towards adipogenic lineage contained small lipid droplets, which stained red with Oil Red O stain; during osteogenic differentiation, the cells formed large nodules and stained positive for the presence of calcium; and the chondriogenic differentiating cells showed the presence of proteoglycans (blue) when stained with Toluidine Blue. We seeded ADSC in 5 channels for each differentiation lineage, and all channels gave positive staining results. We conclude that microfluidic channels support proliferation and differentiation of ADSC. This system uses small amounts of culture medium, experiments with different culture compositions can be efficiently performed, and culture manipulations can be automated using fluid-handling robotics. Because microfluidics can deal with small number of cells, the characteristics of cellular structure and function and the microenvironment of the stem cells can be understood in a more precise manner. The miniaturization of cell culture platforms allows the observation of cellular behaviour at the scale found in living systems.
305 TRANSPOSON-MEDIATED REPROGRAMMING OF LIVESTOCK SOMATIC CELLS TO INDUCED PLURIPOTENT STEM CELLS
Authors: Talluri T. R. , Hermann D. , Barg-Kues B. , Debowski K. , Behr R. , Ivics Z. , Hall V. J. , Rasmussen M. A. , Hyttel P. , Niemann H. , Kues W. A.
Published: December 2012

The elusive nature of embryonic stem cells in livestock makes reprogramming of somatic cells to induced pluripotent stem (iPS) cells a promising approach for targeted genetic modifications. The first attempts to produce iPS cells from livestock species were made using retro- and lentiviral vectors, which are associated with an increased risk of insertional mutagenesis and which are not easily removable after reprogramming. Here, we describe a nonviral method for the derivation of porcine and bovine iPS cells, using Sleeping Beauty (SB) and piggyBac (PB) transposon systems. The transposons encode the murine or primate reprogramming factors OCT4, SOX2, KLF4, MYC, and LIN28, separated by self-cleaving peptide sequences, respectively. In addition, the PB transposon cassette contains a NANOG-cDNA. The SB or PB transposon-reprogrammed porcine iPS cells expressed typical markers of embryonic stem cells (SSEA1, SSEA4, TRA-1-60, and endogenous stemness genes), showed long-term proliferation under feeder-free culture conditions, differentiated into cell types of the 3 germ layers in vitro, and formed teratomas after subcutaneous injection into immune-deficient nude mice. Both transposon systems are currently being tested in bovine fibroblasts. The results are a major step towards the derivation of authentic porcine and bovine iPS cells, in which the transposon transgenes can be eliminated after reprogramming.
316 EVALUATION OF A HORMONAL PROTOCOL FOR LAPAROSCOPIC OVUM PICK-UP IN PREPUBERTAL EWES
Authors: Teixeira P. P. M. , Padilha L. C. , da Silva A. S. L. , Barros F. F. P. C. , Coutinho L. N. , Brito M. B. S. , Flores F. N. , Silva M. A. M. , Medreiros R. M. , Kirnew M. D. , Da Conceição M. E. M. , Pacheco A. M. , Santos I. R. C. , Vicente W. R. R.
Published: December 2012

In vitro embryo production is a cutting-edge technology in constant evolution and has been used routinely. With the objective of improving genetic value, recent studies have been focused on the production of high-performance descendants from early prepubertal animals. The aim of this study was to compare hormonal protocols of ovarian stimulation for laparoscopic ovum pick-up in prepubertal sheep, to determine ovarian response and number of oocytes. For this study, 36 Santa Ines sheep, aged between 4 and 8 weeks, were submitted to a progestogen-based, short-term ovulation induction protocol (without use of prostaglandin F2α), and following 36 h, ovarian stimulation (300 IU of eCG) associated with different regimens of administration of FSH, according to experimental group. The ewes were divided into 1 of 6 groups: 2 groups received 80 mg of FSH by either single administration in the first group (G80U) or multiple constant administrations at 12-h intervals in the second group (G80M); animals belonging to 2 other groups received 160 mg in the same fashion, in a single-dose regimen (group G160U) or in multiple administrations (group G160M). The other 2 groups constituted the control groups: one received no hormonal treatment for ovulation induction and ovarian stimulation (GCN); the other received the ovulation induction protocol, but no ovulation stimulation was carried out (GCI). The animals were submitted to laparoscopic ovum pick-up 48 h following the beginning of the ovarian stimulation. The number of follicles viewed (FV), aspirated (FA), and recovered (OR) laparoscopically was recorded. Data were assessed using the one-way ANOVA test, and the comparison among groups was carried out using the Tukey test. The mean of follicles visualised, aspirated, and oocytes recovered are shown in Table 1. Group G160U had a better result regarding OR compared with the other groups. Moreover, the current study highlights that laparoscopic ovum pick-up for in vitro maturation using prepubertal ewes is viable for commercial purposes. More studies are required in order to improve the quality of oocytes and successful in vitro maturation rates.

Table 1.Results of visualised follicles (VF), aspirated follicles (AF), and oocytes retrieved (OR)
320 INFLUENCE OF PROGESTERONE AND MOMENT OF ESTRADIOL BENZOATE INJECTION ON MULTIPLE-OVULATION EMBRYO TRANSFER RESULTS
Authors: Kmaid S. , Saldaña J. M.
Published: December 2012

Current superovulatory treatment combines progesterone as well as oestradiol injection at the moment of CIDR insertion to improve synchronization of follicular wave emergence [reviewed by Bo et al. 2002 Theriogenology 57, 53–172; and Mapletoft et al. 2003 J. Anim. Sci. 81 (E. Suppl.), E28–E36] The aim of the present work was to study if delaying oestradiol benzoate (EB) injection 24 or 48 h from CIDR insertion would have the same outcome on embryo production after superovulatory treatment as additional progesterone administered at CIDR insertion. One hundred and eleven superovulatory treatments conducted during the same period (5 consecutive collection days) at random stages of the oestrus cycle at the same location were retrospectively evaluated. Holstein cycling heifers of 18 months of age (body condition score 3.2 ± 0.28, 1–5 range; weighing 357 ± 28 kg) were divided according to the following treatments: control group (n = 24): Day 0: CIDR insertion (Pfizer Animal Health, New York, NY, USA), 50 mg of progesterone, and 2 mg of oestradiol benzoate (Benzadiol, Universal Laboratory, Montevideo, Uruguay); Group BE24 (n = 47): Day –1: CIDR insertion and Day 0: 2 mg of oestradiol benzoate; Group BE48 (n = 40): Day –2: CIDR insertion and Day 0: 2 mg of oestradiol benzoate. All heifers were superstimulated on Day 3.5 (Day 0 = EB injection) with 290 NIH units of FSH (Folltropin, BionicheAnimal Health, Belleville, ON, Canada) in twice-daily decreasing doses over 4.5 days. On Day 6, all cows received 2 doses (a.m. and p.m.) of a prostaglandin F2α analogue (800 µg, Delprostenate, Glandinex, Universal Laboratory), and CIDR were removed on the morning of Day 7. Cows were injected with 10 µg of GnRH (Receptal, Intervet, Boxmeer, the Netherlands) on Day 8 a.m. and were inseminated 12 and 24 h later with semen of 3 bulls of proven fertility. On Day 15, ova/embryos were collected nonsurgically and evaluated in accordance with IETS guidelines. Data were analysed with a negative binomial regression model (Table 1). Administration of EB 24 or 48 h after CIDR insertion produced similar numbers of transferable and freezable embryos as well as total ova/embryos compared with addition of progesterone and EB at the moment of CIDR insertion. According to these results, EB could be administrated without progesterone injection either 24 or 48 h after CIDR insertion without compromising embryo production.

Table 1.Embryo production of heifers superstimulated after additional progesterone (P4) and different times (24 or 48 h after CIDR insertion) of oestradiol benzoate administration (results are mean ± SE)
321 PRECISION GENETIC ENGINEERING OF THE PIG GENOME AND SKIN TRANSPLANTATION WITHIN A SYNGENIC CLONE COHORT CARRYING DIFFERENT VITAL REPORTER TRANSPOSONS
Authors: Kues W. A. , Köhler P. , Diederich M. , Ehling C. , Holler S. , Kahle E. , Niemann H. , Ivics Z. , Garrels W.
Published: December 2012

Farm animals have a long-standing tradition as models in transplantation medicine. The implementation of techniques for precision genetic engineering techniques in pigs will allow to assessing safety and efficacy of novel cell therapies in this large animal model (Garrels et al. 2011 PLoS One 6, e235731). Recently, we generated a syngenic clone cohort of Sleeping Beauty (SB) transposon transgenic pigs by active transgenesis. Eight syngenic pigs carried a monomeric transposon with a Venus cDNA, and 4 animals carried a transposon with an mCherry cDNA; both groups of animals had the same genetic background and differed only in regard to the reporter transgene. In addition, half siblings resulting from conventional breeding were used as control animals (heterologous). To assess immunocompatibility within the clone cohort, skin transplantation between mCherry pigs and Venus pigs was performed. The donors (2 mCherry pigs) and the recipients (3 Venus pigs) were anesthetized and 25-mm2 patches of skin were transplanted under sterile conditions. As control, skin patches of half siblings (heterologous) were transplanted to the Venus recipients. Each recipient was transplanted with 6 small skin patches. The fluorescence reporters Venus and mCherry allowed for unambiguous tracking of cell origin, potential cell fusion, or cell migration in the transplants. Skin patches, which were transplanted between animals of the clone cohort, were accepted within 2 to 3 weeks (n = 14), whereas heterologous skin transplants (n = 4) were rejected. Some of the skin transplants were recovered for fluorescence microscopic analysis after 3 to 6 weeks. Fluorescence microscopy allowed unequivocal discrimination between host cells and cells derived from the transplant in the “wound” zone. Major advantages of this approach are the identical genetic background, omission of an immunosuppressive regime, and direct visual discrimination of transplanted and host cells by different fluorophore reporters. Importantly, no signs of transgene silencing were found for the monomeric SB transposon constructs. These data show that precision genetic engineering is possible in the domestic pig, and that the mCherry and Venus reporters are suitable for long-term cell tracking in transplantation situations. This approach is promising for the development of a preclinical model for novel cell therapies.
332 COMBINATION OF ZINC FINGER NUCLEASES AND MODIFIED BACTERIAL ARTIFICIAL CHROMOSOMES REVEALS TREMENDOUS RATES OF HOMOLOGOUS RECOMBINATION
Authors: Fezert P. , Wuensch A. , Wolf E. , Klymiuk N.
Published: December 2012

DNA-based vectors have been used for decades to modify the genomes of mammalian cells by homologous recombination in a specific and site-directed way. Even though various modifications of the procedure have been presented, efficiency is relatively low for many target sites and novel projects still have an unforeseeable outcome. This is in particularly true for site-directed mutagenesis in primary cells intended for use in the generation of large animal models because of their impaired predisposition for homologous recombination compared with stem cells. The recent development of site-specific nucleases is based on a completely different principle: they do not necessarily involve recombination between DNA strands, but rather make use of the inefficient correction of double-strand breaks in the genomic DNA by the cellular DNA repair machinery after such a double-strand break has been introduced by a synthetic enzyme that directed nuclease activity to a defined site in the genome. Here, we intended to evaluate the potential of zinc finger nucleases (ZFN) to introduce a lacZ reporter gene into the CFTR locus. Initially, the efficiency of 3 different ZFN pairs was examined under different conditions revealing modification efficiencies between 0 and 38%. An optimized protocol was used to combine the most efficient ZFN pair with either a bacterial artificial chromosome (BAC) vector or a conventional targeting vector carrying the desired modification. Although the conventional vector failed to introduce the reporter gene in any of more than 200 clones examined, the BAC correctly modified the target site in 32 of 75 clones in a heterozygous way and in 10 out of 75 clones in a homozygous way. However, the introduction of small vector fragments into the CFTR locus in rare cases indicated that the ZFN caused a double-strand break but the vector was not able to act as a recombination donor. On the other hand, transfection of the BAC alone only resulted in 1 modified clone out of 98 and, thus, our data strongly support the hypothesis that the forced introduction of double-strand breaks dramatically increases the rate of homologous recombination, but they also provide indication that the design of the targeting vector has a profound influence on the efficiency.
34 PRE-IMPLANTATION DEVELOPMENT OF HORSE SOMATIC CELL NUCLEAR TRANSFER EMBRYOS ORIGINATED FROM METAPHASE I OOCYTES
Authors: Lagutina I. , Colleoni S. , Lazzari G. , Galli C.
Published: December 2012

Because of the limited availability of horse oocytes and their wide maturation window that leads to the existence of a subpopulation of oocytes with significantly longer maturation period, horse cloning would benefit from the possibility of also using metaphase I oocytes (Choi et al. 2009 Cloning Stem Cells). The scope of this work was to compare the developmental ability of cloned horse embryos constructed using oocytes in metaphase I (MI) and II (MII). Oocytes of slaughtered mares were matured for 27 h in DMEM/F12 with 10% FCS, 1 µL mL–1 of insulin-transferrin-selenium (ITS), 1 mM sodium pyruvate, 50 ng mL–1 of long-epidermal growth factor, 100 ng mL–1 of long-insulin-like growth factor-I, and 0.1 IU mL–1 each of FSH and LH, denuded of cumulus and enucleated using a zona-free method (Lagutina et al. 2005 Reproduction). Oocytes with a visible polar body were classified as MII and those with no polar body as potential MI. During enucleation, oocytes having a metaphase plate only were confirmed as MI; oocytes in anaphase and telophase were classified as MII. Adult skin fibroblasts of passages 2 to 10 were cultured in TCM 199/DMEM with 10% FCS and serum starved for 1 to 2 days before NT. Nuclear-transfer embryos were constructed after washing of zona-free oocytes in 400 µg mL–1 of phytohemagglutinin P and attachment of each to a single cell in HEPES-SOF by fusion with 2 direct-current (DC) pulses of 1.2 kV cm–1 applied for 30 µs in fusion medium. One hour after fusion, embryos were activated by 5 µM ionomycin for 4 min, followed by culture in 5 µg mL–1 of cycloheximide and 1 mM DMAP in SOFaa for 3 h. Embryos were cultured in SOFaa in 5% CO2 and 5% O2 at 38.5°C. Half of the medium was renewed on Day 3 and replaced on Day 5 with DMEM/F12 with 5% FCS and 5% serum replacement. Cleavage was assessed 48 h after activation and the rate of blastocyst formation was recorded at Day 8. The data were compared by chi-square test. The development ability of MI embryos assessed by cleavage and blastocyst formation was significantly lower than of MII embryos (Table 1). The obtained MI blastocysts were smaller than their MII counterparts. These data demonstrate that MI oocytes account for 20% of the total oocytes after 27 h of maturation, have a lower developmental competence to form blastocysts after NT, and the blastocysts obtained are of smaller size and likely less viable. Therefore, the use of MI oocytes can only marginally improve the outcome of horse cloning.

Table 1.Embryo development after SCNT using oocytes in metaphase I or II
MULTI-TRANSGENIC PIGS FOR XENOTRANSPLANTATION
Authors: Ayares David
Published: December 2012

Successful development of somatic cell nuclear transfer (cloning) technology in pigs has allowed for precise genetic manipulation of the pig genome. For xenotransplantation applications, pigs have been produced in which both copies of the α1,3-galactosyl transferase (GT) gene were inactivated (GTKO pigs). Analysis of tissues from GTKO pigs demonstrated a complete lack of immunogenic Galα1,3Gal (Gal) sugars, while in vivo pre-clinical studies in nonhuman primates, using cells (i.e. pancreatic islets) or whole organs (heart, kidney, liver, lung), demonstrated the elimination of hyperacute rejection, and prolonged survival compared to wild-type controls. While survival of GTKO xenografts was extended, challenges including induced antibody responses to non-gal antigens, thrombosis, inflammation, and cell-mediated rejection remained, pointing to the need for further genetic modification of the source pig. Towards this goal, through a combination of cloning and breeding, in combination with GTKO, we have produced multi-transgenic pigs (some with 5 different transgenes) with controlled expression of genes for (1) complement regulation to address the humoral response to anti-non-gal targets (DAF, CD46); (2) inhibition of inflammation and thrombosis (TFPI, CD39, thrombomodulin, EPCR); and (3) local protection against the human cellular response (CTLA4Ig, CIITA-DN). For some transgenes a constitutive promoter system can be used for expression in all tissues, such that one animal can be used for multiple transplant applications, however, our results have shown that for certain transgenes, tissue-specific gene expression is preferred. Since inhibition of thrombosis, complement modulation, and suppression of T-cell responses are important to delayed xenograft rejection of both whole organs and islet cell xenografts, pigs have been produced with tissue-specific transgene expression in either the vascular endothelium or endocrine pancreas compartments, or constitutively in all tissues. In vivo results in nonhuman primates have demonstrated complete normalization of blood glucose for up to 1 year in diabetic monkeys, and 8-month survival of multigenic pig hearts in baboons, evidence for the promise of this technology for human clinical applications.
40 PIG CLONING AND GENE EXPRESSION PATTERNS IN MINIPIG ADIPOSE TISSUE-DERIVED MESENCHYMAL STEM CELLS
Authors: Kim M. J. , Oh H. J. , Park J. E. , Kim G. A. , Park E. J. , Choi J. , Moon J. H. , Rhee S. H. , Kim T. , Lee B. C.
Published: December 2012

In several laboratory animals and humans, adipose tissue-derived mesenchymal stem cells (ASC) are very interesting; they are easy to harvest and can expand to generate millions of cells from a small quantity of fat. The ASC are known as useful materials for clinical applications in human cell therapy and as a donor cell in somatic cell nuclear transfer (SCNT). In this study, we investigated the expression patterns of several genes (Oct-4, Nanog, Sox2, Dnmt1, and Dnmt3b) in minipig ASC, and whether ASC can be a suitable donor cell type for producing cloned pigs. For the study, we respectively isolated ASC, adult skin fibroblast (ASF) and fetal fibroblast (FF) from a 6-year-old female minipig. The ASC were attached to a plastic dish with a fibroblast-like morphology, expressed cell-surface marker characteristics of stem cells, and underwent osteogenic, adipogenic, and neurogenic differentiation when exposed to specific differentiation-inducing conditions. To observe gene expression, total RNA was extracted from ASC, FF, and ASF, respectively, and was used for reverse transcription (RT). After RT, real-time PCR was performed to investigate the expression of Oct-4, Sox2, Nanog, Dnmt1, and Dnmt3b. The expression of β-actin was measured and used as an endogenous control. In the following experiment, we carried out SCNT using ASC, ASF, and FF. The ratio of blastocysts to 2-cell embryos and total cell number of blastocysts were monitored as experimental parameters. Statistical analysis was performed using one-way ANOVA (GraphPad Prism version 5). As a result, the relative abundance of DNMT1 in ASC (1.9 ± 0.9) was significantly higher than that in FF and ASF (0.1 ± 0.2 and 1.0 ± 0.5, respectively; P < 0.05), but no significant difference in expression of the DNMT3b gene was observed. Interestingly, the quantity of Oct-4 was significantly higher in FF and ASC than in ASF (2.8 ± 0.4 and 2.9 ± 0.5 v. 1.0 ± 0.1, respectively; P < 0.05), and Sox2 showed significantly higher expression in ASC (3.7 ± 0.5) than in ASF and FF (1.0 ± 0.1 and 1.4 ± 0.6, respectively; P < 0.05). Nanog expression was similar in ASF, FF, and ASC. After SCNT, the developmental competence to blastocysts did not differ among the 3 groups (ASF: 7.0 ± 0.2%, FF: 16.15 ± 6.1%, and ASC: 11.1 ± 0.7%). However, total cell numbers of blastocysts derived from ASC and FF were significantly higher in ASF (89.0 ± 7.9 and 105.0 ± 5.5 v. 57.5 ± 5.2, respectively). In conclusion, the present study revealed that minipig ASC and minipig FF possess slightly different gene expression patterns and ASC have potential in terms of in vitro development and blastocyst formation ability similar to ASF and FF.
This study was supported by IPET (no. 311011-05-1-SB010), RDA (no. PJ0089752012), RNL Bio (no. 550-20120006), Institute for Veterinary Science, and the BK21 program.
45 EFFECT OF DEACETYLASE INHIBITOR VALPROIC ACID ON BOVINE CULTURED SOMATIC CELLS
Authors: Bai L. , Li X. , Liu Y. , Wei Z. , Li G.
Published: December 2012

Normal development depends on a precise sequence of changes in the configuration of chromatin. Epigenetic modifications, such as histone acetylation and methylation and DNA methylation, control the precise tissue-specific gene expressions. Valproic acid (VPA) is an aliphatic acid compound and a deacetylase inhibitor that influences cellular proliferation, differentiation, apoptosis, and migration. Several studies have shown that treatment of fused, cloned embryos with VPA improved embryo development. However, whether the treatment of donor cells with VPA affected cloned embryo development was not clear. In the present study, bovine fibroblast cells were treated with VPA at 0, 0.25, 0.5, 1.0, 2.0, and 4.0 mM concentrations for 24 and 48 h, respectively. The cell growth, cell cycle, diploid composition, and histone modifications were examined. The results showed that (1) when the cells were treated with VPA at and over 2.0 mM concentrations for 24 or 48 h, cell growth was significantly inhibited; (2) VPA treatment resulted in a decrease in cell diploid composition; (3) VPA induced the arrestment of cells at the G0/G1 stages; (4) VPA treatment increased early cell apoptosis; (5) VPA at a 4.0 mM concentration and treatment of cells for 24 h improved H3K9 acetylation; however, treatment of cells with VPA decreased H3K9 methylation in all experimental groups; and (6) when the cells treated with VPA at 0, 0.25, 0.5, 1.0, and 2.0 mM concentrations for 24 h were used as donor cells for NT, the cleavage rates were 83.2, 80.0, 82.1, 80.5, and 65.5%, respectively, and the percentages of blastocyst development were 30.5, 29.6, 29.2, 25.0, and 15.5%, respectively. When the cells were treated with VPA at 4.0 mM concentration, the cleavage (65.5%, 76/116) and blastocyst development (15.5%, 18/116) significantly decreased as compared with the control [83.2% (158/190) and 30.5% (58/190), respectively]. The cell numbers of the blastocysts derived from VPA treatment at 0, 0.25, 0.5, 1.0, and 2.0 mM concentrations were 89.6, 88.6, 87.6, 75.0, and 71.3, respectively, which decreased with the increase in VPA concentration. In conclusion, VPA affected cell growth and histone modifications in a concentration-dependent manner. The VPA-treated cells did not improve the cloned embryo development and even decreased blastocyst when VPA was at a high concentration.
This work was supported by the National Basic Research Program of China (no. 2012CB22306).
60 THE EFFECT OF L-ASCORBIC ACID DURING CULTURE, CRYOPRESERVATION, OR BOTH ON PORCINE EMBRYOS PRODUCED IN VITRO
Authors: Castillo-Martín M. , Yeste M. , Morató R. , Mogas T. , Bonet S.
Published: December 2012

The benefits of adding l-ascorbic acid during the cryopreservation procedure have been reported before in mouse and bovine. In this study, the effects of l-ascorbic acid (AC) supplementation during culture, cryopreservation, or both procedures on the developmental ability and embryo quality of in vitro produced porcine blastocysts were examined. Embryo quality criteria consisted of total cell number, percentage of apoptosis, and cryotolerance. After in vitro fertilisation, presumptive zygotes were randomly assigned to 2 culture treatments in which the culture medium NCSU23 was supplemented with 100 µM AC (n = 1162) or nonsupplemented (n = 1163) for a 144-h period. On Day 6, blastocyst formation was assessed by stereomicroscopy, and a representative fraction of Grade I- and II-blastocysts of each culture treatment was evaluated using 4′,6-diamidino-2-phenylindole-TUNEL co-staining and considered as fresh-control. The remaining fraction of Grade I- and II-blastocysts was vitrified/warmed following the Cryotop® method. To determine the effect of AC supplementation during cryopreservation procedures, each culture treatment was divided into 2 groups: (1) embryos exposed to 100 µM AC, and (2) nonexposed embryos (vitrified-control). Survival was determined according to reexpansion rates after 24 h of recovery in NCSU23 medium. After 24 h, reexpanded blastocysts were co-stained using the 4′,6-diamidino-2-phenylindole-TUNEL technique, and total number of cells and apoptosis indexes were determined. Experiment was replicated 9 times for each group. Data were analyzed by t-test for independent variables and a 2-way ANOVA. Results are expressed as means ± SE, and the significant level was set at 5% (Table 1). After culture, supplementing NCSU23 medium with AC showed no significant differences in blastocyst formation (fresh-control 11.6 ± 7.8 v. AC 11.6 ± 7.7), in number of cells (fresh-control 36.7 ± 15.8 v. AC 36.1 ± 15.9), or in apoptosis index (fresh-control 2.9 ± 5.7 v. AC 3.5 ± 4.7). On the other hand, only when both culture and vitrified media were supplemented with AC was there a significant increase of blastocyst survival. In contrast, no significant differences in embryo survival were observed when only 1 of these 2 media (culture or vitrification) was supplemented. Supplementing culture media or cryopreservation solutions with AC did not affect the total cell number or apoptosis index in vitrified blastocysts. In conclusion, the addition of 100 µM l-ascorbic acid to the culture and cryopreservation solutions improves the cryotolerance of in vitro-produced porcine blastocysts.

Table 1.Survival of blastocysts (24 h), total cell number, and percentage of apoptosis after vitrification/warming
69 AN EASY-TO-PERFORM METHOD TO ASSESS VIABILITY OF FELINE FREEZE-DRIED SPERM
Authors: Magalhães L. C. O. , Melo-Oña C. M. , Sudano M. J. , Paschoal D. M. , Crocomo L. F. , Ackermann C. L. , Villaverde A. I. S. B. , Landim-Alvarenga F. C. , Lopes M. D.
Published: December 2012

Freeze-drying sperm seems to be the ultimate alternative to preserve sperm cells from endangered species because of its easiness in transportation and storage of samples. This technique has already been used for equine, bovine, murine, rabbit, canine, and feline sperm cells. However, it is still important to verify the DNA integrity of such samples to produce viable offspring. The objective of this study was to verify the reliability of acridine orange (AO) staining to assess DNA damage of feline freeze-dried sperm samples. Three normospermic cats were used as sperm donors, and sperm samples were collected with the use of an artificial vagina without the presence of an oestrous queen. The control group (G1) used only fresh semen. To increase the range of sperm variability before freeze-drying, the sperm samples were supplemented with 2 extenders, namely SOF (G2) and human tubal fluid (G3). These extenders were selected because of their lack of cryoprotectant agents with a view to producing membrane damage and thus putting DNA integrity at risk. Samples were taken to a freeze-dryer (Edwards do Brasil, Brazil) to produce stable free-dried sperm. Over 50 samples were assessed for DNA damage by using AO and alkaline comet assay (CA). Acridine orange taints red/orange the sperm cells that present sperm damage, and CA shows long comet tails for cells with DNA damage. The CA was selected to countercheck the AO results owing to its higher accuracy. Nevertheless, both AO and CA count 100 cells per analysis, and a total of 3 repetitions per sample were sufficient. For the statistical analysis, SAS PROC GLM was used, and the significance level was set at 0.05. The AO staining showed that the G1 samples had a greater DNA damage (27.1%) if compared with G2 (15.2%) and G3 (23.1%; P < 0.0001). Nevertheless, G2 and G3 displayed sufficient DNA integrity throughout the whole lyophilization process. The CA, which is a more precise evaluation technique, proved that the groups had distinguished results but still were ordered in the same way: G1 was the one with more DNA damage (17.7%), G3 was second (14.4%), and G2 was third (3.2%) in DNA alterations. Just like AO, the CA showed difference among treatments (P < 0.0001). Since AO is a technique that requires very little sophistication and presented about the same results as the very elaborate CA method, it might be used on a daily basis to assess feline sperm DNA that underwent the freeze-drying process.
7 LAPAROSCOPIC INTRAUTERINE AND INTRAPERITONEAL INSEMINATION IN FAT-TAILED EWES
Authors: Niasari-Naslaji A. , Akbarinejad V. , Hoseinipajooh K. , Akbari G. , Gharibi S. , Arabha H.
Published: December 2012

The present study was conducted to evaluate laparoscopic intrauterine and intraperitoneal AI in a fat-tailed sheep breed. Multiparous and nulliparous (n = 72) healthy Shall ewes (Iranian native breed) were assigned into 3 experimental groups to be inseminated with 3 laparoscopically insemination methods, including intrauterine insemination with frozen semen (IUIFZ; n = 34), intraperitoneal insemination with fresh semen (IPIFR; n = 21), and intraperitoneal insemination with frozen semen (IPIFZ; n = 17). To perform the experiment easily and control confounding factors, the experiment was conducted in 5 replicates. In each replicate, similar numbers of ewes belonging to each experimental group were included, synchronized, and inseminated with the fresh or frozen semen collected from the same fertile ram. Estrus was synchronized using CIDR for 14 days. Immediately after CIDR removal, all ewes received 400 IU of pregnant mare serum gonadotropin. Inseminations with 300 million total spermatozoa (≥70% progressive forward motility) were performed 51.1 ± 0.64 h after CIDR removal. Intrauterine insemination was conducted at the tip of the uterine horn, ipsilateral to the ovary having a dominant follicle. In the case that both ovaries had a dominant follicle, half of the straw was deposited in each horn. In 2 other experimental groups (IPIF and IPIR) either fresh or frozen semen was deposited on the surface of ovaries having a dominant follicle. Pregnancy was diagnosed with transrectal ultrasonography on Day 30 after AI. Data were analyzed using logistic regression. The pregnancy rate in the IUIFZ group (51.7%) was significantly higher than in IPIFR and IPIFZ experimental groups (17.6 and 0%, respectively; P < 0.01), and there was significant difference between IPIFR and IPIFZ in pregnancy rate (P < 0.05). In conclusion, the laparoscopic intrauterine insemination method can be applied effectively in Shall fat-tailed sheep. Laparoscopic intraperitoneal inseminations with fresh or with frozen semen are not recommended for AI in fat-tailed sheep.
71 BOVINE SPERM DEATH KINETICS: CHANGES IN MOTILITY, ACROSOMES, AND PLASMA MEMBRANE
Authors: Ahmad M. , Ahmad N. , Riaz A. , Anzar M.
Published: December 2012

Extent and timing of alterations in structures and functions of sperm after its placement in the female reproductive tract are important for successful fertilization. To our knowledge, the few reports are available on the kinetics of alterations in bovine sperm structures and functions during pathway to their death. Therefore, the present study was conducted to determine the changes in motility, acrosome and plasma membrane asymmetry in fresh and frozen–thawed semen during incubation at 37°C over the period of 24 h. Semen was collected from 3 breeding beef bulls, pooled, and considered as one replicate (total replicates = 5). Each pooled semen sample was diluted in Tris-citric acid egg yolk glycerol extender (pH 6.8), cooled to +4°C over 90 min, and then cryopreserved by a programmable cell freezer. Fresh (pooled semen) and frozen–thawed semen were incubated at 37°C for 24 h. Each semen sample was evaluated for sperm motility with computer-assisted semen analysis and acrosomal integrity and plasma membrane asymmetry using fluorescein isothiocyanate-peanut agglutinin/propidium iodide and Annexin V/propidium iodide assays, respectively, at 0, 2, 4, 6, 12, and 24 h of incubation at 37°C, with a flow cytometer. Statistical analysis was conducted using PROC MIXED model in statistical analysis system as 2 (semen types) × 6 (times) factorial model, using time as repeated measure. Progressive motility was higher (P < 0.05) in fresh than in frozen–thawed semen until 6 h. Progressive motility declined (P < 0.05) below the threshold level (i.e. 30%) much later (12 h) in fresh as compared with frozen–thawed semen (2 h). However, acrosomal integrity and plasma membrane asymmetry deteriorated (P < 0.05) below threshold at the same time interval (2 h) in both fresh and frozen–thawed semen. Viable sperm (AN–/PI–) remained higher (P < 0.05) during the first 6 h in fresh than in frozen–thawed semen and declined (P < 0.05) below the threshold at 12 h in fresh and at 6 h in frozen–thawed semen. In fresh semen, the necrotic sperm (AN–/PI+) population increased (P < 0.05) over time and reached maximum (97%) at 24 h. In frozen–thawed semen, a mixed population of late apoptotic (53%) and necrotic (34%) sperm was found at 24 h. In conclusion, the alterations in sperm motility, acrosomes, plasma membrane integrity, and asymmetry were slower in fresh than in frozen–thawed semen. Fresh sperm followed necrosis and frozen–thawed sperm underwent necrosis and apoptosis-like pathways, respectively.
This study was supported by the Canadian Commonwealth Scholarship Program by the Canadian Bureau for International Education (CBIE), and Agriculture and Agri-Food Canada.
93 EXPOSURE TIME IS CRITICAL IN BLASTOCYSTS ADAPTATIONS TO BRANCH-CHAIN AMINO-ACID DEPLETION
Authors: Velazquez M. A. , Eckert J. J. , Fleming T. P.
Published: December 2012

Decreased branch-chain amino-acids (BCAA) availability during the preimplantation period has been linked with adverse phenotypes (e.g. high blood pressure) in mouse offspring born to females subjected to protein undernutrition. This study aimed to determine the effects of decreasing (50%, L-bcaa) or omitting (0%, 0-bcaa) BCAA during in vitro culture on preimplantation embryo development and blastocyst outgrowth formation. The control medium (100%, N-bcaa) consisted of KSOM supplemented with serum insulin (1 ng mL–1) and uterine luminal fluid amino-acid concentrations [including BCAA: valine (0.46 mM), isoleucine (0.21 mM), and leucine (0.32 mM)] found in MF1 mice fed with normal levels of protein (18%). Groups of 2-cell (n = 194 per treatment) and 8-cell (n = 180 per treatment) embryos from nonsuperovulated mice were cultured for 66 and 44 h, respectively. Resultant late blastocysts were either subjected to differential cell staining (10 replicates) or individually cultured in N-bcaa medium for a further 96 h for outgrowth formation (6 replicates). Treatment groups were compared within 2-cell or 8-cell embryos by nonparametric tests (Kruskal-Wallis, Mann–Whitney, chi-square, or Fisher exact test). Blastocyst formation was not affected by BCAA depletion in the 2-cell group. However, in the 8-cell group, more (P = 0.016) blastocysts were hatching (27.7%) and less (P = 0.010) at the late blastocyst stage (60.5%) when BCAA were omitted compared with N-bcaa (17.2% hatching; 73.3% late blastocysts). Decreasing BCAA did not affect development to late blastocysts or hatching (68.8 and 23.3%). Blastocysts derived from 2-cell embryos cultured in L-bcaa (n = 69) had less total and trophectoderm cells than did those cultured in N-bcaa (n = 72; total cells: 63.75 ± 1.47 v. 68.28 ± 1.48; P = 0.026; trophectoderm: 53.74 ± 1.41 v. 58.06 ± 1.44; P = 0.025). Omitting BCAA (n = 71) did not affect total (64.7 ± 1.43) or trophectoderm (55.14 ± 1.49) cell number in 2-cell-derived blastocysts. Cell numbers in blastocysts derived from 8-cell embryos were not altered by BCAA treatment. Similarly, cell numbers of the inner cell mass were not affected by BCAA treatment in either embryo category. At 48 h after plating for outgrowth formation under N-bcaa conditions, hatching and attachment were not affected by prior BCAA depletion. Active spreading was slightly advanced after L-bcaa treatment in 2-cell outgrowths compared to N-bcaa (52.1 v. 26.9%; P = 0.086), whereas 8-cell outgrowths were not affected by BCAA treatment. These results suggest that blastocyst phenotype and trophectoderm cell proliferation before implantation are sensitive to BCAA depletion and that initial blastocyst activation might be promoted in embryos sensing a lack of BCAA during the preimplantation period. These adaptations depend on exposure time and duration of BCAA depletion, thus potentially contributing to triggering specific developmental consequences.
Funded by BBSRC BB/1001840/1.