bluuebunny/crossref_metadata_2025 huggingface dataset:
142 EFFECT OF HIGH AND LOW ANTRAL FOLLICLE COUNT IN PUBERTAL BEEF HEIFERS ON IVF
Authors: Chase C. C. , Wright E. C. , McNeel A. K. , Cushman R. A. , Perry G. A. , Cupp A. S. , Vallet J. L. , Sypherd D. D. , Miles J. R.
Published: December 2013

Pubertal heifers can be classified between those with high (n = 25) or low (n = 15) antral follicle counts (AFC). The objective of this study was to determine oocyte development and maturation (e.g. fertility) in an IVF system for high- and low-AFC heifers. From a pool of 120 heifers, 10 high- and 10 low-AFC heifers were determined by transrectal ultrasonography; all heifers with evidence of oestrous cyclicity (i.e. pubertal) were synchronized with two 5-mL injections of prostaglandin F2α 11 days apart. Heifers were euthanized over 4 days on Days 15 to 16 of the synchronized oestrous cycle. A total of 15 heifers (n = 7 high and n = 8 low AFC) were at the appropriate stage of the oestrous cycle. Ovaries were collected and transported to the laboratory. Follicles less than 8 mm in diameter were aspirated. The IVF procedures and media were as previously described (Miles et al. 2004. Biol. Reprod. 71, 1919–1926). Cumulus-oocyte complexes (COC) were identified and washed in oocyte collection medium and then in maturation medium and were cultured (5% CO2; 38.5°C) for 24 h. Following maturation, COC were transferred and washed in fertilization medium. Thawed frozen semen from a crossbred bull was subjected to the swim-up procedure. Motile spermatozoa were collected and added to COC to yield a final concentration of spermatozoa per milliliter of fertilization medium. About 24 h later, presumptive zygotes were washed in development medium, placed in microdrops of development medium, and cultured for 8 days. On Days 3 and 8 after fertilization, cleavage and blastocyst development, respectively, were assessed. Data were analysed using the Proc Mixed procedure of SAS (SAS Institute Inc., Cary, NC, USA) and the model included the effects of day of collection (n = 4), group (n = 7 high- or n = 8 low-AFC heifers), and the interaction. The interaction did not differ (P = 0.10). Day of collection influenced (P < 0.05) the number of COC and the number of oocytes cleaved. High- compared to low-AFC heifers had the greater (P < 0.05) numbers of COC (42.7 ± 4.66 v. 22.1 ± 4.59), oocytes that cleaved (28.1 ± 3.60 v. 15.9 ± 3.55), and developed to blastocysts (13.2 ± 1.71 v. 6.2 ± 1.69). However, there was no difference (P > 0.10) in the percentage of COC that cleaved (65.3 ± 5.58 v. 66.2 ± 5.50%, high v. low, respectively) or that developed to blastocysts (46.7 ± 6.75 v. 42.2 ± 6.65%). In conclusion, AFC did not appear to affect oocyte maturation and development through the blastocyst stage.
15 THERMORESISTANCE SPERM TEST FOR EPIDIDYMAL STALLION SPERM FROZEN WITH TWO CRYOPROTECTANTS
Authors: Mellisho E. , Olmos R. , Ancco E. , Celiz H. , Quispe C.
Published: December 2013

The Peruvian Paso Horse has become a flagship product of Peru, for which it is very important to work on promoting and preserving their genetic material through semen. The objective of this study was to evaluate the thermoresistance of epididymal sperm of stallion frozen with 2 cryoprotectants (dimethylformamide and glycerol). The testes with the epididymides (n = 16) of 8 adult stallions in good health condition were obtained in the slaughter house and transport at isothermal conditions (30°C). Spermatozoa from the epididymis were recovered and incubated with lactated ringer's solution (Hartman solution) for 30 min, centrifuged at 2000 RPM for 15 min, and resuspended with an extender based on sucrose (7%) and egg yolk (3%) with different levels of cryoprotectants: dimethylformamide-DMF (T4%), glycerol-GLI (4%), and a combination of dimethylformamide-DMF (2%) + glycerol-GLI (2%) with an equilibrium time of 20 min at 5°C. The final dilution was 160 million motile spermatozoa placed on 0.5-mL straws. These straws were kept over liquid nitrogen fumes for 15 min over a tripod stand. After cryopreservation, the straws were stored in liquid nitrogen tanks (–196°C). The thawing process of the straws was initiated at 75°C for 7 s, immediately followed by an evaluation of sperm motility at 4 times 0, 30, 60, and 90 min in which sperm was incubated on water bath at 37°C (thermoresistance test). The data was evaluated (ANOVA) with the SAS 9.12 program (SAS Institute Inc., Cary, NC, USA) and, for the media, Tukey's test was carried out. The results showed an effect of the treatment on the motility of the frozen-thawed semen; dimethylformamide, the cryoprotectant, had the best performance.

Table 1.Motility of frozen-thawed stallion epididymal sperm (thermoresistance sperm test)1
154 THE EFFECT OF ZINC ON PORCINE IN VITRO MATURATION AND SUBSEQUENT EMBRYONIC DEVELOPMENT AFTER IN VITRO FERTILIZATION
Authors: Jeon Y. , Yoon J. D. , Cai L. , Hwang S. U. , Kim E. , Lee S. , Hyun S. H.
Published: December 2013

Zinc (Zn) is one of the abundant transition metals in biology and is an essential component of most cells. However, there are few reports about the effect of Zn in porcine oocytes. The objective was to investigate the effects of supplementary Zn during in vitro maturation (IVM) of porcine oocytes. We investigated nuclear maturation, intracellular glutathione (GSH) levels, reactive oxygen species (ROS) levels, and subsequent embryonic development after IVF. Before the experiment, Zn concentrations in IVM medium and body fluids were measured using inductively coupled plasma spectrophotometer (sensitivity: 1 μM) and treatment concentrations were determined. Zinc concentration was 12.6 μM in porcine plasma and 12.9 μM in porcine follicular fluid. We confirmed that Zn was not detected in IVM medium. A total of 541 cumulus–oocyte complexes (COC) were used for the evaluation of nuclear maturation. The COC were matured in TCM-199 medium supplemented with various concentrations of Zn (0, 6, 12, 18, and 24 μM). After 44 h of IVM, no significant difference was observed in all groups (metaphase II rate: 85.7, 88.7, 90.4, 90.3, and 87.2%, respectively). A total of 100 matured oocytes were examined for the effects of different Zn concentrations (0, 6, 12, 18, and 24 μM) on porcine oocyte intracellular GSH and ROS levels, which were measured through fluorescent staining and image analysis program. The groups of 12, 18, and 24 μM showed a significant (P < 0.05) increase in intracellular GSH levels (1.45, 1.67, and 1.78, respectively) compared with the control and 6 μM group (1.00 and 1.08, respectively). The intracellular ROS level of oocytes matured with 12, 18, and 24 μM (0.82, 0.68, and 0.55) were significantly (P < 0.05) decreased compared with the control and 6 μM groups (1.00 and 1.03, respectively). Finally, the developmental competence of oocytes matured with different concentrations of Zn (0, 6, 12, 18, and 24 μM) was evaluated after IVF. There were no significantly different in cleavage rates. However, cleavage patterns and blastocyst (BL) formation were different. Fragmented embryo ratio of the 12 μM group (14.9%) was significantly lower than that of the other groups (control, 6, 18, and 24 μM: 26.4, 17.8, 18.4, and 18.0%, respectively). Oocytes treated with 12 μM Zn during IVM had a significantly higher BL formation rate (28.2%) after IVF compared with the control (19.8%). In conclusion, these results indicate that Zn treatment as body fluid concentration during IVM improved the developmental potential of IVF in porcine embryos by increasing the intracellular GSH concentration and decreasing the ROS level.
This work was supported, in part, by a grant from the Next-Generation Bio Green 21 Program (No. PJ00956901), Rural Development Administration, and the National Research Foundation of Korea Grant funded by the Korean Government (NRF-2012R1A1A4A01004885, NRF-2013R1A2A2A04008751), Republic of Korea.
16 SPERM DISTRIBUTION AFTER LAPAROSCOPIC INTRAUTERINE INSEMINATION OF FROZEN GOAT SEMEN
Authors: Anakkul N. , Suwimonteerabutr J. , Tharasanit T. , Khunmanee S. , Diloksumpan P. , Berg D. K. , Techakumphu M.
Published: December 2013

Generally, laparoscopic intrauterine insemination (LII) provides a higher success rate than that of cervical insemination in goat. However, the sperm distribution following LII in goat remains unknown, especially when frozen semen is used. This study aimed to evaluate the distribution of frozen-thawed goat sperm after LII. In experiment 1, the frozen-thawed sperm were stained with CellTrackerTM Green CMFDA (CT-Green) or CellTrackerTM Red CMPTX (CT-Red), and then evaluated in vitro for viability and motility at 0, 3, 6, and 9 h after staining. In experiment 2, the CT-Green and CT-Red labelled sperm (30 × 106 sperm in 0.125 mL in each side) were laparoscopicaly inseminated into the left and right uterine horns, respectively (n = 4). After ovariohysterectomy at 6 h post-insemination, the distributions of green and red coloured sperm were assessed by tissue section and sperm flushing. The comparison of semen parameters among groups (control, CT-Green, and CT-Red) were statistically analysed by a general linear model. The results revealed that the fluorochromes used in this study did not impair the sperm motility and viability (P > 0.05). The frozen-thawed goat sperm transuterine-migrated following LII, as both CT-Green (left insemination) and CT-Red labelled sperm (right insemination) were found in both sides of the reproductive tracts. This study concludes that LII in goat would be simpler and consume less time if the semen was unilaterally deposited.
Supported by a grant from Chulalongkorn University Centenary Academic Development Project and RGJ-PhD-industrial link program, Thailand Research Fund (PHD/0156/2550).
18 DEVELOPMENTAL COMPETENCE OF CLONED PORCINE EMBRYOS PRODUCED WITH DIFFERENT CLONING PROCEDURES
Authors: Liu Y. , Lucas-Hahn A. , Petersen B. , Li R. , Hassel P. , Ziegler M. , Li J. , Larsen K. , Niemann H. , Callesen H.
Published: December 2013

Two nuclear transfer (NT) techniques are routinely used to produce cloned animals, traditional cloning (TC) and handmade cloning (HMC). The TC embryos keep their zona and can be transferred at early stages, whereas HMC embryos are zona-free and must be cultured to the morula/blastocyst stage before transfer. Some studies have shown that in vitro culture reduces embryo development and quality, but it is not known whether embryos produced by TC or HMC differ because of the NT method or the in vitro culture. Therefore, we investigated the developmental competence and histone acetylation (H3K18ac) of porcine NT embryos produced by TC and HMC with (Day 5 and 6) or without (Day 0) in vitro culture. Nuclear transfer experiments were performed on same day (Day 0), using same batch of porcine oocytes and donor cells and same in vitro culture conditions. Cloning procedures were previously described (TC : Cloning Stem Cells 10 : 355; HMC : Zygote 20 : 61). Parthenogenetically activated embryos (PA) were used as control of activation and culture conditions. Embryos from all groups were collected for immunostaining of H3K18ac on Days 0, 5, and 6. The normalized H3K18ac level was calculated as previously described (Epigenetics 6 : 177). Cell numbers per blastocyst in each group were counted on Days 5 and 6. The cleavage rate (Day 2) and blastocyst rates (Days 5 and 6) between groups were analysed by Chi-squared test, whereas cell number per blastocysts and H3K18ac level between groups and days were analysed by ANOVA (SAS version 9.2; SAS Institute Inc., Cary, NC, USA). Cleavage rate of HMC embryos was lower than that of TC embryos, but blastocyst rate and cell number per blastocyst were higher in the HMC group compared with TC (Table 1). Differences of H3K18ac level between HMC, TC, and PA groups were only observed on Day 6 but not on Day 0 or Day 5. Within HMC and TC groups, there was no difference in H3K18ac level between Day 0 and Day 5, but the level was lower on Day 6 compared with Day 5 in the HMC group, whereas the TC group displayed the opposite pattern. In conclusion, NT embryos produced by HMC show higher blastocyst rate and cell number per blastocyst compared with TC embryos. Both in vitro culture and the NT method result in differences of the normalized H3K18ac levels. Further study is needed to investigate putative differences between NT embryos produced by HMC and TC compared to in vivo embryos also after transfer to recipients.

Table 1.Cleavage and blastocyst rate, cell numbers, and normalized H3K18ac level for handmade cloning (HMC), traditional cloning (TC), and parthenogenetically activated (PA) embryos1
188 PLURIPOTENCY OF PORCINE EMBRYONIC AND INDUCED PLURIPOTENT STEM CELLS RELATED WITH THE EXPRESSIONS OF EPITHELIAL–MESENCHYMAL TRANSITION AND APOPTOTIC MARKERS
Authors: Kim Y.-S. , Yi B.-R. , Hyun S.-H. , Lee C.-K. , Choi K.-C.
Published: December 2013

In transgenic pig production for generating animal models of human diseases, apoptosis of an early implantation embryo disturbs the transgenic pig production. Porcine embryonic stem cells (pESC) and porcine induced pluripotent stem cells (piPSC) have an advantage for the generation of transgenic pigs; however, porcine stem cells have not yet been established. In addition, epithelial–mesenchymal transition (EMT) may play a critical role in embryo development and apoptosis. Thus, in this study we generated pESC and pIPSC and further examined the changes in EMT and apoptotic markers. We cultured pESC and piPSC in pESC media containing basic fibroblast growth factor (bFGF), doxicyclin, and leukemia inhibitory factor (LIF), and performed RT-PCR and alkaline phosphatase (AP) test to measure pluripotency markers. The RT-PCR results show that OCT-4, NANOG, and SOX2 were expressed in these stem cells, characteristic of stem cells. AP-positive cells were observed in pESCs and piPSC. In addition, we performed immunocytochemistry (ICC) to examine the expression of surface markers, such as SSEA-1 and SSEA-4. We found that pESC and piPSC expressed these markers, indicating that they have a stem cell property similar to rodent and human stem cells. Second, we treated pESC and piPSC with transforming growth factor beta (TGF-β) to examine the relationship between EMT and apoptotic markers, and confirmed a significant variation of EMT and apoptotic markers, i.e. bcl-2, bax, E-cadherin, and vimentin, by Western blot analysis. In a future study, we will examine the effect(s) of various endocrine hormones secreted by the ovary, such as E2 or P4, on the expressions of EMT and apoptotic markers in pESC and piPSC. Consequently, this study will contribute to elucidate underlying mechanism(s) of EMT and apoptosis by endocrine factors to prevent early apoptosis of pig embryos in these porcine stem cells.
This work was supported by a grant from the Next-Generation BioGreen 21 Program (No. PJ009599), Rural Development Administration, Republic of Korea.
198 ISOLATION, CHARACTERIZATION, AND IN VITRO DIFFERENTIATION OF GOAT ADIPOSE-TISSUE-DERIVED MESENCHYMAL STEM CELLS INTO PANCREATIC ISLETS-LIKE CELLS
Authors: Dubey A. , Malik H. N. , Singhal D. K. , Saugandhika S. , Boateng S. , Singhal R. , Fatima S. , Sharma V. , Saini S. , Kumar S. , Guha S. K. , Malakar D.
Published: December 2013

The present study was carried out for isolation of goat (Capra hircus) adipose-tissue-derived stem cells (gADSCs) from adipose tissue, their characterization, and in vitro differentiation of gADSCs into pancreatic islets-like cells by giving conditioned medium. Goat ADSCs were isolated from goat adipose tissue by the enzymatic digestion method and were enriched by filtering through a 41-μm filter. Thus, filtered cells resuspended in a cell culture flask containing growth enriching medium and cultured in 5% CO2 in air at 38.5°C. Goat ADSCs were characterised by amplification of mesenchymal stem cell specific markers i.e. CD29, CD34, CD44, CD90, and CD166 as positive markers and CD41 and CD71 as negative markers. Immunocytochemistry of mesenchymal stem cell was also carried out with specific markers CD44 and CD90. Goat ADSCs were further characterised by in vitro differentiating them into osteocytes, chondrocytes, and adipocytes. For in vitro differentiation of gADSCs into osteocytes gADSCs were supplemented with conditioned medium i.e. DMEM containing fetal bovine serum (FBS), dexamethazone, B-glycerol phosphate and L-ascorbic acid. Osteogenic differentiation was confirmed by positive Alizarin red S staining and amplification of Osteopontin and Collagen I genes. For differentiation into chondrocytes cells, gADSCs were incubated in DMEM/F12 containing dexamethazone, ITX, BMP-4, and FBS for 21 days. Differentiated cells were confirmed by positive Safranin O staining and expression of chondrocytes specific Collagen III and Aggrecan genes. For adipogenesis, gADSCs were incubated with DMEM/F12 containing FBS, dexamethasone, and ITX and differentiated cells were confirmed by positive Oil Red O staining and amplification of adipocytes specific genes i.e. LPL, PPRγ and PPRα. For in-vitro differentiation gADSCs into pancreatic islets-like cells on the third or fourth passage gADSCs were incubated in conditioned medium containing serum-free DMEM/F12 medium with glucose (17.5 mM) in the presence of nicotinamide (10 mM), activin-A (2 nM), exendin-4 (10 nM), pentagastrin (10 nM), retinoic acid (10 μM) and mercaptoethanol (20 μM). The in vitro differentiation gADSCs into pancreatic islets-like cells was confirmed by amplification of pancreatic endoderm specific genes i.e. igf-1, sst, ngn3, pdx-1, isl-1, c-kit, thy-1, and Glut-2, and no expression was detected for above endoderm specific genes in undifferentiated gADSCs. Pancreatic islets-like cells were further characterised by immunostaining and Western blotting of Pdx-1, insulin, and Islets-1 specific protein. It could be concluded that gADSCs was differentiated into different lineages and secretory insulin was produced from pancreatic islets-like cells.
204 ISOLATION, CHARACTERIZATION, AND DIFFERENTIATION OF ADIPOSE TISSUE DERIVED MESENCHYMAL STEM CELLS: AN AUTOLOGOUS TRANSPLANTATION TO PATIENTS
Authors: Malik H. N. , Dubey A. , Singhal D. K. , Saugandhika S. , Boeteng S. , Fatima S. , Singhal R. , Sharma V. , Saini S. , Kumar S. , Guha S. K. , Malakar D.
Published: December 2013

Adult stem cells derived from all possible sources of livestock serve as the best possible alternative to embryonic stem cells. The discovery of mesenchymal stem cells has provided the new horizon to stem cell therapy. Adipose tissue derived mesenchymal stem cell (ADSCs), an easy source of adult stem cell has created a lot of interest among researchers as patient specific treatment and autologous transplantation in animals is becoming a viable option. The proposed study was carried out for 1) isolation of ADSCs from dogs, suffering from hip dysplasia or from paraplegia, 2) ADSC characterisation and in vitro differentiation ability into osteocytes, chondrocytes, adipocytes and neurocytes specific cells. Adipose tissues were collected from belly/umbilical cord region. ADSCs were isolated by enzymatic digestion method followed by enriching through a 41 μm filter. Filtered cells were then resuspended in cell culture flasks containing growth enriching medium and cultured in 5% CO2 in air at 37°C for 5 days. ADSCs were characterised by amplification of mesenchymal stem cell specific markers i.e. CD29, CD44, CD90, and CD166 and by immunocytochemistry of mesenchymal stem cell specific protein i.e. CD44 and CD90. ADSCs were further in vitro differentiated. ADSCs derived osteocytes, chondrocytes, and adipocytes were validated through the amplification of specific markers of osteocytes (Osteopontin, Collagen I); chondrocytes (Aggrecan and Collagen II) and adipocytes (LPL, PPARα, PPARγ). Dog ADSCs were further autogenic transplanted into hip dysplasia and paraplegic patients. These patients recovered well one month from transplantation and were able to move freely. It may be concluded that these findings may have implications for defining the physiological roles of ADSCs in arthritis; orthopaedic ailments, joint regeneration, neuronal disorders, and several other applications leading to novel therapeutic opportunities.
38 ROCKY MOUNTAIN BIGHORN SHEEP (OVIS CANADENSIS CANADENSIS) EMBRYOS PRODUCED USING SOMATIC CELL NUCLEAR TRANSFER
Authors: Stroud T. , Xiang T. , Romo S. , Kjelland M. E.
Published: December 2013

The present objective was to determine if interspecies somatic cell nuclear transfer (SCNT) could be a viable option for producing cloned Rocky Mountain bighorn sheep (RMBS) embryos, a model for other endangered ovine species or sheep of high genetic value. A primary question was whether viable cells from a harvested 5.5-year-old male RMBS (Ovis canadensis canadensis) could be cultured 4 days postmortem for SCNT embryo production. Domestic sheep ovaries from an abattoir were placed in TCM199 holding medium containing Hanks’ salts, kept at ambient temperature, and transported to the laboratory in less than 3 h. Oocytes were collected using 2 methods, aspirating visible follicles (G1) or slicing ovary tissues (G2), and matured in TCM199-based medium. At 20 h postmaturation, oocytes were stripped of cumulus cells and those with an extruded polar body underwent SCNT using ViaGen's protocol. Tissue samples were collected from the RMBS, placed in plastic bags, and shipped to the laboratory for cell culture; that is, tissue disinfection and mincing, placement in cell culture flasks with cell culture medium and into an incubator at 38.5°C in a humidified atmosphere of 5% CO2. At confluency, cells having fibroblast-like cell morphology were cryopreserved using DMSO-containing medium and stored at –196°C. For SCNT, 4-day postmortem donor cells were thawed and cultured for at least 3 days. Matured oocytes were enucleated using a micromanipulation system and Hoechst 33342 to ensure nucleus removal. A donor cell was inserted into an enucleated cytoplast and fusion pulses were applied to reconstruct embryos. Cloned embryos were cultured in modified SOF in 5% CO2, 5% O2, and 90% N2 for 6 days before shipment in a portable incubator at 38.5°C in SOF for analysis on Day 7. Pearson's χ2 test and Fisher's exact test were used to determine statistical differences (α = 0.05). The 60 ovaries yielded 324 oocytes (5.4 oocytes/ovary) and 217 matured (67%) of which 204 were enucleated, 156 fused (77%), and 102 cleaved (65%), producing 25 blastocysts and morulae (25/156 = 16%). G1 resulted in 62 cleaved embryos of 91 fused, whereas G2 resulted in 40 cleaved embryos of 65 fused (χ2 = 0.728; P = 0.393). Using the IETS stage and grading system, 3 blastocysts (one 5–2, one 6–1, one 6–2) and 22 morulae (4–1) appeared viable, but the rest had degenerated. Specifically, G1 produced 3 blastocysts and 8 stage 4–1 embryos versus 14 stage 4–1 embryos in G2 (based on fused or cleaved embryos: χ2 = 2.516, P = 0.113 and χ2 = 3.914, P = 0.048, respectively). Fibroblast-like cell cultures were obtained from 4- and 5-day postmortem tissue samples; however, only Day 4 cultured cells were used for RMBS SCNT embryo production. Although G1 produced more blastocysts than G2, the difference was not statistically significant (P > 0.05).
39 EFFECT OF FOLLICULAR SIZE OF CYTOPLAST DONOR ON CLONING EFFICIENCY IN GOATS
Authors: Hall J. , Yang M. , Meng Q. , Dai J. , Polejaeva I. A.
Published: December 2013

In goats, oocytes derived from small follicles (<3 mm in diameter) have low developmental competence after IVF (Crozet et al. 1995). However, their performance is compatible to oocytes derived from large follicles (>3 mm) when intracytoplasmic sperm injection is used (Jimenez-Macedo et al. 2006). No data are currently available on the ability of oocytes derived from small follicles to support development to term after somatic cell nuclear transfer (SCNT) in goats. Here we evaluated the effect of follicular size of cytoplast donor on cloning efficiency in goats. Seven rounds of oocyte maturation and nuclear transfer were carried out on separate days. During each replicate, oocytes were collected separately from large follicles (LF, n = 378) and small follicles (SF, n = 981) from the same group of ovaries. First, only large follicles (>3 mm) were dissected and rinsed, and then an ovary was moved to another dish to dissect the rest of the follicles. The groups of oocytes derived from LF and SF were treated separately throughout the process including maturation, SCNT, embryo culture and embryo transfers. Neonatal skin fibroblasts derived from transgenic goats were used as nuclear donors in this study. Nine embryo transfers were conducted for LF group and 12 for the SF group. Oocytes from SF had significantly lower rate of nuclear maturation compared to oocytes derived from LF (33.5% ± 3.7 v. 54.3% ± 5.4; P < 0.01). There was no difference in fusion rate between these 2 groups (66 v. 69%; 215/325 v. 138/199; SF v. LF). Additionally, we found no statistical difference in Day 30 pregnancy rate (42% v. 33%; SF v. LF group) or birth rate (2 healthy kids produced from each of the groups, 16.7% v. 22.2%; SF v. LF). The results demonstrate that oocytes from small follicles have similar competence to support development to term as oocytes derived from large follicles following SCNT in goats.
This work was supported by the Utah Multidisciplinary Arrhythmia Consortium and Utah Agricultural Experiment Station project #1100.
5 DISRUPTION OF THE HIGH MOBILITY GROUP AT-HOOK 2 (HMGA2) GENE IN SWINE REDUCES POSTNATAL GROWTH
Authors: Chung J. , Zhang X. , Colins B. , Howard K. , Simpson S. , Salmon C. , Koh S. , Sper R. , Byrd C. , Piedrahita J.
Published: December 2013

The high mobility group AT-hook 2 (HMGA2) protein has been shown to be a crucial gene for cell growth, proliferation, and apoptosis; HMGA2 is also a strong biological candidate for growth, because mutations in this gene alter body size in mice and humans. Compared with wild-type controls, adult mice lacking HMGA2 are 60% smaller, and adult heterozygous mutants are 20% smaller. In humans, HMGA2 has been associated with adult and childhood height without any other deleterious effect. Additionally, a microdeletion in the HMGA2 gene in a human patient resulted in short stature, with no dysmorphologies and normal puberty. In order to determine the effect of HMGA2 on fetal and adult growth in pigs, a transgenic pig line deficient in HMGA2 expression was generated by gene targeting in fetal fibroblasts (FF). Using a targeting vector carrying a reporter gene, and homology arms specific to HMGA2, heterozygous mutant cell lines were generated. The cell lines were then used to generate 6 heterozygous females by somatic cell nuclear transfer (SCNT). Bodyweights and lengths from snout to base of tail were measured every 2 weeks for a year for mutant (n = 6) and wild-type farm gilts (n = 6). Data were analysed by one-way ANOVA. As in mice, disruption of one allele of the HMGA2 gene resulted in 25% reduction in weight (P < 0.0001) and 14% reduction in length (P < 0.0001). Early in postnatal growth (2 months), weights of mutants were not different than wild-type. However, mutants were 20 to 35% lighter (P < 0.05) during mid stages (6 months) and 25 to 30% (P < 0.0001) in late stages (3 months). The same insertional mutation generated 8 heterozygous male clones by SCNT. In addition, 7 nontransgenic males from the same FF line were generated as SCNT controls. Bodyweights and lengths were measured every 2 weeks for 30 weeks for HMGA2 heterozygous mutants (n = 8), control SCNT (n = 7) and wild-type farm boars (n = 5). The weight curve of boars showed similar pattern as for mutant gilts. At 30-week postnatal stage, mutants were 17% (P < 0.05) and 16% (P < 0.05) lighter in weight compared with littermate and wild-type animals, respectively. We are presently developing homozygous HMGA2 mutant lines. Currently, 3 of 6 heterozygous gilts have been bred with heterozygous boars, with 1 confirmed pregnancy. The expectation is that the homozygous animals will, like mice, be 60% smaller than the wild-type animals. The approach described here will result not only in a valuable large-animal model of dwarfism, but also in a tool to reduce the size of existing transgenic and nontransgenic swine lines. This, in turn, will increase the receptivity of valuable transgenic lines by the biomedical community.
Funding for this work was provided by NIH grant R21-OD010553 to JP.
59 EVALUATION OF CRYOPRESERVED CHICKEN SEMEN WITH DIFFERENT CRYOPROTECTANTS
Authors: Choi J. S. , Shin D.-B. , Ko Y.-G. , Do Y.-J. , Seong H.-H. , Kim D.-H. , Kong I.-K. , Kim S. W.
Published: December 2013

The purpose of this study was to evaluate the viability, acrosome integrity, mitochondrial activity, and fertility of frozen-thawed fowl semen using different cryoprotective agents. The experiments were carried out on 10 sexually adult roosters of the Ogye breed (Korean native black chicken). The semen was collected twice per week and diluted in a 1 : 1 ratio with EK extender without cryoprotectant at 5°C. After equilibration for 30 min, diluted semen was added with an equal volume of diluents containing 14% dimethylacetamide (DMA), 14% dimethylformamide (DMF), or 15% methylacetamide (MA). The semen were packed into 0.5-mL plastic straws, frozen for 30 min by exposure to liquid nitrogen vapor 4 cm above the surface of liquid nitrogen, and then plunged into liquid nitrogen at –196°C. Frozen semen was thawed in a cold water bath at 5°C for 2 min. For fluorescence-assisted cell sorting (FACS) analysis, the frozen-thawed semen was adjusted to a final concentration of 90 million spermatozoa per milliliter with EK extender. Sperm membrane integrity was evaluated by the live-dead staining method with SYBR-14 dye and propidium iodide (PI). Acrosome integrity was measured with fluorescein isothiocyanate-labelled Pisum sativum agglutinin (PSA) and PI. The percentage of functional mitochondria was estimated using Rhodamine 123 (R123) dye and PI. After intravaginal AI was performed twice per week by injecting 0.2 mL of thawed semen directly into the vagina within 2 min after thawing, the resulting eggs were incubated for 7 days to confirm fertilization. The survival rates of cryopreserved sperm with DMF, DMA, and MA were 52.1 ± 5.5, 46.9 ± 5.1, and 36.6 ± 4.7%, respectively. The survival rate of DMF was significantly higher than those of DMA and MA (P < 0.05). The percentages of sperm that had damaged acrosomal membranes using DMF, DMA, and MA were 36.6 ± 1.4, 47.5 ± 1.9, and 61.2 ± 1.9% (P < 0.05), respectively. The percentages of live sperm with intact mitochondrial membranes cryopreserved with DMF, DMA, and MA were 52.7 ± 1.1, 44.5 ± 1.0, and 38.4 ± 1.9%, respectively, with significant differences (P < 0.05). The fertilization rates of resulting eggs after AI were 68.4% in DMF, 67.9% in DMA, and 61.9% in MA, without significant differences. These results indicate that cryopreserved rooster semen with 7% DMF showed a significantly higher survival rate and mitochondrial functionality but a lower rate of damaged acrosomes. As a cryoprotective agent, DMF has the lowest influences on Ogye rooster sperm membranes and acrosome integrity and thus could be used as a conservation method for poultry genetic resources.
65 ASSOCIATION BETWEEN ALDOSTERONE AND CORTISOL LEVELS DURING THE OVULATORY PERIOD IN SPANISH PUREBRED MARES
Authors: Satué K. , Montesinos P. , Gardon J. C.
Published: December 2013

Preovulatory increased aldosterone (ALD) has been described as one of the physiological events that characterise the oestrous cycle in relation to the dynamics of the renin angiotensin aldosterone system. Within the mechanisms associated with this response are the stimulating effect of oestrogen on the synthesis of angiotensinogen, haemodynamic variations of renal blood flow, changes in sodium concentration, changes in the local sympathetic activity, and the effect of cortisol (CORT). In fact, CORT via adrenocorticotropic hormone contributes primarily to preovulatory ALD, and it is the primary regulator factor synthesis ALD (Chidambaram et al. 2002; Pechère-Bertschi et al. 2002; Szmuilowicz et al. 2006; Sealy and Laragh 2011). This mechanism has been described in several animal species, but remains unknown in the mare. The aim of this study was to investigate the association of endogenous CORT increases, with the changes in the synthesis of ALD during the ovulatory period in the mare. This research was approved by the Animal Ethics Committee of the CEU-Cardenal Herrera University. The study was carried out on 25 healthy Spanish purebred between 4 and 17 years old. Venous blood samples were taken daily from Day –2 to 2 of the oestrous cycle, considering the time 0, the day of ovulation identified by transrectal ultrasound. Serum CORT and ALD concentrations were determined to by competitive immunoassay. To analyse the effect of cycle day on concentrations of CORT and ALD, ANOVA was used. The interrelations between both parameters were expressed by Pearson correlation coefficient. A significant rise of CORT and ALD were observed on the day of ovulation (CORT: 87.68 ± 10.71 ng mL–1; ALD: 535.3 ± 71.49 pg mL–1) with respect to previous 2 days (CORT: 67.13 ± 3.165 ng mL–1; ALD: 380.3 ± 36.20 pg mL–1), decreasing to 2 days postovulation (CORT: 61.61 ± 1.766 ng mL–1; ALD: 495.2 ± 14.75 pg mL–1; P < 0.05). Significant correlations between CORT and ALD (r = 0.53) were founded. The results of this study show that, as has been reported in cyclic females of other species, in Spanish purebred mares, the ALD and CORT concentrations increase significantly during the period of ovulation. Similarly, although CORT is involved in the synthesis of ALD, its contribution is not important enough to induce activation of the renin angiotensin aldosterone system by itself, so other CORT-independent factors must also be involved in the synthesis of ALD.
67 CORRELATION OF ARCHITECTURAL AND FUNCTIONAL REPROGRAMMING OF NUCLEI DURING EMBRYONIC GENOME ACTIVATION OF EARLY BOVINE EMBRYOS GENERATED BY IVF AND SCNT
Authors: Popken J. , Koehler D. , Brero A. , Wuensch A. , Thormeyer T. , Guengoer T. , Wolf E. , Zakhartchenko V. , Cremer T.
Published: December 2013

Development of mammalian pre-implantation embryos provides an excellent model to explore interactions of nuclear organisation and nuclear functions. Based on light optical sectioning with confocal laser scanning microscopy and structured illumination microscopy, we performed a quantitative three-dimensional image analysis of nuclei in early bovine embryos generated by in vitro fertilization (IVF) and somatic cell nuclear transfer (SCNT) of bovine fibroblast nuclei. The same sequence of changes was observed in nuclei of both IVF and SCNT embryos during embryonic genome activation (EGA) is that typically achieved in embryos between 8 and 16 cells. In both pre-EGA IVF and SCNT embryos, chromosome territories (CT) were assembled as spatially distinct entities at the nuclear periphery, whereas the nuclear interior was typically occupied by a mostly chromatin free lacuna enriched with splicing factors. Detection of H3K4m3 demonstrates the presence of transcriptionally competent chromatin before EGA, which was correlated with large-scale movements of CT into the nuclear interior and a several-fold decrease of nuclear volumes. Post-EGA nuclei are characterised by a conventional nuclear architecture with chromatin distributed throughout the nuclear space, heterochromatin enriched with histone markers for transcriptionally silent chromatin beneath the nuclear lamina and around nucleoli, as well as heterochromatin clusters and chromocenters throughout the nuclear interior. Pre- and post-EGA nuclei were recorded with the superior resolution of structured illumination microscopy to allow a quantitative analysis of the nuclear topography of H3K4me3 and RNAP II signals. These signals were highly significantly enriched in the perichromatin region (PR) surrounding the compact, transcriptionally silent interior of megabase-sized chromatin domains, which form the basic structural units of CT. The PR is in direct contact with interchromatin compartment (IC) channels starting at nuclear pores, permeating the nuclear space and harboring nuclear bodies in IC lacunas. Our findings support a model for the functional nuclear architecture based on spatially distinct, but co-aligned three-dimensional networks of an active and an inactive nuclear compartment. The active nuclear compartment is built up from the structurally and functionally interacting IC and PR, whereas the inactive nuclear compartment consists of the compact, transcriptionally silent core of chromatin domain clusters.
This work is supported by the DFG (ZA 425/1-3, CR 59/29-2).
80 DEVELOPMENT OF A SYNTHETIC MEDIUM FOR THE IN VITRO CULTURE OF BOVINE EMBRYOS
Authors: Moreno D. , Neira A. , Dubreil L. , Liegeois L. , Destrumelle S. , Amirat-Briand L. , Bencharif D. , Tainturier D.
Published: December 2013

In the majority of media for embryo culture, 2 of typical components used are FCS or BSA; however, the presence of FCS in the culture medium has been shown to have a negative effect on embryo quality and the use of animal-derived proteins in culture media increases the risks of disease transmission through in vitro embryo production. The aim of this study was to develop an in vitro embryo culture medium free from FCS and BSA, but with the addition of various growth factors and cytokines (GF-CYK: IGF-I, IGF-II, bFGF, LIF, GM-CSF) 50 ng mL–1 and (TGF-β1) 100 ng mL–1 supplemented with hyaluronan (HA) and recombinant albumin (RA). Bovine oocytes (n = 1043, 6 replicates) from abattoir ovaries were matured in TCM-199 medium with 60 μg mL–1 penicillin, 60 μg mL–1 streptomycin, and 10 ng mL–1 EGF for 24 h at 39°C and 5% CO2 in humidified air. Afterward, the oocytes were fertilized in IVF-TALP medium with 6 mg mL–1 fatty acid-free BSA and 1.7 IU mL–1 heparin for 18 h under the same conditions. After fertilization, presumptive zygotes were divided into two groups and cultured in 30 μL droplets of SOF supplemented with (1) 0.4% BSA + 5 μg mL–1 insulin, 5 μg mL–1 transferrin, and 5 ng mL–1 selenium (ITS) as a control; or (2) GF-CYK + 0.5 mg mL–1 HA + 0.15% RA (M1). Droplets were preserved under mineral oil in a humidified atmosphere of 5% CO2, 5% O2, and 90% N2 at 39°C. Blastocyst development and blastocyst diameter was observed at 7 and 8 days post-fertilization (dpf). Developmental and diameter data were analysed using the Wilcoxon test by using R software. The blastocyst rates were not significantly different between the control and M1 medium: at 7 dpf (22.9% ± 4.8 and 30.2% ± 3.0), and at 8 dpf (29.6% ± 5.1 and 37.4% ± 2.0 respectively; P > 0.05). The blastocyst diameter obtained with the M1 medium was significantly greater (P < 0.05) than that of the control at 7 dpf (173.3 μm ± 4.9 and 157.2 μm ± 4.1, respectively); however, no significant differences were observed at 8 dpf (190.3 μm ± 5.2 and 179.7 μm ± 5.3, respectively). In conclusion, the FCS- and BSA-free medium with GF-CYK, HA, and RA (M1) showed a comparable development rate to the control medium at 7 and 8 dpf. These growth factors and cytokines in association with hyaluronan and recombinant albumin have a synergistic action by promoting an increase in the blastocyst diameter at 7 dpf. This is fully synthetic method of embryo culture; it presents a valuable tool to reduce the risks of disease transmission via embryo transfer.
91 EFFECT OF LEUKEMIA INHIBITORY FACTOR (LIF) ON MATURATION OF PORCINE OOCYTES IN VITRO MATURATION AND DEVELOPMENT OF PARTHENOGENETIC EMBRYOS
Authors: Choi Y. B. , Kim S. J. , Park E. J. , Song K. Y. , Moon J. H. , Lee B. C.
Published: December 2013

Cytokines have been suggested to have an important role for successful implantation. Among the cytokines, leukemia inhibitory factor (LIF) is a pleiotropic cytokine of the interlukin-6 family that has been confirmed for its significance in implantation in human and animal studies. Furthermore, it has been reported that LIF enhanced in vitro maturation (IVM) in cattle and sheep, blastocyst formation and hatching rate of embryos and pregnancy rate in mouse, human, cattle and sheep. However, to our knowledge, there has been no report on the effects of human LIF (hLIF) on pig oocytes IVM and embryos development. Therefore, we designed and performed this study to examine the effect of hLIF treatment on pig IVM and oocyte developmental competence. We investigated the effect of hLIF treatment during pig oocyte in vitro maturation (IVM) and in vitro culture (IVC) on parthenogenetic embryos. Three groups of different hLIF concentrations were used: 0, 0.5, and 1.0 ng mL–1. In experiment 1, hLIF was contained on IVM media, in experiment 2, hLIF was contained on IVC media for 7 days, and experiment 3, hLIF was contained on IVM and IVC media. In experiment 1, hLIF in IVM media significantly increased cleavage rate in 0.5 ng mL–1 group [hLIF 0 ng mL–1; 46.56 ± 3.1 (%), 0.5 ng mL–1; 58.43 ± 3.6 (%), 1.0 ng mL–1; 52.05 ± 2.7 (%)] and total cell number of blastocysts in hLIF 1.0 ng mL–1 group (hLIF 0 ng mL–1; 35.00 ± 2.3, 0.5 ng mL–1; 40.71 ± 3.1, 1.0 ng mL–1; 51.06 ± 3.7) but no significant differences were found in oocyte maturation rate or blastocyst formation rate. In experiment 2, total cell number of blastocysts showed significant difference in hLIF 1.0 ng mL–1 in IVC media (hLIF 0 ng mL–1; 43.81 ± 1.6, hLIF 0.5 ng mL–1; 45.97 ± 2.0, hLIF 1.0 ng mL–1; 52.10 ± 2.9). Finally, total cell number of blastocysts increased in hLIF 0.5 ng mL–1 in IVM and IVC media [hLIF 0 ng mL–1; 46.50 ± 2.3 (%), 0.5 ng mL–1; 55.11 ± 2.9 (%)]. In conclusion, hLIF supplementation to IVM and IVC medium improved porcine embryo development in terms of increasing total cell number of blastocysts.
This study was supported by Korean MKE (#10033839), the Research Institute for Veterinary Science.
Evidence for European brown hare syndrome virus introduction with translocated brown hares (Lepus europaeus): implications for management of restocking operations
Authors: Spyrou Vassiliki , Stamatis Costas , Birtsas Periklis , Psychas Vassilios , Manolakou Katerina , Billinis Charalambos , Mamuris Zissis
Published: December 2013

Context Criticisms of wildlife restocking operations typically focus on concerns that translocations can lead to the introduction of pathogens, and risk the integrity of locally adapted genetic diversity. Restocking programs aiming to stabilise population declines of European brown hares using captive-bred individuals have been carried out in several European countries, including Greece. Aims To assess the potential for imported hares to introduce novel strains of European brown hare syndrome virus (EBHSV) during restocking operations, by (1) inferring the origin of wild Greek hares on the basis of their mitochondrial DNA (mtDNA) haplotype, (2) screening the hares to detect and characterise EBHSV, and (3) determining whether certain hare origin–EBHSV combinations occur in the wild. Methods RNA extraction, polymerase chain reaction (PCR) amplification, and sequence and phylogenetic analyses of EBHSV were performed on 53 hares. Diagnostic RFLP markers of the mtDNA were used to infer the origins of sampled hares. Key results Thirty-three hares had ‘typical’ native Greek haplotypes and 20 had mtDNA haplotypes matching those found in imported and released hares. Twelve of the latter and none of the former were positive for EBHSV. Phylogenetic analysis showed that nine virus isolates formed a single genetic lineage distinct from northern–central European ones. Three virus sequences from three imported reared-and-released hares, from Chalkidiki, were closely related to the northern–central European EBHS viruses. Conclusions Alien strains of EBHSV are co-introduced with released captive-bred animals, possibly resulting in negative impacts on populations of Greek hares that have not evolved resistance to these novel virus strains. Implications The identification of these allopatric EBHSV strains has led the authorities to ban captures and transportations of local brown hares for any restocking operation. We consider it imperative to reinforce microbiological and genetic controls before further releases of captive-bred game species in the wild in Greece.
Cohesion promotes nucleolar structure and function
Authors: Harris Bethany , Bose Tania , Lee Kenneth K. , Wang Fei , Lu Shuai , Ross Rhonda Trimble , Zhang Ying , French Sarah L. , Beyer Ann L. , Slaughter Brian D. , Unruh Jay R. , Gerton Jennifer L.
Published: December 2013

The cohesin complex contributes to ribosome function, although the molecular mechanisms involved are unclear. Compromised cohesin function is associated with a class of diseases known as cohesinopathies. One cohesinopathy, Roberts syndrome (RBS), occurs when a mutation reduces acetylation of the cohesin Smc3 subunit. Mutation of the cohesin acetyltransferase is associated with impaired rRNA production, ribosome biogenesis, and protein synthesis in yeast and human cells. Cohesin binding to the ribosomal DNA (rDNA) is evolutionarily conserved from bacteria to human cells. We report that the RBS mutation in yeast (eco1-W216G) exhibits a disorganized nucleolus and reduced looping at the rDNA. RNA polymerase I occupancy of the genes remains normal, suggesting that recruitment is not impaired. Impaired rRNA production in the RBS mutant coincides with slower rRNA cleavage. In addition to the RBS mutation, mutations in any subunit of the cohesin ring are associated with defects in ribosome biogenesis. Depletion or artificial destruction of cohesion in a single cell cycle is associated with loss of nucleolar integrity, demonstrating that the defects at the rDNA can be directly attributed to loss of cohesion. Our results strongly suggest that organization of the rDNA provided by cohesion is critical for formation and function of the nucleolus.
Cascade: a R package to study, predict and simulate the diffusion of a signal through a temporal gene network
Authors: Jung Nicolas , Bertrand Frédéric , Bahram Seiamak , Vallat Laurent , Maumy-Bertrand Myriam
Published: December 2013

Summary: Temporal gene interactions, in response to environmental stress, form a complex system that can be efficiently described using gene regulatory networks. They allow highlighting the more influential genes and spotting some targets for biological intervention experiments. Despite that many reverse engineering tools have been designed, the Cascade package is an integrated solution adding several new and original key features such as the ability to predict changes in gene expressions after a biological perturbation in the network and graphical outputs that allow monitoring the spread of a signal through the network.

Availability and implementation: The R package Cascade is available online at http://www-math.u-strasbg.fr/genpred/spip.php?rubrique4.

Contact:  fbertran@math.unistra.fr

Supplementary information:  Supplementary data are available at Bioinformatics online.
The dual role of HOP2 in mammalian meiotic homologous recombination
Authors: Pezza Roberto J. , Voloshin Oleg N. , Volodin Alexander A. , Boateng Kingsley A. , Bellani Marina A. , Mazin Alexander V. , Camerini-Otero R. Daniel
Published: December 2013

Deletion of Hop2 in mice eliminates homologous chromosome synapsis and disrupts double-strand break (DSB) repair through homologous recombination. HOP2 in vitro shows two distinctive activities: when it is incorporated into a HOP2–MND1 complex it stimulates DMC1 and RAD51 recombination activities and the purified HOP2 alone is proficient in promoting strand invasion. We observed that a fraction of Mnd1−/− spermatocytes, which express HOP2 but apparently have inactive DMC1 and RAD51 due to lack of the HOP2–MND1 complex, exhibits a high level of chromosome synapsis and that most DSBs in these spermatocytes are repaired. This suggests that DSB repair catalyzed solely by HOP2 supports homologous chromosome pairing and synapsis. In addition, we show that in vitro HOP2 promotes the co-aggregation of ssDNA with duplex DNA, binds to ssDNA leading to unstacking of the bases, and promotes the formation of a three-strand synaptic intermediate. However, HOP2 shows distinctive mechanistic signatures as a recombinase. Namely, HOP2-mediated strand exchange does not require ATP and, in contrast to DMC1, joint molecules formed by HOP2 are more sensitive to mismatches and are efficiently dissociated by RAD54. We propose that HOP2 may act as a recombinase with specific functions in meiosis.