bluuebunny/crossref_metadata_2025 huggingface dataset:
134 TRANSCRIPTOME PROFILE OF BOVINE BLASTOCYSTS DERIVED FROM ALTERNATIVE IN VIVO AND IN VITRO CULTURE CONDITIONS AT SPECIFIC PHASES OF EARLY EMBRYONIC DEVELOPMENT
Authors: Gad A. , Besenfelder U. , Havlicek V. , Hölker M. , Cinar M. U. , Rings F. , Dufort I. , Sirard M. A. , Schellander K. , Tesfaye D.
Published: December 2011

An understanding of gene expression patterns due to altered environmental conditions during different time points of the pre-implantation period would improve our knowledge on regulation of embryonic development and improve success of embryo culture. The aim of this study was to examine the effect of alternative in vivo and in vitro culture conditions at specific phases of early embryonic development on transcriptome profile of bovine blastocysts. Using nonsurgical endoscopic oviducal transfer technology, 5 different blastocyst groups were produced. The first 2 groups were matured in vitro and then either transferred after maturation or after in vitro fertilization to synchronized recipients. The other 3 groups were matured, fertilized and cultured in vitro until 4-cell, 16-cell and morula stage before transfer. Blastocysts from each group were collected by uterine flushing at Day 7 and pooled in groups of 10. Complete in vitro (IVP) and in vivo blastocysts were produced and used as controls. A unique custom microarray (Agilent) containing 42 242 oligo probes (60-mers) was used over 6 replicates of each group vs the in vivo control group to examine the transcriptome profile of blastocysts. Compared with the in vivo control group, clear dramatic shifts were found in the number of differentially expressed genes (DEG, fold change ≥2) at 2 different time points. The first shift occurred for blastocyst groups that were transferred after in vitro fertilization and before embryonic genome activation (EGA). The second shift occurred for blastocyst groups that were transferred after EGA, as well as for the IVP group. Ontological classification of DEG showed that the more time spent under in vitro conditions, the higher the percentage of DEG involved in cell death and apoptotic processes. Moreover, lipid metabolism was the most significant process affected in the blastocysts transferred after in vitro maturation and blastocysts transferred at 16-cell stage. Most DEG involved in this process were down-regulated. Pathway analysis revealed that signalling pathways were the dominant pathways in all groups except the group that was transferred after in vitro maturation. That group showed significant down-regulation for genes involved in retinoic acid receptors activation pathways. These results showed that fertilization and EGA were the most critical developmental stages influenced by in vitro culture conditions and subsequently affect blastocyst quality, as measured in terms of gene expression patterns. Moreover, we identified molecular mechanisms and pathways that were influenced by altered culture conditions. These findings will enable the examination of strategies for modifying in vitro culture conditions at critical stages that will allow more efficient production of developmentally competent blastocysts.
141 DEVELOPMENT OF rLENTIVIRAL VECTORS FOR MALE GERMLINE-SPECIFIC Cre RECOMBINASE EXPRESSION IN LIVESTOCK
Authors: Peoples M. , Golding M. , Long C. , Westhusin M.
Published: December 2011

Transgenic livestock have been used as biomedical models and have the potential to increase production characteristics. Unfortunately, some of the tools used to confirm genetic modification (transgenesis) are unacceptable in terms of public image. One key component is a fluorescent marker confirming foreign gene insert. The fluorescent protein benefits the researchers producing and selecting transgenic animals but is not required for the enhancement of the animal. Removal of the fluorescent marker can be accomplished by employing the Cre-lox recombination system. By using this system one can produce male genetically modified animals that express the enhanced trait in addition to the fluorescent marker, but their sperm only contain the portion of the transgene that represents the enhanced production trait. As a result, offspring derived from these animals exhibit the desired production trait but not the fluorescent marker. The goal of this research was to develop rlentiviral vectors that use the gamete-specific promoter stimulated by retinoic acid 8 (Stra8) to drive expression of Cre recombinase. The base rlentiviral vector we chose to use was pLB. It is ideal for this research because it has a U6 promoter to drive expression of a short hairpin RNA for an enhanced production trait, as well as Lox P sites flanking a cytomegalovirus-green fluorescent protein (CMV-GFP) expression cassette. Initially we identified and PCR amplified a 400-bp mouse Stra8 promoter and a 1.5-kb promoter region of the pig. The Stra8 promoters were integrated into the pLB vector directly upstream of the green fluorescent protein (GFP). These intermediate vectors should have germline-specific expression of GFP and are the first vectors using a pig Stra8 promoter. Next, we PCR amplified and inserted the coding sequence for Cre recombinase into these vectors for germline-specific Cre expression resulting in the first pig Stra8-Cre expression vector. The Lox P sites of this vector are flanking the GFP expression cassette as well as the Str8-Cre expression cassette. To confirm the functionality of the Cre-lox recombination system, the pLB vectors were transfected into human embryonic kindey 293T cells and fluorescence was measured. After Day 2, a Cre expression plasmid was transfected and 3 days post-Cre transfection, fluorescence was measured again. A decrease in fluorescence and GFP positive cell numbers was observed, thus confirming the functionality of the Cre-lox recombination event for these vectors. These vectors will be used to produce transgenic animals by lentiviral transgenesis. Our laboratory has been successful in using this method to produce transgenic livestock. The transgenic animals will be analysed to confirm male germ-cell-specific expression of Cre as well as removal of the GFP fluorescence. If successful, these will be the first transgenic animals using a pig Stra8 promoter for Cre expression for a novel single rlentiviral vector Cre-lox recombination system.
146 DIFFERENTIAL EXPRESSION OF TRANSCELLULAR CALCIUM TRANSPORT AND PARACELLULAR TIGHT JUNCTION GENES IN THE PLACENTAE OF CALBINDIN-D9k, -D28k AND -D9k/D28k KNOCKOUT MICE
Authors: Hwang I. H. , Jeung E. B.
Published: December 2011

The placenta has many essential roles in the maintenance of pregnancy and homeostasis. Calcium transport and regulation are also controlled by the placenta. In general, calcium transport is divided into an active transcellular pathway and a passive paracellular pathway. Transient receptor potential cation channel subfamily V member 5/6 (TRPV5/6), calbindin-D9k/-28k (CaBP-9k/-28k) and Na+/Ca2+ exchanger (NCX1) are involved in the transcellular pathway. The paracellular pathway is determined by the expression of tight junction genes, such as occludin, claudins and ZO-1. In this study, we analysed the difference in calcium transport in the placentae of CaBP-9k and CaBP-28k knockout (KO) mice compared with that of wild-type (WT) mice. Placentae were collected and used for mRNA and protein evaluation from 9 mice of each type (a total of 36 mice). All mice were killed on gestational Day 19. We confirmed mRNA expression by RT-qPCR and protein expression by Western blot analysis. The data were statistically analysed by one-way ANOVA using Tukey's test. In the transcellular pathway, the expression levels of NCX1 and TPRV6 were shown to be significantly increased in KO mice compared with WT mice. In the paracellular pathway, occluding, which is directly related to permeability, mRNA and protein expression was significantly increased in single KO mice compared with WT, but not in double KO mice. Claudin-4, which is a cation barrier, mRNA and protein expression patterns were significantly decreased in single KO mice compared with WT, but not in double KO mice. These results imply that the disability of calcium buffering due to CaBP-9k or CaBP-28k KO may lead to an accelerated transcellular pathway. In addition, a single KO of calcium-binding proteins may lead to decreased paracellular permeability to weaken the leakage of calcium through the tight junction and may also lead to increased cation selectivity of tight junctions. Taken together, these results might indicate that KO of calcium-binding proteins may induce activation of a compensating transcellular pathway in the placenta of mice and a paracellular pathway may support the maintenance of calcium homeostasis.
149 PRODUCTION OF KNOCK-DOWN MICE WITH shRNA CYTOCHROME P-450 4F16 GENE AND REDUCTION OF THE IMMUNE SYSTEM
Authors: Yang B. C. , Hwang K. C. , Kim K. W. , Lee H. C. , Chung H. J. , Jung H. K. , Oh K. B. , Hwang S. S. , Kim N. H. , Park J. K.
Published: December 2011

The putative mouse homologue of cytochrome P-450 4F16 (Cyp4f16) is induced by interleukin-1 (Il-1), interleukin-6 (Il-6) and tumour necrosis factor-α (Tnf-α) and repressed by interleukin-10 (Il-10) and lipopolysaccharide (LPS). The Cyp4F16 is a subfamily of Cyp4F and it is also related to eicosanoids that are important mediators in the inflammatory cascade (Cui et al. 2001). To investigate the role of Cyp4F16, in the present study, we report the production of Cyp4f16 gene knock-down mice in 2 strains of mice, namely A/J and C57BL/6. The A/J is susceptible to infection and it is associated with Cyp4F16, whereas C57BL/6 is relatively resistant to infection. An shRNA-Cyp4F16 expression vector was microinjected into pronuclei of fertilized mouse oocytes and the embryos were transferred into pseudopregnant recipients. As a result, 25 and 50 mice were produced in the A/J and C57BL, respectively. Two mice in the A/J strain and 6 in the C57BL strain were confirmed by PCR as transgenic. Organs were collected in each of the lines produced by inbreeding and screened with real-time PCR for Cyp4f16 transcripts. The Cyp4f16 gene was expressed in a tissue-specific manner with high expression in the pancreas, spleen and lung and a lower level of transcription in the heart, muscle, thymus, kidney, testis and liver. In the spleen of transgenic Cyp4f16 knock-down mice, Cyp4f16 mRNA and protein expression levels were significantly lower than those of wild-type mice. The A/J Cyp4f16 knock-down mice suffered an inflammatory skin disease and tumours, but wild-type A/J mice and knock-down C57BL mice did not. Taken together, these results suggest that Cyp4f16 may play a regulatory role in the immune system and point to the use of the Cyp4f16 knock-down mouse as an experimental animal model for the study of the inflammatory process.
This work was supported by a grant PJ0070762010 from BioGreen 21 Program, Rural Development Administration, Republic of Korea.
153 HEPARIN-BINDING OVIDUCTAL SECRETORY PROTEINS AS A MEDIUM SUPPLEMENT FOR IN VITRO EMBRYO PRODUCTION IN CATTLE
Authors: Sharma A. K. , Mohanty A. K. , Das S. K.
Published: December 2011

The purpose of this study was to determine the effect of heparin-binding oviducal secretory proteins as a media supplement on IVF and embryo development in cattle. The native proteins were isolated from oviducts collected from a slaughterhouse by repeated freeze-thawing and precipitated by ammonium sulfate (60%) followed by dialysis overnight in 10 mM phosphate buffer solution containing 1 mM PMSF at pH 7.0 at 4°C. The dialyzed product was then passed through a high-performance liquid chromatography system with a HiTrap™ heparin prepacked column (GE Healthcare, Piscataway, NJ) equilibrated with 10 mM phosphate buffer containing 1 mM EDTA. The collected protein fractions [heparin-binding protein (HBP), heparin-unbinding protein (HUBP)] and total protein (TP) were run on SDS-PAGE. The SDS-PAGE profile of the eluted HBP fraction contained 5 major protein bands visible between approximately 66 to 97 kDa, the profile of the HUBP fraction contained 2 bands nearer to approximately 66 kDa and in TP, all 7 bands were visible between approximately 60 to 95 kDa. All 3 fractions (i.e. HBP, HUBP and TP) were used in 3 different concentrations (0, 1, 10 and 30 μg mL–1) for in vitro maturation, sperm preparation, IVF and in vitro culture of cleaved embryos. Cumulus–oocyte complexes were collected from slaughterhouse ovaries, washed 4 to 5 times and cultured in maturation media for 24 h in a 5% CO2 incubator at 38.5°C with maximum humidity. In vitro-matured oocytes were co-incubated with capacitated sperm in Fert-BO media at 38.5°C in 5% CO2 in air with maximum humidity. After 18 h, oocytes were washed thoroughly and cultured in embryo development media for cleavage. After 40 to 42 h, cleavage was observed and embryos were transferred into the replacement media for further development. In the HBP group, overall cleavage rates (%) were 63 ± 3.0, 53 ± 2.6, 65 ± 4.7 and 43 ± 0.6 and blastocyst formation (%) was 17 ± 1.6, 23 ± 2.5, 25 ± 2.2 and 24 ± 1.7 in 0, 1, 10 and 30 μg mL–1 concentrations, respectively. However, in the HUBP group, the overall cleavage rates (%) were 66 ± 4.3, 56 ± 2.0, 60 ± 2.7 and 32 ± 0.8 and blastocyst formation (%) was 18 ± 1.1, 25 ± 1.8, 24 ± 2.2 and 14 ± 0.8 in 0, 1, 10 and 30 μg mL–1 concentrations, respectively. In the TP group, cleavage rates (%) were 67 ± 4.9, 60 ± 4.1, 73 ± 3.2 and 41 ± 1.6 and blastocyst formation (%) was 19 ± 1.1, 18 ± 1.9, 27 ± 2.2 and 20 ± 1.6 in 0, 1, 10 and 30 μg mL–1 concentrations, respectively. These results indicate that 10 μg mL–1 of the total oviductal secretory protein fraction used as a media supplement significantly increased (P < 0.01) the cleavage rate and blastocyst formation as compared with the HBP and HUBP fractions.
The authors sincerely acknowledge the director and joint director (R), National Dairy Research Institute, Karnal and Incharge, Eastern Regional Station, National Dairy Research Institute, Kalyani, India, for providing the facilities to carry out the work.
156 EFFECT OF DURATION POSTPARTUM ON OOCYTE QUALITY IN LACTATING HOLSTEIN COWS FOLLOWING TRANSVAGINAL FOLLICLE ASPIRATION AND IN VITRO FERTILIZATION
Authors: Matoba S. , O'Hara L. , Carter F. , Kelly A. K. , Fair T. , Rizos D. , Lonergan P.
Published: December 2011

The objective of this study was to examine the effect of duration postpartum on oocyte quality in lactating dairy cows, as assessed by oocyte morphology and development after fertilization and culture in vitro. Holstein-Friesian spring-calving cows were used (n = 16; parity 3.0 ± 0.36, weight at calving 611 ± 16.2 kg, previous 305-day milk yield 6454.0 ± 276.4 kg). Body weight (BW) and body condition score (BCS) were recorded at approximately 2 weeks before the expected calving date, at calving and then weekly until the end of the experiment (approximately 80 days postpartum). Blood plasma samples were collected weekly, beginning 2 weeks before the expected calving date and continuing until the end of the experiment and were analysed for nonesterified fatty acids (NEFA), β-hydroxybutyrate (BHBA), insulin, insulin-like growth factor I (IGF-I) and glucose. There was a linear decline in BW from calving (Day 0) to Day 28, after which it remained stable. The BCS declined from 14 days precalving, reaching a nadir at approximately Day 35 to 42, after which it increased to the end of the period. Nonesterified fatty acids were significantly elevated from the week before calving until Day 42 postcalving, whereas BHBA was significantly elevated from calving to Day 49. The IGF-I declined dramatically from Day –14 to a nadir on Day 7. There was a significant increase in glucose from Day –7 to Day 0, followed by a precipitous decline to Day 7. Based on the assumption that uterine involution (UI) is complete by approximately Day 42 postpartum in most cows, data from Day 0 to 42 postpartum (pre-UI) were compared with corresponding data from Day 42 to 80 (post-UI). Apart from BCS, all the physiological parameters measured (milk yield, BW and blood metabolites) differed significantly between the 2 periods. In particular, IGF-I, insulin and glucose concentrations were higher after Day 42, whereas BHBA and NEFA were lower compared with before Day 42. The number of oocytes recovered per session and oocyte quality grade did not differ between periods. Positive associations of follicles aspirated and insulin, BHBA and NEFA were detected. Number of oocytes recovered was positively associated with milk yield, BW, glucose and NEFA. Number of cleaved oocytes was positively associated with BW and NEFA. In conclusion, the data do not provide evidence of an effect of lactation-induced metabolic stress on oocyte developmental competence in the postpartum dairy cow assessed in terms of morphological quality and ability to develop after IVF.
This work was supported by Science Foundation Ireland (07/SRC/B1156).
166 EFFECT OF INDIVIDUAL CULTURE SYSTEM ON IN VITRO DEVELOPMENT OF IN VITRO-MATURED - IN VITRO-FERTILIZED BOVINE EMBRYOS
Authors: Nishii R. , Imai K. , Koyama H. , Dochi O.
Published: December 2011

An individual in vitro culture system for bovine embryo needs to be developed for the study of embryo developmental competence. The objective of this study was to examine the effect of individual culture systems on the development of in vitro-matured–in vitro-fertilized bovine embryos. Two individual culture systems were compared. Cumulus–oocyte complexes (COC) were collected by aspiration of ovarian follicles (diameter, 2 to 5 mm) obtained from a local abattoir. The COC were matured in TCM-199 supplemented with 5% calf serum and 0.02 AU mL–1 of FSH. Groups of 20 COC were incubated in 100-μL droplets of IVM media at 38.5°C under an atmosphere of 5% CO2 for 20 h. After 18 h of gamete co-culture (3 × 106 sperm mL–1), the presumptive zygotes were cultured in CR1aa medium supplemented with 5% calf serum at 38.5°C under an atmosphere of 5% CO2, 5% O2 and 90% N2 for 9 days (fertilization = Day 0). The presumptive zygotes were randomly assigned to 1 of the following 3 treatments: single culture (1 zygote was cultured in a 5-μL droplet), well-of-the-well (WOW; Sugimura et al. 2010 Biol. Reprod. 83, 970–978) culture (25 zygotes were cultured individually in each 125-μL droplet) and control culture (25 zygotes were cultured in a 125-μL droplet). Embryo development was evaluated for cleavage and blastocyst rates, on Day 2 and Day 7 to 9 after IVF, respectively. The rates of development up to the blastocyst stage and total cell number in the blastocysts, determined by an air-drying method, were investigated. The cleavage and blastocyst rates were analysed by the chi-square test and the total cell numbers were analysed by ANOVA. The cleavage rates were significantly higher in the control and WOW groups than in the single-culture group (P < 0.01) and the blastocyst rates were significantly lower in the single-culture group than in the control culture group (P < 0.05; Table 1). The total cell numbers (mean ± s.d.) of blastocysts did not significantly differ between the single culture (154.6 ± 21.8), control culture (155.2 ± 22.5) and WOW culture (159.8 ± 27.0) groups. These results indicate that although the blastocyst rate was lower in the single-culture system than in the WOW or group culture system, in vitro-matured–in vitro-fertilized bovine embryos can be cultured using the single-culture system. Moreover, the quality of blastocysts developed by the single-culture system is the same as that of blastocysts developed using the other 2 culture systems.

Table 1.Effect of different culture methods for bovine embryo development
17 CELL CYCLE SYNCHRONIZATION OF BOVINE FIBROBLASTS BY AZADIRACHTA INDICA EXTRACTS
Authors: Quintão C. C. R. , Rabelo N. C. , Pereira M. M. , Giacomini S. B. , Moreira A. P. , Raposo N. R. B. , Carvalho B. C. , Iguma L. T. , Viana J. H. M. , Camargo L. S. A.
Published: December 2011

One requirement for somatic cell nuclear transfer (NT) is the coordination between donor cell cycle and recipient cytoplasm. As an alternative to commercially available substances to synchronize the cell cycle in G0/G1, we tested 2 extracts, aqueous and hexane obtained from the plant Azadirachta indica A. Juss (popularly called Neem). Extracts from this plant have shown antiviral, antibacterial and anticancer activities, widely described in the literature (Kumar et al. 2009 Invest. New Drugs 27, 246–252). The hexane extract was prepared in the Soxhlet apparatus until total collapse and then submitted to rotary evaporation. The aqueous extract was prepared by dynamic maceration and was subsequently lyophilized. Bovine fibroblasts collected from Gyr cows were cultured in DMEM (Sigma) supplemented with 10% fetal cow serum (FCS) and incubated at 37°C, 5% CO2 and 95% humidity. After obtaining 70% of cell confluence, the extract was added to cells at the following concentrations: 0 μg mL–1 (negative control), 50 μg mL–1, 100 μg mL–1, 200 μg mL–1 and 300 μg mL–1, for 12 and 24 h. Simultaneously, a group with serum starvation (positive control; cells cultured in DMEM plus 0.5% FBS for 3 days) was prepared. Three repetitions were performed in triplicate for each concentration and control groups. Cell cycle readings were performed by flow cytometry (Facs Callibur, Becton Dickinson, San Jose, CA, USA) and DNA histograms were analysed by WinMDI software to determine the percentage of cells in G0/G1 phase, S and G2 cell cycle, so that 10 000 cells were analysed in each reading on a flow cytometer. Data were analysed by analysis of variance and means were compared by Student-Newman-Keuls test. The percentages of cells at G0/G1 phase for aqueous extracts were lower (P < 0.05), regardless of the concentration and exposure time, than the 0 μg mL–1 (83.73 ± 1.14%) and serum starvation (86.64 ± 1.44%). In contrast, the percentages of cells synchronized at G0/G1 with 50 μg mL–1 for 12 h (84.23 ± 0.56%), 50 μg mL–1 for 24 h (85.66 ± 0.57%), 100 μg mL–1 for 12 h (87.85 ± 0.51%) and 200 μg mL–1 for 12 h (85.87 ± 0.45%) using hexane extracts were higher (P < 0.05) than with 0 μg mL–1 (81.44 ± 0.29%), but lower (P < 0.05) than the serum starvation (91.33 ± 0.31%). In conclusion, hexane extracts from the plant Azadirachta indica A. Juss can synchronize mammalian cell cycle at G0/G1 despite the low proportion when compared with serum starvation. Studies to evaluate efficiency of cell cycle resuming and viability after somatic cell nuclear transfer are ongoing.
Financial support: FAPEMIG, CNPq and Project 01.07.01.002.
174 OXIDATIVE STRESS AND LIPID PEROXIDATION IN BOVINE IN VITRO-PRODUCED EMBRYOS WITH DIFFERENT DEVELOPMENTAL SPEEDS
Authors: Di Francesco S. , Rubessa M. , Boccia L. , De Blasi M. , Stiuso P. , Gasparrini B. , Balestrieri M. L.
Published: December 2011

In vitro-produced embryos are less viable than their in vivo counterparts. It is known that the developmental speed is a reliable marker of embryo viability. One of the major factors impairing in vitro embryo development is oxidative stress. The aim of the study was to evaluate oxidative stress and lipid peroxidation in bovine in vitro-produced embryos that reached different developmental stages at the end of culture. Abattoir-derived oocytes were matured in vitro in TCM-199 with 15% bovine serum, 0.5 μg mL–1 of FSH, 5 μg mL–1 of LH, 0.8 mM L-glutamine and 50 mg mL–1 of gentamicin. Mature cumulus–oocyte complexes (COC) were fertilized in Tyrode's modified medium, supplemented by 5.3 SI mL–1 of heparin, 30 μM penicillamine, 15 μM hypotaurine, 1 μM epinephrine and 1% of bovine serum. Both in vitro maturation and IVF were carried out at 39°C and 5% CO2 in air. After 20 to 22 h of gamete co-incubation, presumptive zygotes were denuded and cultured in SOF for 7 days at 39°C under humidified air with 5% CO2, 7% O2 and 88% N2 in air. At the end of culture, embryos were assessed according to the stage of development as tight morulae (TM), early blastocysts (eBl), blastocysts (Bl), expanded blastocysts (XBl) and hatched blastocysts (HBl). For each stage of development, an average of 20 embryos were used to determine manganese superoxide dismutase (MnSOD) activity and levels of nitric oxide (NO2–) and thiobarbituric acid-reactive substances (TBARS). The SOD activity was determined by a colourimetric method (Caraglia M et al. 2011 Cell Death Dis. 2, 150, doi:10.1038/cddis.2011.34) whereas NO2– and TBARS were measured by a spectrophotometric method (Balestrieri et al. 2011 J. Cell. Physiol. doi:10.1002/jcp.22874). Data were analysed by t-test. Greater (P < 0.05) MnSOD activity was observed in faster developing embryos (i.e. XBl and HBl) compared with slower ones (i.e. TM, eBl and Bl; 0.46 ± 0.04, 0.46 ± 0.03, 0.14 ± 0.01, 1.66 ± 0.01 and 3.26 ± 0.3 U μg–1 of protein, in TM, eBl, Bl, XBl and HBl, respectively). At the same time, XBl and HBl showed the lowest NO2– levels. However, NO2– values were lower in TM compared with eBl and Bl (0.04 ± 0.002, 0.07 ± 0.005, 0.06 ± 0.003, 0.01 ± 0.002 and 0.01 ± 0.001 nM μg–1 of protein, in TM, eBl, Bl, XBl and HBl, respectively). Similarly to NO2–, TBARS levels were lower in XBl and HBl compared with the other stages (0.0059 ± 0.002, 0.009 ± 0.003, 0.006 ± 0.002, 0.001 ± 0.0001 and 0.0009 ± 0.0002 μM μg–1 of protein, in TM, eBl, Bl, XBl and HBl, respectively). In conclusion, these results clearly indicate developmental stage-dependent changes in MnSOD activity and levels of NO2– and TBARS, suggesting that oxidative stress and lipid peroxidation are reduced in faster developing embryos.
176 CYTOCHALASIN PREVENTS SPERM HEAD INCORPORATION INTO FERTILIZED PIG OOCYTES
Authors: Machaty Z. , Wang C. , Lee K.
Published: December 2011

At fertilization, an acrosome-reacted sperm fuses to the plasma membrane of the oocyte. It is then internalized into the ooplasm and its head forms the male pronucleus that moves toward the female pronucleus. Experiments using cytochalasin, an inhibitor of actin polymerization, suggest that filamentous actin is necessary for spermatozoa incorporation deep into the oocyte cytoplasm. However, the exact role of actin in the process is not entirely clear and the consequences of inhibiting actin polymerization on embryo development are not known. In the present study, we investigated the effect of cytochalasin B on fertilization and subsequent embryo development in the porcine model. In vitro-matured oocytes were rinsed in fertilization medium, a modified Tris-buffered medium supplemented with 0.1% BSA and 1 mM caffeine. Groups of 30 oocytes were placed into 50-μL droplets of the medium covered with mineral oil. Fresh semen was collected from both a wild-type boar and a transgenic boar heterozygous for the green fluorescent protein (GFP), washed 3 times in Dulbecco's PBS and added at a final concentration of 5 × 105 cells mL–1 to each droplet containing the oocytes. The gametes were co-incubated for 5 h at 39°C under 5% CO2 in air. In the treatment group, 10 μg mL–1 of cytochalasin B was supplemented into the fertilization droplets; control groups received an equivalent amount of dimethyl sulfoxide, the solvent used to dissolve the inhibitor. After gamete co-incubation, potential zygotes were transferred to PZM-3 medium for culture. In experiment 1, mature oocytes were fertilized with the wild-type boar sperm. Embryos at the 6- or 8-cell stage were then collected, the blastomeres were separated and DNA from each blastomere was isolated for PCR analysis to monitor the presence of the male-specific SRY gene. In experiment 2, the GFP transgenic sperm was used for fertilization. Fertilized oocytes were cultured for 7 days and the resultant embryos were examined for GFP expression using an epifluorescence microscope. The developmental stages of the embryos were also determined by staining their nuclei with Hoechst 33342. We found that after cytochalasin B treatment, only 19.1% (21/110) of the blastomeres were positive for the SRY gene, whereas in the control group, SRY was detected in 54.5% (61/112) of the cases. In addition, in the cytochalasin B-treated group, 1.9% (7/376) of the embryos expressed GFP, as indicated by green fluorescence; this percentage was 35.4% (146/412) in the control embryos. The frequency of 2-cell and blastocyst-stage embryos was similar between the control and cytochalasin B-treated groups (84.1 vs 81.0%; and 11.6 vs 12.5%, respectively). The results indicate that the presence of cytochalasin B during fertilization effectively blocks the incorporation of the male nucleus into the newly formed zygote without affecting the developmental potential of the pre-implantation embryo.
179 EFFECTS OF DEHYDROEPIANDROSTERONE ON SPERM FERTILIZABILITY IN VITRO AND TESTICULAR GENE EXPRESSION
Authors: Suzuki O. , Koura M. , Noguchi Y. , Uchio-Yamada K. , Matsuda J.
Published: December 2011

Strain differences of in vitro fertilizability still constitute a serious problem in mouse reproduction. To improve the in vitro fertilizability of mouse sperm, we examined the effects of implanting time-release pellets of dehydroepiandrosterone (DHEA), a testosterone precursor, on sperm fertilizability and testicular gene expression. DHEA pellets (5 mg pellet–1, 21-day release form; Innovative Research of America) or placebo pellets were implanted subcutaneously in 9-week-old male mice from 2 strains: C57BL/6CrNSlc (B6) and 129X1/SvJJmsSlc (129X1). After 21 days, in vitro fertilization was conducted using epididymal sperm from these males and oocytes from superovulated 4-week-old Slc:ICR females. The percentages of 2-cell embryo formation in the placebo and DHEA groups were 78.2 ± 14.2% vs 89.3 ± 2.5%, respectively (mean ± standard error of the mean, n = 4 per group) using B6 sperm and 40.9 ± 8.8% vs 41.3 ± 3.1%, respectively (n = 4 per group) using 129X1 sperm, indicating no significant effect of DHEA treatment (P > 0.05 by 2-way ANOVA). However, a strain difference was quite evident (P < 0.05 by 2-way ANOVA). Quantitative Western blotting using testicular protein extracts with glyceraldehyde-3-phosphate dehydrogenase as an internal control showed that the expression of androgen receptor protein (AR) was significantly higher in B6 than in 129X1 males (P < 0.05 by 2-way ANOVA), whereas there was no significant effect of DHEA on the amount of AR in either strain (P > 0.05 by 2-way ANOVA). In B6 testes, 2-dimensional electrophoresis indicated that 1 protein spot was denser in DHEA-treated males than in placebo-treated males. In 129X1 testes, DHEA did not change the density of the corresponding spot. Mass spectrometry of the spot suggested that it was Cu/Zn superoxide dismutase (SOD1). Thus, 21-day-release pellets of 5 mg DHEA had no significant effect on sperm fertilizability in vitro in 2 strains, but the DHEA pellets induced a strain-dependent testicular protein expression, which might be because of the differential AR expression. Although DHEA treatment of male mice has the potential to improve sperm fertilizability in vitro, a more detailed study is needed to determine the optimal dose, age and duration of DHEA treatment.
This work was supported by a grant from the Ministry of Health, Labour and Welfare, Japan.
18 TOTAL PARENTERAL NUTRITION DURING NEONATAL CARE OF THE FIRST BITRANSGENIC FEMALE BOVINE CLONE
Authors: Mucci N. , Kaiser G. , Mutto A. , Wacholder S. , Aller J. , Hozbor F. , Manes J. , Alberio R.
Published: December 2011

This work describes the treatment with total parenteral nutrition (TPN) of a 37-day cloned calf after suffering ruminal acidosis by ingestion of milk. Cells for SCNT were obtained by using a bicistronic vector for human lysozyme and lactoferrin. We obtained 7 embryos and 2 pregnancies. Only one fetus was born alive weighing 47 kg and it was presented to the neonatal unit of INTA showing a deep depression, diarrhoea, dehydration (10%), hyperthermia and inability to stand (Table 1). A 20-cm-length 20-Ga-diameter catheter (Arrow) was placed in the jugular vein. Blood samples from catheter and brachial artery showed leukocytosis, hypoglycemia and metabolic acidosis (Table 1). Esophageal tube was placed to remove 5 L of ruminal content and for the administration of 2 L of a solution of sodium bicarbonate (40 g L–1 of water). Saline (NaCl, 9 g L–1), sodium bicarbonate (8.4 g/100 mL) and 10% dextrose were administered IV until dehydration; blood pH and glucose were corrected. Ceftriaxone 1 g IM/12 h (Acantex, Roche) to prevent bacterial translocation, 1.175 mg of flunixin meglumine (PharmaVet) as anti-endotoxic dose and 80 mg of ranitidine IV/12h (Vetanco) to prevent laminitis were also administered. Two litres of bovine plasma were administered during the first 2 days and, after this, we began with a TPN regimen due to lack of sucking reflex and animal anorexia. Kabiven (Fresenius Kabi AV) was administered at 1 g of lipids/kg/24 h by a regimen of 18 drops min–1 to prevent hyperlipidemia at the recommended dose for humans. For this reason, we also administered dextrose 25% 12.5 drops min–1 and amino acids 11.5% 504 mL (Rivero), to reach a dose of 10 g/kg/24 h and 2 g/kg/24 h, respectively. Saline (NaCl, 9 g L–1) and vitamin complex (Rivial Paediatrics, Rivero) was also administered to cover water and vitamin requirements. The TPN therapy lasted 24 days during which the animal regularized its metabolic functions, reversed signs of ruminal acidosis and learned to eat a balanced ration and hay. To our knowledge, no information is available on such a long period of TPN in bovine neonates. This work shows that TPN can increase the survival chances of high risk animals and thus the final efficiency of cloning and transgenesis.

Table 1.Clinical and haematological responses to 24-day total parenteral nutrition in a 37-day cloned female calf

The authors thank Fresenius Kavi and Rivero Laboratories for their support.
184 INHIBITION OF CALCIUM EFFLUX EXTENDS THE DURATION OF CALCIUM SIGNALS IN PIG OOCYTES
Authors: Yan L. Y. , Wang C. , Luo H. L. , Machaty Z.
Published: December 2011

Calcium signaling involves the transient elevation in the intracellular free-calcium concentration, which is responsible for controlling a great number of biological functions. In many cell types, a signal is generated when calcium stored in the endoplasmic reticulum is released into the cytoplasm, followed by an influx of calcium across the plasma membrane. At the same time, calcium is removed from the cytosol by ATPases, which pump it back into the intracellular store or out of the cell. The size of the calcium signal is thus determined by the amount of calcium moving into and out of the cytoplasm. In the present study, we investigated the effect of inhibiting the movement of calcium across the plasma membrane on a calcium-signal that was artificially induced in pig oocytes. In vitro-matured pig oocytes were loaded with the calcium-indicator dye, fura-2. The release of calcium from the cytoplasmic stores was stimulated by adding ethanol at a final concentration of 7% and changes in the intracellular free-calcium levels were monitored by using InCyt Im2, a dual-wavelength fluorescence imaging system. In the control group, fluorescent measurements were performed in the presence of extracellular calcium. In additional treatment groups, the ethanol treatment was performed in (1) a calcium-free medium (prevents calcium influx); (2) the presence of 1 mM gadolinium (limits calcium fluxes across the plasma membrane in both directions); and (3) the presence of gadolinium in a calcium-free medium. In each group, 15 oocytes were measured; the data were subjected to 1-way ANOVA and differences between treatment means were compared by the Tukey's test. We found that in control oocytes, 7% ethanol induced a rise of 722.1 ± 49.2 nM in the cytoplasmic calcium levels and the average duration of the calcium transient was 309.1 ± 11.3 s. In the calcium-free medium, this increase was significantly smaller (only 117.9 ± 4.8 nM; P < 0.01), probably because the calcium release was not followed by an influx of calcium across the plasma membrane. In the presence of 1 mM gadolinium in the regular calcium-containing medium, the intracellular calcium levels climbed by 278.8 ± 45.4 nM and dropped to baseline levels only after 773.3 ± 79.9 s. Finally, in the calcium-free medium and in the presence of 1 mM gadolinium, 7% ethanol induced only a small rise (64.5 ± 6.3 nM) in the cytoplasmic calcium levels. These latter increases were both significantly lower (P < 0.01) compared to that in the control group. The results indicate that (1) intracellular calcium signals are sustained by a calcium influx from the extracellular medium and (2) preventing calcium efflux extends the duration of the cytoplasmic calcium elevation in the oocyte. This may have relevance in the manipulation of intracellular calcium levels in oocytes for the development of novel parthenogenetic activation methods.
209 EPIGENETIC MODIFICATION WITH ZEBULARINE AND VALPROIC ACID AND EXPRESSION OF PLURIPOTENCY GENES IN BOVINE ADIPOSE STEM CELLS
Authors: Addison M. K. , Coley L. W. , Gentry G. T. , Godke R. A. , Bondioli K. R.
Published: December 2011

Bovine adipose-derived stem cells (ASC), a form of adult stem cells, are somatic cells that have similar characteristics of embryonic stem (ES) cells. ASC are multipotent and have been found to express genes associated with pluripotency, Oct4, Sox2 and Nanog. The unique properties of ASC make them a desirable source for reprogramming experiments. When somatic cells are reprogrammed, there are certain epigenetic changes or modifications that must occur. Epigenetic modifications will change the chromatin configuration without changing the DNA sequence. Somatic cells can be exposed to small molecules that induce some of these changes. Two epigenetic modifying factors are a DNA methyltranferase inhibitor, zebularine (Zeb) and a histone deacetylase inhibitor, valproic acid (VPA). By altering gene expression with these epigenetic modifiers, the cells may be stimulated to reprogram more efficiently than untreated cells. Three lines of bovine ASC were isolated from the stromal fraction of adipose tissue and expanded through 3 passages in DMEM (high glucose) supplemented with 10% fetal bovine serum. Cells were cultured by addition of 5 mM VPA or 100 μM Zeb to the culture medium and cultured for 5, 7, 10 or 14 days before mRNA was isolated and converted to cDNA. Control groups, consisting of untreated bovine ASC from the same cell lines, were cultured for the same time periods as the treatment groups. Quantitative RT-PCR was performed to determine transcript levels for Oct4, Sox2, Nanog and poly adenylate polymerase (PAP) as a reference gene. Transcript levels were quantified by relative quantification using the ΔΔCT method and expressed as ratios of the target genes (Oct4, Sox2 and Nanog) to the reference gene (PAP) and normalized against a calibrator consisting of untreated bovine ADS cells. Normalized ratios were log-transformed before analysis by ANOVA to correct for lack of normality. A one-way ANOVA was completed to test the statistical significance among the control and treatment groups. Pairwise comparison was conducted to test expression levels between individual treatments. When cells were treated for 5 days, Oct4 and Nanog expression levels were increased (P ≤ 0.05) between the control and both treatment groups. Expression of Sox2 was not different (P = 0.06) across treatment groups. Based on pair wise comparisons gene expression in VPA and Zeb treated groups were not different. Treatment for 7, 10, or 14 days did not result in any difference in transcript levels between the treatment groups and control for any of the genes analysed. VPA and Zeb treatment for 5 days may have produced a partial reprogramming. This partial reprogramming could aid in the bovine ASC reaching complete pluripotency when combined with other reprogramming techniques.
This work was financed in part by a grant from the LSU System for the ACRES/LSU Collaborative Research Program.
22 MALFORMATIONS FOUND BY AUTOPSY OF CLONED AND TRANSGENIC PIGLETS OF DIFFERENT BREEDS
Authors: Schmidt M. , Winter K. D. , Li J. , Kragh P. M. , Du Y. , Lin L. , Liu Y. , Li R. , Vajta G. , Callesen H.
Published: December 2011

Viability of cloned and transgenic piglets is seriously compromised and one obvious reason could be malformations. The aim of the present study was therefore to describe gross pathological conditions in dead pre-weaned piglets born after transfer to Large White (LW) recipients of cloned (LW donor cells) or transgenic (Yucatan or Göttingen donor cells) embryos. Donor cells were fibroblasts and the Göttingen and Yucatan cells were made transgenic with 1 of 5 genes known to dispose for different human diseases. Handmade cloning was used to produce embryos that after 5 to 6 days of in vitro culture were transferred surgically to 108 LW sows 4 days after their natural heat. Of these, 21 sows delivered cloned LW piglets, whereas 17 and 16 sows, respectively, delivered transgenic Göttingen and Yucatan piglets. Stillborn and dead pre-weaned piglets were necropsied and malformations registered. Data were analysed by Fisher's exact test with a significance level of P < 0.05. In the 54 litters, total litter size ranged from 1 to 22 piglets (mean 5.4 ± 0.5) and the overall mortality rate until weaning on day 28 was 59%. Malformations were found in piglets from 38 litters where an average of 35% of the piglets showed malformations (between 8 and 100%). In those litters, 1 to 7 piglets had 1, 2, or several malformations (Table 1). The malformation rate in the autopsied transgenic Göttingen was 58% and in Yucatan 46%; these were significantly higher than in the autopsied cloned LW piglets with 18%. Some of the malformations seemed to be related to breed and/or transgene; for instance, heart malformations were most frequent in Yucatan litters (70%) independent of the transgene, whereas gallbladder and gonad malformations were more frequent in various litters with the same transgene. These results show that the use of cloning in pigs results in a considerable loss of piglets due to malformations and transgenic transformation of the cells used for cloning superimpose on this problem. In combination, these elements could seriously compromise the use of pigs as a model for human diseases and the choice of breeds and also transgenes for this kind of work should be considered carefully. However, further improvements in production of cloned/transgenic embryos may ultimately reduce the incidence of malformations.

Table 1.Number of malformations in 54 litters of cloned or transgenic piglets
227 SUPEROVULATION AND EMBRYO COLLECTION IN WOOD BISON DURING THE ANOVULATORY SEASON
Authors: Palomino J. M. , McCorkell R. B. , Anzar M. , Woodbury M. R. , Hawkins N. , Prentice J. , Toosi B. , Adams G. P.
Published: December 2011

Brucellosis and tuberculosis are endemic in Wood Buffalo National Park, the largest reserve of wood bison (Bison bison athabascae) in Canada. Our goal is to produce and preserve disease-free embryos for the purpose of wood bison repopulation. This study was designed to determine if embryo collection is feasible in wood bison during the anovulatory season (May–July) and to test if progesterone priming is required for superovulation. A 2-by-2 design was used to determine the effectiveness of LH (Lutropin) or hCG (Chorulon) for induction of ovulation with or without intravaginal progesterone releasing device (PRID) in 32 wood bison cows. Follicular wave emergence was synchronized among bison by transvaginal ultrasound-guided follicle ablation. Synchronized bison were assigned to 4 groups: PRID+LH (n = 12), PRID+hCG (n = 4), no-PRID+LH (n = 12) and no-PRID+hCG (n = 4). A PRID was inserted on the day of follicular ablation in the respective groups. A single SC dose of 400 mg FSH (Folltropin) in a slow-release formulation was given the day after follicular ablation (i.e. on the expected day of a new follicular wave emergence, Day 0). The PRID was removed on Day 4 and either 25 mg LH or 2000 IU hCG was given IM on Day 5. Artificial insemination was done at 24, 36 and 48 h after LH or hCG treatment. Embryos were collected nonsurgically on Day 13 using commercial bovine equipment. Transrectal ultrasonography was done on Days 0, 5, 6, 7, 8 and 13 to record follicular and ovulatory responses. Count data (mean ± SEM) were analysed by two-way ANOVA and proportions by chi square. The number of ovulatory-sized follicles (≥10 mm) on Day 5 did not differ among groups (P = 0.33; Table 1). Ovulation rate (number of ovulations/number of follicles ≥10 mm) was greater in bison treated with hCG (P < 0.05; Table 1). The number of corpora lutea (CL) on Day 13 was greater in bison treated with hCG without a PRID (P < 0.05; Table 1). No differences in number of ova/embryos and transferable embryos were found among groups (P = 0.36 and P = 0.52, respectively; Table 1). In conclusion, progesterone priming (PRID) had no effect on ovarian superstimulation in wood bison in the anovulatory season. The ovulatory response was satisfactory only in bison treated with hCG. Embryo collection is feasible in wood bison, but the reasons for a low embryo collection rate in all groups remain unclear.

Table 1.Response to superovulation and embryo collection in wood bison

Funded by Advancing Canadian Agriculture and Agri-Food and Agri-Food Innovation.
228 DOMINANT FOLLICLE ABLATION IMPROVES SUPEROVULATORY RESPONSE AND EMBRYO YIELD IN BUFFALOES SUBJECTED TO INTEGRATED SINGLE-OVULATION EMBRYO TRANSFER - MULTIPLE-OVULATION EMBRYO TRANSFER PROGRAM
Authors: Singh I. , Sharma R. K. , Phulia S. K. , Kumar D. , Saxena O. , Verma R. , Khanna S.
Published: December 2011

Effect of the removal of inhibitory consequences of a dominant follicle (DF) on the recruitment, growth and ovulation of follicles in cyclic buffaloes induced to superovulate, was evaluated. Eight elite, multiparous, donor Murrah buffaloes, 80 to 120 days postpartum, were included in an integrated single-ovulation embryo transfer–multiple-ovulation embryo transfer (SOET-MOET) schedule. Superovulation with FSH (Folltropin® NIH-FSH-P1, Bioniche Animal Health, Belleville, ON, Canada) was preceded by nonsurgical single embryo (SOET) recovery on Day 5 or 6 after prostaglandin F2α (Lutalyse®, Pfizer, Madison, NJ, USA)-induced oestrus and AI (Day 0). For MOET, Folltropin was administered from Day 9 p.m. through to Day 13 p.m. in a twice-daily descending dose schedule (0–5, 5–4, 4–3, 3–2, 2–1 mL; total dose of 580 mg of NIH-FSH-P1). A dose of 25 mg of Lutalyse was administered on Day 12 a.m. and p.m. Donors were inseminated with proven semen on Day 14 a.m. and p.m. and Day 15 a.m. Nonsurgical embryo collection was carried out on Day 19 or 20 followed by administration of Lutalyse on Day 25. Following sexual rest for one complete oestrous cycle, Day 5 or 6 SOET was repeated. On Day 9, ultrasound-guided follicle aspiration was used to ablate the DF and the second MOET treatment was initiated at the same time. Mean diameter of DF at the start of superovulation treatment on Day 9 was similar between the first (12.5 ± 0.66 mm) and second (12.2 ± 0.33 mm) MOET cycles. At AI, mean number (9.6 ± 1.43 vs 13.4 ± 1.87) of follicles >9 mm were higher (P < 0.05) in the second (ablation) MOET cycle, though their mean diameters were similar (11.7 ± 0.21 vs 12.3 ± 0.21 mm; P = 0.17). On the day of ova/embryo collection, almost similar numbers of large follicles (4.1 ± 0.93 and 4.4 ± 0.84), with mean diameters of 12.9 ± 0.74 and 13.4 ± 0.83 mm, were observed for the first and second MOET, respectively. Mean (± SEM) numbers of corpora lutea (3.9 ± 0.81 and 4.9 ± 0.61; P = 0.19) and transferable quality embryos (0.6 ± 0.37 and 1.6 ± 0.37; P = 0.077) were counted in the first and second (ablation) MOET cycles, respectively. These preliminary findings suggest that ablation of DF before start of superovulatory treatment may be beneficial in improving the yield of transferable embryos in a buffalo MOET program.
Director, CIRB, is acknowledged for the experimental facilities.