bluuebunny/crossref_metadata_2025 huggingface dataset:
177 ANTHOCYANIN ISOLATED FROM PURPLE SWEET POTATO IMPROVES DEVELOPMENT AND REDUCES OXIDATIVE STRESS OF BOVINE PRE-IMPLANTATION EMBRYOS EXPOSED TO HEAT SHOCK
Authors: Sakatani M. , Suda I. , Oki T. , Kobayashi S.-I. , Kobayashi S. , Takahashi M.
Published: December 2006

Development of cleavage-stage pre-implantation embryos is disrupted by exposure to heat shock. Heat shock also increases intracellular reactive oxygen species (ROS) in pre-implantation embryos. Therefore, reduction of intracellular ROS levels might improve the development of heat-shocked embryos. Recently the antioxidative activities of polyphenols have been widely reported to reduce the oxidative stress. In this study, we investigated the effect of purple sweet potato anthocyanin, a kind of polyphenol that is a strong ROS scavenger, on development and intracellular redox status of bovine pre-implantation embryos exposed to heat shock. Experiment 1: In vitro-produced 8-16-cell-stage embryos on Day 2 after fertilization were exposed to 41.5�C for 6 h in CR1aa containing 0, 0.1, 1, and 10 �g/mL anthocyanin at 5% CO2, 5% O2, and 90% N2. After heat shock, embryos were cultured at 38.5�C at 5% CO2, 5% O2 until Day 8. On Day 8, the proportion of embryos developing to the blastocyst stage was evaluated. Blastocyst total cell number and the ratio between inner cell mass and tropheoderm were evaluated by differential staining. The experiment was replicated five times with more than 70 embryos used in each treatment. Experiment 2: Heat shock treatment of in vitro-produced 8-16-cell-stage embryos was carried out as described in experiment 1. After heat shock, intracellular ROS and glutathione (GSH) levels were measured in individual 8-16 cell stage embryos with fluorescent probes (22,72-dichlorodihydrofluorescein diacetate for ROS and CellTracker" Blue (Invitrogen Japan K. K., Tokyo, Japan) for GSH). The fluorescence emissions of each treatment were normalized to those of 8-16 cell stage embryos cultured at 38.5�C without anthocyanin to obtain the relative fluorescence emission. This experiment was replicated four times. Embryos treated with heat stress without anthocyanin (0 �g/mL) showed low development (14.6 � 3.6%) and blastocyst total cell number (88.2 � 9.4). However, embryos treated with 0.1 �g/mL anthocyanin improved development (31.7 � 4.5%, P < 0.05) and increased the total cell number (96.5 � 11.3). The higher concentrations of anthocyanin (1 and 10 �g/mL) did not affect development and cell number. The intracellular ROS levels in heat-shocked embryos were significantly reduced by all concentrations of anthocyanin (P < 0.05). In addition, anthocyanin increased GSH levels at all doses tested (P < 0.05). These results indicate that an appropriate concentration of anthocyanin improves development by regulating intracellular redox balance in bovine embryos exposed to heat shock.
178 IMPROVED DEVELOPMENTAL COMPETENCE OF BOVINE EMBRYOS BY PGI2 ANALOG TREATMENT
Authors: Song B. S. , Kim J. S. , Koo D. B. , Park J. S. , Lee K. K. , Han Y. M.
Published: December 2006

The microenvironment of the follopian tube, in which the oviductal fluid contains a variety of cytokines and growth factors, affects pre-implantation development of fertilized embryos in mammals. Prostaglandin I2 (PGI2, prostacyclin) exists in oviductal fluid and is synthesized from arachidonic acid by prostacyclin synthetase. PGI2 also enhances the implantation rate of mouse embryos. In this study, the effect of PGI2 analog on the development of bovine embryos was examined. Bovine cumulus oocytes complexes (COCs) were matured in TCM-199 medium supplemented with 10 IU/mL pregnant mare serum gonadotropin (PMSG), 10 IU/mL hCG, and 10 ng/mL epidermal growth factor (EGF) at 39�C, 5% CO2 in air for 20-22 h. Following in vitro maturation, COCs were fertilized in Fert-TALP medium containing 0.6% BSA using frozen semen. Also, oocytes matured in vitro were enucleated, individually reconstructed with bESF cells, fused, and then activated by treatment with 5 �M ionomycin for 5 min and 2 mM 6-DMAP for 4 h. In vitro-fertilized (IVF) and nuclear-transferred (NT) eggs were cultured in 50 ��L drops of CR1-aa medium supplemented with 0.3% BSA in the absence or presence of 1 �M PGI2 analog at 39�C, 5% CO2 in air, respectively. At 3 days of culture, cleaved embryos were further cultured in the same culture media supplemented with 10% FBS for 4 days. Allocations of blastocysts to inner cell mass (ICM) and trophoblast (TE) cells were investigated to assess embryo quality. All experiments were repeated more than three times. All data were analyzed by using the Duncan test of ANOVA by the Statistical Analysis System (SAS Institute, Inc., Cary, NC, USA) and numbers of nuclei in blastocysts were expressed as mean � SE. No difference was detected in the cleaved rate of the eggs between the treated- and nontreated groups. IVF zygotes treated with PGI2 analog represented a higher developmental rate (33%, 122/418) to the blastocyst stage than nontreated controls (24%, 107/456) (P < 0.05). Among IVF-derived blastocysts, interestingly, the proportion (46%, 84/181) of expanded blastocysts was significantly higher in the PGI2 analog-treated group compared with that in the nontreated group (28%, 46/164). The number of nuclei in (165 � 6.1, n = 15) in blastocysts in the PGI2 analog-treated group was higher than that (146.12 � 5.7, n = 18) in the nontreated group (P < 0.05). No difference was detected in the ratio of ICM to total cells between PGI2 analog-treated (42.0 � 3.0%) and nontreated groups (41.9 � 2.9%). Like the IVF embryos, NT embryos in the PGI2 analog-treated group showed a higher in vitro developmental rate (33.6%, 43/128) than the nontreated embryos (24.2%, 32/132) (P < 0.05). Our results indicate that PGI2 analog improves the kinetics of embryo development in cattle.
185 EMBRYONIC LOSS IN PIGS ASSOCIATED WITH OVIDUCT TRANSPLANTATION OF EARLY-STAGE EMBRYOS WITH DAMAGES IN THE ZONA PELLUCIDA
Authors: Ueno S. , Kurome M. , Tomii R. , Hiruma K. , Maeda N. , Saito H. , Nagashima H.
Published: December 2006

It is assumed that if porcine early-stage embryos with damages in their zonae pellucidae are transplanted to the recipient's oviduct, they may suffer from mechanical and immunological stresses by oviduct contraction and the recipient's immune response. This study aimed to examine the impact of zona pellucida damages, which might arise during nuclear transfer and intra cytoplasmic sperm injection (ICSI), on the development and survival of transplanted embryos. Cumulus-oocyte complexes were collected from ovaries obtained at a local slaughterhouse and matured in vitro in NCSU23 to prepare MII-stage oocytes. The zonae pellucidae of these oocytes were either penetrated with 8- to 10-�m square-ended microinjection pipettes or incised with 35- to 40-�m beveled enucleation pipettes. Intact oocytes were used as controls. The oocytes were electroactivated to induce parthenogenesis and transplanted to the oviducts of estrus-synchronized recipient gilts (estrus-synchronized with 1000 IU eCG and 1500 IU hCG). After 5 to 7 days, the recipient uteri were flushed with PBS supplemented with 1% fetal bovine serum (FBS) to collect embryos, and their development (morula-blastocyst stage embryos/collected embryos) and survival (viable embryos/collected embryos) were determined. In total, 221 zona-penetrated, 129 zona-incised, and 57 intact embryos were transplanted to four, two and two gilts, respectively. The efficiency of embryo recovery was similar in all groups (59.0 to 81.8%). However, the zona-penetrated and zona-incised embryos showed inconsistent development and survival compared with controls; the development and survival rate were 92.6% (25/27) to 96.7% (29/30) and 77.8% (21/27) to 96.7% (29/30) in control embryos, respectively, whereas those of zona-penetrated embryos were 57.1% (28/49) to 95.7% (22/23) and 8.2% (4/49) to 78.3% (18/30), and those of zona-incised embryos were 47.6% (30/63) to 92.3% (36/39) and 23.8% (15/63) to 92.3% (22/23), respectively. Large foci of cells that appeared to be macrophage giant cells were observed at the surface or inside of the degenerated zona-damaged embryos. These results indicate that the recipient's immune response may impair development after transplantation of the embryo to the oviduct, when there is damage in the zona pellucida. This may be one of the factors attributable to the reduced efficiency of live progeny production by ICSI and nuclear transfer.
This work was supported by PROBRAIN.
19 PREGNANCY RATES IN DAIRY COWS TREATED WITH INTRAVAGINAL PROGESTERONE DEVICES AND DIFFERENT FIXED-TIME AI PROTOCOLS
Authors: Veneranda G. , Filippi L. , Racca D. , Romero G. , Balla E. , Cutaia L. , Bo G. A.
Published: December 2006

Two experiments were designed to evaluate the effect of different treatments for the synchronization of ovulation on pregnancy rates following fixed-time AI (FTAI) of lactating dairy cows. In Experiment 1, 394 Holstein cows that were 61.7 � 13.6 days postpartum (range 35 to 94 days), with a milk yield of 30.7 � 6.8 liters per day (range 12.0 to 52.4 liters) and a body condition score (BCS) between 2.5 to 3.5 out of 5, were used. Cows were blocked by days postpartum and milk yield and randomly assigned to one of four treatment groups. Cows in P4+EB treatment groups received an intravaginal DIB device (1 g P4; Syntex, Argentina) and 2 mg of estradiol benzoate (EB; Syntex) i.m. on Day 0. On Day 8, DIB devices were removed, and cows received PGF (150 �g D+cloprostenol: Ciclase; Syntex), and were subdivided to receive 400 IU eCG (Novormon 5000, Syntex) i.m. or no further treatment at that time. On Day 9, all cows received 1 mg of EB and were FTAI 60 h after DIB removal. Cows in P4-Synch groups received a DIB device and 50 �g of GnRH (Lecirelina, Gonasyn; Syntex) i.m. on Day 0. On Day 7, DIB devices were removed; cows received PGF and were divided to receive 400 IU eCG i.m. or no further treatment. On Day 9, all cows received a second GnRH treatment and were FTAI 60 h after DIB removal. Blood samples were taken on Days -10 and 0 to determine plasma P4 concentrations; 93% of the cows had >1 ng/mL P4 in at least one sample. In Experiment 2, 200 lactating cows from the same farm were treated with the P4+EB+eCG and P4-Synch without eCG treatments with either a DIB or a CIDR-B (1.9 g P4; Pfizer Animal Health, Groton, CT, USA) in a 2 � 2 factorial design. Cows were examined by rectal palpation 50 days after FTAI to determine pregnancy status, and data were analyzed by chi-square Mantel-Haenszel test. There was an EB/GnRH by eCG interaction (P < 0.05) which was attributed to a higher pregnancy rate in the P4+EB+eCG group (44/98; 44.9%) than in the P4+EB without eCG group (30/100; 30.0%) and P4-Synch+eCG group (30/98; 30.6%); the P4-Synch without eCG group had an intermediate pregnancy rate that did not differ from the other treatment groups (37/98; 38.8%). In Experiment 2, no significant differences (P = 0.40) in pregnancy rates were detected between cows treated with DIB (51/100; 51.0%) or CIDR-B (42/100; 42.0%), and there was no difference (P = 0.18) between P4+EB+eCG (52/100; 52.0%) and P4-Synch without eCG (41/100; 41.0%) treated groups. Results suggest that the addition of eCG will improve pregnancy rates following FTAI in lactating dairy cows treated with EB but not in those treated with GnRH at the time of insertion and after removal of a P4 releasing device. Although treatment with P4+EB+eCG resulted in numerically higher pregnancy rates, results were not different from those obtained in the GnRH-based treatment, without the addition of eCG.
201 CHARACTERIZATION OF CANINE EMBRYONIC GERM CELLS: AN EXPERIMENTAL MODEL FOR CELL THERAPY
Authors: Martins D. S. , Saraiva N. Z. , Garcia J. M. , Ambrósio C. E. , Zatz M. , Miglino M. A.
Published: December 2006

Dogs represent excellent models to test different approaches before use for human therapy. Studies with animal models have suggested that the transplant of stem cells may have success in the treatment of degenerative diseases such as Parkinson's disease, diabetes, Duchenne muscular dystrophy (DMD), and acquired lesions. Embryonic stem cells are pluripotent and therefore have the potential to form all tissues. Our research aims to contribute to the treatment of DMD through the isolation and identification of embryonic germ cells and the development of the methodology of cellular differentiation for future transplantation into dystrophic dogs. Mongrel female dogs were ovariehysterectomized between 25 and 30 days of pregnancy. For recovery of embryos, the excised uterine horns were flushed with heparinized PBS. Samples collected from somites near the mesonephros area of four embryos recovered at 22 to 24 days of pregnancy and designated as A1 through A4 were dissociated and placed in culture. Isolated embryonic cells were allowed to plate onto monolayers of canine fibroblast cells. Flow cytometry was used to identify CD34+ markers. Isolated compact colonies of embryonic germ cells were seen growing around tissue fragments at 7 days of culture and remained in the undifferentiated stage until approximately 21 days in culture. At 14 days of explant, cell colonies were analyzed by flow cytometry. Cells from the A2 embryos contained the highest number of CD34+ cells, whereas no cells from A4 embryos showed specificity for the marker. A small proportion (2.12%) of cells from embryos A1 and A3 showed specificity for the CD34 marker. A quantity of A2 embryo cells that had maintained stem cell characteristics were frozen for future studies. Our results suggest that although spontaneous differentiation occurred, a small population of cells maintain the characteristics of stem cells. We are currently trying to improve the methodology to maintain cells undifferentiated for longer periods and to better control the specific differentiation process.
212 TRYPLE SELECT (10X) EFFECTIVELY REMOVES BOVINE HERPESVIRUS-1 ASSOCIATED WITH IN VIVO-DERIVED EMBRYOS
Authors: Marley M. , Looney C. , Givens M. , Galik P. , Riddell K. , Stringfellow D.
Published: December 2006

Porcine-origin trypsin will effectively remove bovine herpesvirus-1 (BHV-1) from in vivo-derived embryos. It is not known if TrypLE (Invitrogen, Carlsbad, CA, USA) could be used to remove BHV-1, but this recombinant porcine sequence trypsin-like protease would be an attractive alternative because it is highly stable at room temperature and does not pose the same threat for contamination as animal-origin trypsin. Thus, the objective of this study was to determine if TrypLE Express (1X) for 1.5 min of exposure or TrypLE Select (10X) for 10 min of exposure would be effective at removing BHV-1 from Day 7 zona pellucida-intact, in vivo-derived embryos after they had been exposed to the virus. Day 7 bovine in vivo-derived morulae and blastocysts and non-fertile degenerate (NFD) embryos were collected and shipped overnight to our facility. Upon arrival, the zona pellucida intact embryos were washed according to the International Embryo Transfer Society protocol. Developed embryos were washed separately from NFD embryos. One group of 5 or 10 NFD or developed embryos was not exposed to virus and served as the negative control. The remaining embryos and 10 NFD were exposed to 106 PFU/mL BHV-1 (Colorado strain) for 1 h. Following exposure, one group of 5 or 10 NFD or developed embryos was washed and served as the positive control. One group of 10 developed embryos was washed and treated with porcine origin trypsin. The remaining developed embryos were divided into groups of 5 or 10 and washed and treated with TrypLE Express for 1.5 min or TrypLE Select (10X) for 10 min. Following treatment, the embryos were sonicated in groups of 5 or 10 and assayed by virus isolation. The negative control embryos, porcine origin trypsin treated embryos, and TrypLE Select treated embryos were negative for virus. The positive control embryos and the TrypLE Express treated embryos were positive on virus isolation (Table 1). When it was determined that TrypLE Express was not effective at 1.5 min, TrypLE Select (10X) was used for 10 min. These preliminary results indicate that use of TrypLE Select (10X) for 10 min is effective for removal of BHV-1 associated with Day 7, zona pellucida-intact, in vivo-derived embryos. In addition, TrypLE Select has the advantage of being an animal-origin-free product. However, use of TrypLE Express (1X) for 1.5 min was not effective. Because it is not practical to expose embryos to trypsin for 10 min, further research is needed to determine the ideal treatment concentration and time that will effectively remove BHV-1 without harming in vivo-derived bovine embryos.

Table 1.
Virus isolation results
217 QUALITY CHANGES IN POST-THAW SPRINGBOK (ANTIDORCAS MARSUPIALIS) EPIDIDYMAL SPERM MAINTAINED IN FERTILIZATION MEDIUM
Authors: Chatiza F. P. , Pieterse G. M. , Bartels P.
Published: December 2006

The availability of gametes from the cropping of excess wildlife species provides the opportunity for the advancement of knowledge into assisted reproductive technology for possible future conservation measures. Little is known about the longevity of springbok (Antidorcas marsupialis) spermatozoa maintained in fertilization medium. The aim of this project was to determine the quality changes of post-thawed springbok spermatozoa incubated in fertilization medium by measuring plasma membrane integrity over time. Testes (n = 12) were obtained from two geographically distinct free-ranging springbok populations in South Africa. Spermatozoa were flushed from the cauda epididymides within three hours of the animals' death. Samples from an individual male were pooled, diluted to 400 × 106 sperm/mL with Biladyl (Minitüb, Tiefenbach, Germany) fraction A (no glycerol) and equilibrated in a water bath for 6 h at 4°C. An equal volume of Biladyl fraction B (containing 12% glycerol) was added to the sample to make a final concentration of 200 × 106 sperm/mL. Samples were loaded into 0.25-mL straws and frozen in liquid nitrogen vapor (5 cm above the liquid nitrogen level) for 20 min after which they were plunged into liquid nitrogen. Straws from each sample were thawed for 20 s at 36°C in a water bath. Thawed spermatozoa (100 μL) was added to 1 mL IVF-TALP medium containing heparin and PHE (Vajta et al. 1996 Theriogenology 45, 683–689) in 2-mL Nunc tubes (AEC, Amersham, South Africa) and incubated at 38.7°C, in humidified 5% CO2 balance air for 30 h. Aliquots were extracted from the incubating spermatozoa to determine plasma membrane integrity at 6-h intervals. Propidium iodide (Sigma, South Africa) at 50 ng/mL (10 min at RT) was used to evaluate membrane integrity under fluorescence microscopy at ×400, with a 450-nm excitation filter, a 510-nm dicroic beam splitter, and a 520-nm barrier filter. Cells with damaged plasma membrane have nuclei that fluorescence red. Eosin/nigrosin was also used to evaluate membrane integrity under ×400 bright-field microscopy. Cells with damaged plasma membrane stain purple-red, whereas the balance of cells remain translucent. The average post-thaw motility of spermatozoa in populations A and B was 69% (n = 6) and 68% (n = 6), respectively. Plasma membrane integrity of post-thawed springbok spermatozoa deceased steadily in IVF-TALP medium over the 30-h period (Table 1). Cryopreserved epididymal sperm derived from free-ranging springbok populations survive in IVF-TALP media and may be useful in future conservation activities where an isolated gene pool requires genetic supplementation through one or more assisted reproduction techniques such as IVF or AI. Further research is required to confirm and extend these findings.

Table 1.
Percentage plasma membrane integrity of post-thawed springbok sperm over time
234 POTENTIAL SEASONAL INFLUENCE ON THE RECOVERY OF IN VIVO-MATURED CANINE OOCYTES BY FLUSHING FALLOPIAN TUBES
Authors: Hossein M. S. , M. K. K  , Jang G. , Fibrianto Y. H. , Oh H. J. , Kim H. J. , Kang S. K. , Lee B. C. , Hwang W. S.
Published: December 2006

Reproductive physiology, especially the gamete physiology of bitches, hinders the success of in vitro maturation procedure. Assisted reproductive techniques (ART) in canine greatly depend on in vivo matured oocytes. The study was designed to evaluate the potential of flushing fallopian tubes to collect in vivo-matured oocytes for ART by laparotomy and to study the seasonal effects and the parity of donor bitches on the success of oocyte retrieval. A total of 124 bitches, 39, 44, 25, and 16 in spring, summer, autumn and winter, respectively, were used. The average temperature (�F) and relative humidity (%) were 55.29 and 57.17, 75.67, and 72.83, 59.65 and 64.67, and 33.55 and 58.67 for spring, summer, autumn, and winter, respectively. Eighteen nulliparous and 18 multiparous bitches were used to determine the effect of parity. Vaginal cytology and serum progesterone concentration were checked daily to determine ovulation time. Laparotomy was performed under general anesthesia after 72 h when progesterone initially reached 4.0 nm/mL or more. About 7 mL of TCM-199 supplemented with HEPES (Gibco, Grand Island, NY, USA) was used to flush each individual fallopian tube. After morphological assessment under a stereomicroscope, the oocytes were denuded in 0.5% (W/V) hyaluronidase solution (Sigma, St. Louis, MO, USA) and stained with 5 �g/mL bisbenzimide (Sigma). Nuclear status was then observed under ultraviolet light. Based on the nuclear stage, cumulus cell layers, color, and homogeneity of ooplasm, the oocytes were categorized as good, fair, poor, immature, or aged. The good and fair oocytes were considered usable and poor, aged, and immature oocytes were considered unusable for ART. In vivo-matured oocytes can be collected all year around by flushing fallopian tubes. A significantly higher average number of oocytes per bitch were observed during the spring (11.21 � 0.56), compared to the other seasons (7.88 � 0.63 to 9.27 � 0.35) (P < 0.05). A comparatively higher percentage of usable oocytes were collected during autumn. The percentage of oocyte recovery was 74.67, 67.51, 71.57, and 62.70 for autumn, spring, summer, and winter, respectively (P > 0.05). Both the number and the quality of oocytes were influenced by the parity of the donor bitch. Significantly more oocytes were collected from the multiparous bitches compared to nulliparous bitches (10.4 � 0.54 and 7.7 � 0.86, respectively). The percentage of usable oocytes was larger in multiparous bitches compared to nulliparous (70.75 and 64.74, respectively) (P > 0.05). Collection of in vivo-produced oocytes could be a potential source of matured oocytes for ART in canine.
24 GLOBAL TRANSCRIPT PROFILING OF CLONED BOVINE BLASTOCYSTS USING AFFYMETRIX GENECHIP TECHNOLOGY
Authors: Beyhan Z. , Ross P. , Iager A. , Kocabas A. , Cunniff K. , Maserati M. , Page R. , Cibelli J.
Published: December 2006

Identification of genes implicated in the biological processes of somatic cell nuclear transfer will improve our understanding of reprogramming events, i.e. the transformation of a lineage-committed cell into a pluripotent one. In addition, the gene expression profile of cloned embryos can help explain the widely reported developmental failures in cloned animals. In this study, we investigated global gene expression profiles of bovine in vitro-fertilized and cloned embryos using Gene Chip Bovine Genome Arrays (Affymetrix, Inc., Santa Clara, CA, USA). For the generation of cloned bovine blastocysts from two adult fibroblast lines (C and D), we employed methods previously proven to generate live offspring and compared these offspring to in vitro-produced blastocysts. Total RNA isolated from groups of 10 blastocysts was amplified by a template-switching PCR. Amplified cDNAs were used to synthesize biotin-labeled antisense RNAs (aRNAs) during and in vitro transcription reaction. Labeled aRNAs were hybridized to microarrays as described by the manufacturer. Experiments were performed in four replicates. Expression data were analyzed using the Significance Analysis of Microarrays (SAM; Tusher et al. 2001 Proc. Natl. Acad. Sci. 98, 5116-5121) procedure and software. Overall, 48.4% and 46% of 23 000 bovine transcripts spotted on the arrays were present in cloned and in in vitro-produced control blastocysts, respectively. The SAM procedure identified 43 genes that changed at least 1.5-fold, with an estimated false discovery rate (FDR) of 20%. Comparison of gene expression between NT embryos produced from two different cell lines and IVF controls with the same criteria revealed 6 (clones from cell line C vs. IVF) and 46 (clones from cell line D vs. IVF) differentially expressed genes. The number of transcripts expressed differentially between the cloned embryos with different donor cell origin was 437. Of the 43 differentially expressed transcripts in cloned blastocysts, 13 have unknown functions and the rest of the genes related to cell structure (tuftelin, desmoplakin), cell cycle/mitosis (Kinesin like 4, katanin, stathmin, PCNA), energy metabolism (lactate dehydrogenase, ATPsynthase, lipid-binding protein, keto acid dehydrogenase E1, metallothionein), and cell signaling (GTP-binding protein1, GTP binding stimulatory protein). Our results indicate that expression profiles of cloned blastocysts could be affected by somatic donor cell.
241 EFFECT OF INTRODUCTION OF THE INVERDALE FECUNDITY (FecXI) GENE INTO A TEXEL × SCOTTISH HILL EWE (CHEVIOT) FLOCK ON LITTER SIZE, BIRTHWEIGHT, AND LAMB SURVIVAL
Authors: Alink F. M. , Robinson J. J. , Mylne M. J. A. , Kenyon P. , Watt R. G. , Wood M. J. , McEvoy T. G.
Published: December 2006

The naturally occurring Inverdale fecundity gene (FecXI), first identified in a Romney flock in New Zealand is located on the X-chromosome and is a point mutation of the bone morphogenetic protein-15 (BMP-15) gene. Although homozygous carriers are infertile due to abnormal development (hypoplasia) of the ovaries, heterozygotes exhibit an increase in ovulation rate and numbers of lambs born, thus making the gene a candidate for improving prolificacy of crossbred hill ewes in a stratified hill and lowground sheep industry. Heterozygous Inverdale gene-carrying crossbred (Texel � Cheviot) ewes (n = 76) and noncarrier controls (n = 18) were mated by non-carrier Texel rams at approximately 18 months of age. Ultrasound pregnancy scanning took place at approximately 70 days of gestation. Lambing records comprised litter size and birthweight data and all losses, including mummified fetuses. Regression analysis tested effects of ewe genotype, pre-mating liveweight, and body condition score on litter size. ANOVA and Kruskal-Wallis tests were used to compare genotype and litter size category effects on dam and offspring data. Mean (� SEM) litter size was 2.6 � 0.10 and 1.9 � 0.11 for FecXI carrier and noncarrier ewes (P < 0.001). Although carrier ewes had lower pre-mating liveweights (60 � 5.2 vs. 63 � 3.8 kg; P < 0.05) and body condition scores (3.1 � 0.29 vs. 3.3 � 0.24; P < 0.01) than noncarrier ewes, litter size was not affected by either factor. Ultrasound scanning was a good predictor (r2 = 0.86; P < 0.001) of the eventual lamb crop. Incidences of singletons, twins, triplets and higher multiples were 8, 38, 42 and 12%, respectively, among FecXI carrier dams. Corresponding incidences among noncarriers were 17, 78, 5, and 0%. Peri- and neonatal losses increased with litter size. Of lambs from single, twin or triplet births, losses were 20% and 6% for FecXI carrier and noncarrier ewe groups, respectively (P = 0.049). Of 32 quadruplets (mean live weight = 2.65 kg), 9 did not survive; the sole set of quintuplets (mean = 1.52 kg) also succumbed. Birth weights of lambs influenced survival (P < 0.001); among quadruplets, for example, survivors were 0.5 kg heavier than counterparts that died. Surviving triplets from FecXI carrier dams were 0.8 kg heavier than non-survivors (3.66 kg vs. 2.86 kg). Overall, in this study mortality was four times greater among lambs below 3 kg live weight than among their heavier counterparts. Consequently, further studies are needed to investigate how Inverdale gene-carrying ewes can be managed during the peri-conception period to optimize litter size, placental function, and prenatal growth for maximum lamb survival.
This work was funded by HIE, ANM Group, Britbreed, Ltd., and Harbro Ltd.; F. M. A. is a Genesis Faraday Associate and is sponsored by the SAC Trust, Harbro, Ltd., and Innovis.
243 REPROGRAMMING OF FIBROBLASTS TO EXPRESS MALE GERM CELL OR MAST CELL FUNCTIONS USING TESTIS OR MAST CELL-DERIVED CELL EXTRACTS
Authors: Choi H.-Y. , Kim B.-W. , Lee E.-R. , Roh S. , Cho S.-G.
Published: December 2006

The concept of epigenetic reprogramming of a somatic nucleus was supported by the birth of cloned animals and the derivation of embryonic stem cells after nuclear transplantation into oocytes. Moreover, recent studies reported that the direct transformation of one differentiated somatic cell type into another is possible and would be advantageous for producing isogenic replacement cells. In this study, we were able to modulate the cell fate of fibroblasts by introducing cell extracts derived from a mast cell line, RBL-2H3. NIH-3T3 cells were treated with streptolysin O (SLO; 230 ng/mL), which reversibly permeablizes plasma membrane, and incubated for 30 min with the mast cell- derived cell extracts (4 mg/mL). After resealing the membrane of the cells, we incubated the cells for 3 weeks and then analyzed them for the expression of mast cell-specific genes such as MAFA (mast cell function-associated antigen) and FceRI (high-affinity IgE receptor). Our results showed that the cell extracts can activate the expression of mast cell-specific genes, implying that cell extracts can provide regulatory components required for reprogramming the cell fate to initiate a transcriptional program specific for the cell type. Moreover, mast cell-specific degranulation and cell morphology changes were observed in cultured mouse fibroblasts. We could detect mast cell-specific functions even after 15 days of incubation. Next, to induce porcine fibroblasts to take on testis sertoli cell-specific properties, we reversibly permeablized porcine primary fibroblasts with SLO (230 ng/mL) and incubated the cells with porcine testis extracts (4 mg/mL). As expected, in the reprogrammed primary porcine fibroblasts, the porcine testis extracts activated the expression of porcine testis sertoli cell-specific genes including protamine 1 and 2, SOX9, MIS (mullerian inhibitory substance), preproacrosine (ACR), phosphoglycerate kinase-2 (PGK-2), protein C, and c-kit ligand. The male germ cell functions were sustained for more than 10 days after the reprogramming process. Taken together, our data suggest that testis- or mast cell-derived cell extracts can reprogram fibroblasts to express male germ cell or mast cell functions, respectively, supporting the concept that cell extracts can reprogram the genome activity to activate cell-specific gene expression.
This work was supported by ARPC (Grant no. 204117-03-1-HD110) in Korea, and by Biogreen 21 program (Grant no. 20050401034658).
253 GENE SILENCING OF CYCLOOXYGENASE-2 mRNA BY RNA INTERFERENCE IN BOVINE CUMULUS - GRANULOSA CELLS
Authors: Kobayashi S.-I. , Sakatani M. , Kobayashi S. , Okuda K. , Takahashi M.
Published: December 2006

Recently, interference RNA (RNAi), inducing inhibition of the specific gene expression, attracted a lot of attention. Many researchers have reported that the 21-mer small interference RNA (siRNA) is introduced into target cells and then siRNA can suppress the gene expression. RNAi is a useful tool for functional analysis of specific genes. However, there is little information about RNAi for the analysis of gene function in reproductive physiology in ruminants. Thus, this study was aimed at evaluating RNAi for the analysis of cyclooxygenase-2 (Cox-2) mRNA expression in bovine cumulus-granulosa (CG) cells as well as prostaglandin F2� (PGF2�) production. We investigated both the effective concentration of Cox-2 siRNA and the effect of the introduction time of siRNA on Cox-2 mRNA expression. Bovine CG cells were collected at slaughterhouse and cultured in 4-well dishes. After the cells reached confluency, two experiments were conducted. In the present study, Cox-2 siRNA was simply added to culture medium with lipofectamine" 2000 (Invitrogen Japan K. K., Tokyo, Japan) as the transfection reagent. In experiment 1, the concentration of 0, 100, 250, and 500 pM of Cox-2 siRNA was introduced into the CG cells. After 24 h of introduction, the amount of mRNA expression for Cox-2 was measured by reverse transcription polymerase chain reaction (RT-PCR) and real-time PCR. In experiment 2, 250 pM siRNA for Cox-2 was introduced into CG cells for 0, 3, 6, 12, and 24 h. After culture, the amount of mRNA expression for Cox-2 was measured and the culture medium was collected to determine PGF2� concentration by enzyme immunoassay. The Cox-2 mRNA expression was not affected by 100 pM siRNA introduced into CG cells for 24 h, but 250 and 500 pM Cox-2 siRNA significantly reduced the Cox-2 mRNA expression (10% of that of the 0 pM siRNA group). Moreover, the suppressive effect of 250 pM siRNA was observed at 6 h after introduction (60% of that of the 0 pM siRNA group at 0 h) and the reduction of mRNA expression by RNAi became more obvious over 12 h (10% of that of the 0 pM siRNA group at 0 h). On the other hand, the PGF2� concentration in the culture medium was not significantly different at 12 h after siRNA introduction, however, the PGF2� concentration of the RNAi treatment group at 24 h was significantly lower than that of the 0 pM siRNA group at the same time point. These results suggest that gene silencing by Cox-2 siRNA is a means of analyzing the function and expression of specific genes in bovine CG cells.
This study was supported by the Japan Society for the Promotion of Science for Young Scientists.
254 DIFFERENTIAL TRANSCRIPTION AND CYSTOPLASMIC POLYADENYLATION ELEMENTS IN PORCINE GERMINAL VESICLE AND METAPHASE II OOCYTES
Authors: Korte S. , Springer G. , Spollen W. , Patel R. , Whitworth K. , Bivens N. , Forrester L. , Murphy C. , Green J. , Prather R.
Published: December 2006

Identification of transcripts produced during porcine oocyte maturation is one of the first steps in defining pathways important to development. Practically, this information will aid in the refinement of in vitro culture processes, allowing for more efficient in vitro embryo production. To this end, mRNA was isolated from 50 in vitro-matured sow metaphase II stage oocytes (Bomed, Inc., Madison, WI, USA) (MIIs), 50 in vitro-matured gilt metaphase II-stage oocytes (MIIg), and 50 gilt germinal vesicle stage oocytes (GVO) by using the Dynabead® system (Dynal, Inc., Lake Success, NY, USA) and amplified by using the SMART system (Clontech, Palo Alto, CA, USA). The PCR products were ligated into a pSport vector and transformed into electro-competent E. coli. Colonies were randomly picked and sequenced at the University of Missouri DNA Core. Sequences were clustered with similar sequences derived from a larger expressed sequence tag (EST) project (http://genome.rnet.missouri.edu/Swine/) by using the tlcluster program developed at the University of Iowa. Following clustering, individual clusters in the cDNA libraries were compared by using Fisher's exact test (P < 0.01) to determine if they were differentially represented. Two sets of comparisons were performed, one between the MIIs and MIIg libraries, and another between the GVO library and the combination of both metaphase II libraries (MII). The number of clusters per number of clones in the library was 966/1668 (GVO), 458/820 (MIIg), and 158/819 (MIIs). There were 15/419 clusters that were different between the MIIs and MIIg libraries, and 26/1269 that were different between the MII and GVO libraries. Potential cytoplasmic polyadenylation elements (CPEs) identified from the literature were found in the GVO and MII libraries by using a custom pattern-matching program. Of the clusters with differential expression, 4/15 (MIIs vs. MIIg) and 7/26 (MII vs. GVO) contained CPEs. Table 1 contains a partial list of differentially expressed genes and the sequence of their cytoplasmic polyadenylation elements. Many genes were found to be differentially expressed in both (MII vs. GVO and MIIs vs. MIIg) comparisons. Collectively, these findings will facilitate the elucidation of important developmental pathways in swine and other animals.

Table 1.
Comparison of mRNA expression and cytoplasmic polyadenylation elements

This work was partially funded by a University of Missouri Life Sciences Mission Enhancement grant and Food for the 21st Century.
290 THE FACTORS ON RATES OF ABNORMALITY, DISEASE AND MORTALITY OF CALVES DERIVED FROM IN VITRO EMBRYOS OF KOREAN NATIVE CATTLE
Authors: Park Y.-S. , Kim S.-S. , Park M.-C. , Park H.-D.
Published: December 2006

In Korea, in vitro production and transfer of bovine embryos has advanced remarkably and applied commercially. However, in vitro-produced embryos result in lower pregnancy and higher abortion rates and in some cases increased rates of abnormality and mortality in calves. The present study was conducted to investigate the effects of various factors such as recipient parity, delivery season, offspring number, pregnancy period, delivery type, midwifery type, dystocia and vaccination, on the viability of calves derived from embryos produced in vitro. Korean Native Cow ovaries were obtained from local slaughterhouse and cumulus-oocyte complexes (COCs) were aspirated from 2 to 8 mm follicles. Selected COCs were matured in TCM-199 supplemented with 10% fetal calf serum (FBS), 1 �ML FSH, 10 �ML LH and 1 �ML Estradiol-17� for 20-22 h. In vitro-matured oocytes were fertilized using frozen-thawed percoll separated semen (Day 0) in fer-TALP medium for 20 h. The presumptive zygotes were cultured in CR1aa medium supplemented with 0.3% BSA (before Day 3) or 10%FBS (After Day 3). All types of cultures were made in an incubator at 38.5�, 5% CO2 in air. Statistical analysis was performed using the Chi-square test. Two blastocysts were transferred to the Holstein recipients (n = 1888). The parturition was occurred in total 755 recipients. There was no difference in the abnormality of calves among treatments. The incidence of disease was significantly higher in single calf than twin calves (18.4 vs. 6.7%), in multiparous than nulliparous group (40.0 vs. 9.9%), in eutocia than dystocia group (20.0 vs. 4.8%), in spring and winter groups than summer and autumn groups (20.3, 22.7 vs. 4.3, 0.0%), and in non-vaccinated than vaccinated group (22.7 vs. 1.6%), respectively (p < 0.05). The rate of mortality was significantly higher when transferred into nulliparous than multiparous (22.3 vs. 0.0%), when were dystocia than eutocia group (71.4 vs. 14.1%), when were non-midwifery than midwifery (45.0 vs. 13.6), when delayed midwifery than earlier midwifery (31.6 vs. 11.5%), and when were non-vaccinated than vaccinated group (28.0 vs. 9.8%), respectively (P < 0.05). The present study suggested that the viability of bovine calves derived from in vitro was affected by the recipient parity, parturition treatment technique and vaccination.
This study was supported by the BIO-GREEN 21 PROGRAM.
296 INFLUENCE OF SHORT-TERM HEAT STRESS ON THE BLASTOCYST RATE OF BOVINE EMBRYOS (INDICUS VS. TAURUS) FROM OOCYTES OBTAINED BY OVUM PICKUP
Authors: Sartorelli e Sartorelli E. , Barcelos A. C. Z. , Satrapa R. A. , Martins D. F. , Nogueira M. F. G. , Potiens J. R. , Seneda M. M. , Barros C. M.
Published: December 2006

There is evidence that the deleterious effects of heat stress (HS) on fertility are less pronounced in Bos indicus than in B. taurus breeds, due primarily to differences in their thermoregulatory capacity. In the present work, the resistance to heat stress of Nelore embryos (B. indicus) was compared to either a breed not adapted (Angus; B. taurus) or adapted to high temperatures (Bonsmara; 5/8 B. indicus × 3/8 B. taurus). In Experiments (Exp.) 1 (Nelore vs. Angus) and 2 (Nelore vs. Bonsmara), oocytes obtained by ovum pickup OPU (during autumn) were matured (TCM-199), fertilized, and cultured (SOFaaci) in vitro. Ninety-six hours post-insemination (hpi), embryos with more than 16 cells were randomly allocated in two main groups: Group Control (embryos were maintained at 39°C all of the time) and Group HS (embryos were maintained at 41°C during 12 h and afterwards returned to 39°C). Blastocyst rates were determined on the 7th day of culture. In Exp. 1, 294 oocytes from Nelore and 144 from Angus cows had a cleavage rate of 67.9 and 59.4%, respectively. Ninety-six-hpi embryos (>16 cells) were distributed in four groups: Nelore Control (n = 97), Nelore HS (n = 95), Angus Control (n = 34) and Angus HS (n = 25). The blastocyst rates were 39/97 (40.2%), 23/95 (24.2%), 19/34 (55.9%), and e 8/25 (32.0%), respectively. The difference in rate of blatocyst formation caused by heat stress on Nelore (16.0%) and Angus (23.9%) was not significantly different (P < 0.05), and suggests, from oocytes obtained by OPU, that Nelore embryos may be more tolerant to HS than Angus embryos. However, it is necessary to increase the number of blastocysts per group in order to better characterize the effects of heat stress on these embryos. In Exp. 2, 294 oocytes from Nelore and 101 from Bonsmara cows had a cleavage rate of 41.2 and 51.2%, respectively. Ninety-six-hpi embryos (>16 cells) were distributed in four groups: Nelore Control (n = 44), Nelore HS (n = 49), Bonsmara Control (n = 22), and Bonsmara HS (n = 22). The blastocyst rates were 35/44 (79.5%), 30/49 (61.2%), 10/22 (45.5%), and 6/22 (27.3%), respectively. In spite of the fact that Bonsmara embryos had a lower blastocyst rate as compared to Nelore, the decline on blastocyst rate caused by HS was very similar in Nelore (18.3%) and Bonsmara embryos (18.2%). Additional OPU are underway to test the hypothesis that thermotolerance of Nelore embryos is similar to that in embryos from a breed adapted to high temperatures (Bonsmara), and superior to embryos from a non adapted breed (Angus).
E. S. S., R. A. S., and M. F. G. N. were supported by a fellowship from FAPESP, and A. C. Z. B. by a fellowship from CAPES of Brazil.
302 VIABILITY OF BOVINE SPERMATOZOA INCUBATED WITH FOREIGN DNA
Authors: Feitosa W. B. , Milazzoto M. P. , Rovegno M. , Martins L. F. , Visintin J. A. , Assumpção M. E. O. A.
Published: December 2006

Several studies have been performed over the years to promote the understanding and improvement of the sperm-mediated gene transfer technique. However, little is known about the effect of exogenous DNA in the sperm cells. The aim of this study was to evaluate the effect of the incubation period and exogenous DNA addition on mitochondrial activity and acrosomal status of bovine spermatozoa. Frozen-thawed semen was separated by Percoll gradient (45/90%) at 600g for 30 min, and the pellet was resuspended and washed by centrifugation in sperm-TALP at 200g for 5 min. The spermatozoa were resuspended in fertilization medium (without heparin) at a concentration of 5 × 106 spermatozoa/mL and incubated at 39°C, 5% CO2 in air with 500 ng pEYFP-Nuc/mL (Clontech, Mountain View, CA, USA) (DNA group) for 1 and 2 h or without DNA (control group) for 0, 1 and 2 h. JC-1 (Molecular Probes; Invitrogen Brasil, Ltd., Sao Paulo, Brazil) was used to determine mitochondrial potential and fluorsecein isothiogyanate (FITC-PSA; Sigma-Aldrich, Brazil) was used to assess acrosomal status. Two microliters of JC-1 (76.5 μM in DMSO) and 50 μL of FITC-PSA (100 μg/mL in DPBS) were added to 150 μL of IVF medium + 5 × 106 spermatozoa/mL; the mixture was incubated at 25°C for 10 min. DNA incorporation was evaluated by p-EYFP-Nuc PCR amplification. All treatments were repeated ten times and data were analyzed by ANOVA and Tukey test (P < 0.05). The results are described in Table 1. The sperm were classified as: Class 1 (intact acrosome and high mitochondria potential), Class 2 (intact acrosome and low mitochondria potential), Class 3 (reacted acrosome and high mitochondria potential) and Class 4 (reacted acrosome and low mitochondria potential). In both groups, the Class 1 sperm decreased over time, whereas an increase in number was verified for Class 2 sperm. There was no significant difference in numbers among the incubation periods in both groups for Class 3 sperm. However, there was an increase in Class 4 sperm number over time, indicating that the main effect of the incubation period was the loss of mitochondria potential. There was no effect of the exogenous DNA addition on sperm viability in relation to the control group, indicating that the exogenous DNA had no effect on mitochondrial activity and acrosomal status of bovine semen.

Table 1.
Effect of incubation period and exogenous DNA addition on sperm
viability

This work was supported by FAPESP 03/10234–7; 03/07456–8.
310 NUCLEAR AND MICROTUBULE REMODELING AND IN VITRO DEVELOPMENT OF NUCLEAR TRANSFERRED CAT OOCYTES WITH SKIN FIBROBLASTS OF DOMESTIC CAT AND LEOPARD CAT
Authors: Yin X. J. , Choi E. G. , Cho S. J. , Jin J. Y. , Kim N. H. , Lee H. S. , Kong I. K.
Published: December 2006

The leopard cat (Prionailurus bengalensis), a member of the felidae family, is a threatened animal in South Korea. In terms of endangered felids, nuclear transfer is a potentially valuable technique for assuring the continuation of species with dwindling numbers. The protocol for nuclear transfer has been described previously (Yin et al. 2005 Reproduction 129, 245-249). In this experiment we evaluated nuclear and microtubule remodeling and the in vitro developmental potential of enucleated cat oocytes reconstructed with nuclei from either domestic cat fibroblasts (DCF) or leopard cat fibroblasts (LCF). Microtubule aster was allocated to decondensed chromatin at 6 h post-activation following nuclear transfer of fibroblast cells from both DCF and LCF (3/3 in DCF, 2/3 in LCF), suggesting the introduction of a somatic cell centrosome (Kim et al. 1996 Mol. Reprod. Dev. 43, 248-255; Park et al. 2004 Mol. Reprod. Dev. 68, 25-34). At 12 h following nuclear transfer, the nucleus swelled into a large pronucleus-like structure in most reconstructed oocytes (5/9 in DCF and 4/6 in LCF), which showed further enlargement until 18 h after nuclear transfer (4/6 in DCF, 4/6 in LCF). Two microtubule asters were seen near the swollen nucleus. At 18 h following nuclear transfer, the mitotic metaphase (1/6 in DCF) or two cell divisions (1/6 in DCF, 2/6 in LCF) were observed. The percentage of blastocyst formation from nuclear transfer embryos derived from DCF (4/46, 8.6%) was not significantly different from that for nuclear transfer embryos constructed with LCF (4/52, 7.6%). Statistical significance was established at the P < 0.05 level by a X2-test (SAS; SAS Institute, Inc., Cary, NC, USA). These results indicate that nuclear and microtubule remodeling processes and in vitro developmental ability are similar in cat oocytes reconstructed with both domestic cat and leopard cat nuclei.
This work was supported by KOSEF (grant # M10525010001-05N2501-00110).