bluuebunny/crossref_metadata_2025 huggingface dataset:
408 HOLSTEIN DONORS RESPONSE USING OR NOT USING ESTRADIOL 17-β IN THE SUPEROVULATORY TREATMENT
Authors: Fuentes S. , Ruiz D. , Vera P. , Castro A. , Moreno I. , Liebana E. , de la Fuente J.
Published: August 2010

Because of new regulations banning the use of estradiol in the European Union and other countries, the present study was designed to determine the efficacy of an alternative to the use of estradiol 17-β in superovulation of Holstein cows and heifers. A total of 61 lactating cows and 49 heifers (15-18 months old) were divided into 2 groups: Group A (32 cows and 22 heifers) and Group B (29 cows and 27 heifers). Animals in Group A received a CIDR device (Pfizer, Madrid, Spain) for 13 days. At the same time of CIDR insertion and 5 days later, all animals received 2 mL (0.15 mg) of d-cloprostenol (PGF; Veteglan®, Calier, Spain). Gonadotropin-releasing hormone (0.5 mg of gonadorelin; Fertagyl®, Schering-Plough, Carbajosa de la Sagrada, Spain) was administered twice, 36 h after the second PGF and 24 h after CIDR removal. Superovulation treatments were initiated 36 h after the first GnRH, with a total dose of 15 mL (750 IU) of FSH (Pluset®, Calier, Spain) for cows and 11.5 mL (575 IU) for heifers, given in 10 twice-daily injections that decreased in dose. Luteolysis was induced with 0.15 mg of PGF given twice, 12 h before and also at the time of CIDR removal. Animals in Group B received a CIDR device for 7 days. Twenty-four hours after CIDR insertion, all animals received 100 mg of progesterone (Smithkline, Tres Cantos, Spain) and 5 mg of estradiol 17-β (Sigma, St. Louis, MO, USA). Beginning on Day 5, animals were superovulated with similar doses of FSH to those used in Group A. Luteolysis was induced with 0.15 mg of PGF given twice, 12 h before CIDR removal and also at the time of removal. Gonadotropin-releasing hormone (0.5 mg) was administered at the time of AI. Animals from both groups were inseminated with frozen-thawed semen at 12 and 24 h after the onset of standing estrus. Embryos were recovered nonsurgically 7 days later. A total of 299 frozen-thawed embryos (Freeze Control® CL5500, Bioniche, Belleville, Ontario, Canada) from both groups were transferred in synchronized Holstein heifers. Pregnancy was confirmed by rectal palpation on Day 40 to 45 of gestation. Data were analyzed by two-way ANOVA and results are shown in the table. No significant differences were found between cows and heifers, nor was there a difference between groups. These results showed that the previous superovulatory treatment using progesterone and estradiol can be successfully replaced with the treatment protocol described herein.

Table 1.Mean number of recovered ova/embryos, transferable embryos, and pregnancy rates after embryo transfer (ET) in Holstein cows and heifers superovulated with 2 treatment protocols
42 SEMEN FERTILITY OF THE BULL EVALUATED BY SECOND-ORDER STATISTICS
Authors: Silva T. F. , Barreto Filho J. B. , Macedo R. S. , Ferraz Jr M. V. C. , Braga Jr R. A. , Rabelo G. F. , Nogueira M. O. G.
Published: August 2010

The biospeckle (BSL) is based on the incidence of laser light upon active biological materials, followed by analysis and manipulation of secondary images, and then, application to those images of a second-order statistics technique called the inertial moment (IM) that results in information about the activity of the specimen. This technique was previously used to analyze sperm motility. A bull’s fertility could be evaluated by several semen parameters in vitro and more precisely by conception rates of AI heifers. The objective of this work was to evaluate the fertility of bull’s frozen semen showing different levels of activity in an AI program. Semen of 6 mature IA donor bulls (Bos taurus indicus) was previously divided in 2 groups (group I: motility ≥50%; group II: motility <50%), each group comprising 3 animals. Sperm concentration was 30 to 35 × 106 cells per straw. Semen was thawed at 37°C for 30 s in a water bath. Samples were illuminated by nonpolarized He-Ne laser (632 nm, 10 mW) for 40 s, the time needed to take 512 images under a time rate of 0.08 s. Images were acquired by a CCD camera and analyses were conducted by building space-time speckle matrices from center columns of 512 images of the dynamic speckle and then the IM was calculated. Beef heifers (n = 109) under the same nutrition and management conditions underwent AI with the semen samples. All inseminations were done by the same technician and donor bulls were used consecutively throughout the breeding season. Pregnancy diagnosis was done by ultrasound images (Falco 100, 6 MHz, Pie Medical, Crawley, UK) 28 days after insemination. The SISVAR software was used for variance analysis and comparison of means by Tukey test at a nominal level of 5%. The IM (194.13 ± 31.99) obtained from 10 illuminations per sample of each bull and the conception rate (CR) of group I (CR = 0.63 ± 0.49) differed (P < 0.05) compared with group II (IM = 142.03 ± 30.62; CR = 0.43 ± 0.50). Pearson correlation coefficient (P < 0.05) between IM and CR was r = 0.21, showing coherence with IM and CR, despite its low value. These data showed that second-order statistics are correlated to semen fertility and could be used to evaluate ejaculates of the bull.

Financial support: FAPEMIG grant EDT 94/07 and CNPq.
422 GENERATION OF REPORTER TRANSGENIC MICE FOR THE CHEMOKINE CXCL2 USING TWO DIFFERENT DNA CONCENTRATIONS
Authors: Crispo M. , Cárdenas-Rodriguez M. , Schlapp G. , Fernández G. , Rumbo M.
Published: August 2010

Transgenic mice have important implications in biomedicine, and are widely employed to understand gene functions and their regulation. The improvement of transgenic efficiency is relevant because of the low rate of success for this technology. CXCL2 is a chemokine secreted by macrophages and epithelial cells under proinflammatory stimulus of the innate immune response such as bacterial endotoxins. The main effect of CXCL2 is the recruitment of neutrophils to the site of production to fight infections. The objective of this study was to evaluate the effect of 2 DNA concentrations in the efficiency of the transgenesis process. To this aim we used a luciferase reporter under the control of CXCL2 promoter for the generation of a transgenic line to report activation of innate immune response. A total of 1727 1-cell embryos were divided into 2 experimental groups to be microinjected with 0.5 or 1.0 ng μL-1 of DNA in 25 sessions. Three-week-old B6SJL F1 females (n = 131) were superovulated with 5 IU of eCG i.p. (Novormon, Syntex, Buenos Aires, Argentina) and 5 IU of hCG i.p. (Ovusyn, Syntex) 46 h later, and mated with B6SJL F1 stud males. At the moment of hCG treatment, foster females were mated with vasectomized males to induce pseudogestation. Donor females were euthanized by cervical dislocation 20 h after hCG treatment, and embryos were recovered from the ampulla, denuded in 300 μg mL-1 hyaluronidase (Sigma, St. Louis, MO, USA) and incubated at 37°C with 5% CO2, in drops of M16 media (Sigma) under mineral oil, until microinjection. DNA construction consisted of the luciferase reporter gene under the control of the murine CXCL2 gene promoter. Embryos were microinjected into 1 pronucleus under an inverted microscope (Nikon, NY, USA) using glass microtools and mechanic micromanipulators (Eppendorf, Hamburg, Germany). Intact/injected embryos were assessed 30 min after microinjection. Fifteen to 20 embryos per foster female were transferred in both oviducts. Birth rate, survival of pups at Day 7 after birth, number of transgenic pups assessed by standard PCR, and overall transgenic efficiency was registered for each group. Data were analyzed by Yates-corrected chi-square test. No statistical differences were founded except for a higher number of pups alive/embryo transferred in the lower DNA concentration, suggesting the advantage of using 0.5 ng μL-1 v. 1.0 ng μL-1.

Table 1.Effect of DNA concentration in the generation of CXCL2-luc transgenic mice
428 PRODUCTION OF GERM-LINEAGE CELL AND FUNCTIONAL GAMETES FROM MULTI-POTENT SPERMATOGONIAL STEM CELLS
Authors: Lim J. E. , Eum J. H. , Kim H. J. , Lee H. S. , Kim J. H. , Lee J. E. , Shin D. H. , Chung H. M. , Yoon T. K. , Lee D. R.
Published: August 2010

Multi-potent spermatogonial stem cells (mSSC), derived from uni-potent SSC, are a type of reprogrammed cells with similar characteristics to embryonic stem cells (ESC). Similar to ESC, mSSC are capable of differentiating into 3-germ layers in vitro and teratoma formation in vivo. Additionally, mSSC proliferate rapidly and can be transfected more easily than SSC. In contrast to previous reports, we have found that mSSC also have germ-cell-specific micro (mi)RNA and gene expression profiles. Therefore, the aims of this study were to compare the efficiency of mSSC v. ESC to differentiate into germ lineage and produce male gametes, as well as to develop a novel system for the production of genetically modified mice. Mouse mSSC were transfected with a lentiviral vector expressing green fluorescent protein (GFP) and testis-specific gene and maintained in the ESC-culture medium containing leukemia inhibitory factor (LIF). Embryonic bodies (EB) were formed after the cells were detached from the feeder cells. Bone morphogenetic protein (BMP)-4 (10 ng mL˜1) and retinoic acid (RA, 0.1 μM) were added to the ESC-culture medium for 3 days in order to induce differentiation into germ lineage cells. Then, these cells were changed to germ cell-culture medium (Stem-Pro™ containing GDNF; Invitrogen, Carlsbad, CA, USA) and cultured for 3 days. After 6 days, cultured cells were sorted by magnetic activating cell sorting system using specific marker for germ cells, CD-9. Isolated germ lineage cells were transplanted into a busulfan-treated mouse testis for the production of male germ cells. Three to 6 weeks later, the testis and epididymis were collected, and half of the sample was used to perform histological analysis and the other half for the production of intracytoplasmic sperm injection (ICSI)-derived embryos. The statistical significance of differences between the 2 groups was evaluated by Student’s t-test Immunocytochemical and flow cytometrical analysis performed 6 days after differentiation showed that the ratio of germ cell-specific markers in EB derived from mSSC was higher than those from ESC. Moreover, after 3 to 6 weeks of transplantation the testis produced sperms and germ cells expressing GFP. We have successfully produced embryos by ICSI and offspring by embryo transfer into uteri of poster mothers. These results demonstrate that mSSC can be easily differentiated into germ lineage cells compared with ESC and have the potential to generate functional gametes. Therefore, the differentiation and transgenesis of mSSC may be a useful model for production of genetically modified mice.

This work was supported by a grant of the Korea Healthcare Technology R&D Project, Ministry for Health, Welfare & Family Affairs, Republic of Korea (A084923).
429 GENERATION OF Oct4-EGFP TRANSGENIC PIGS FOR MONITORING REPROGRAMMING AND PLURIPOTENCY
Authors: Nowak-Imialek M. , Kues W. A. , Petersen B. , Lucas-Hahn A. , Herrmann D. , Lemme E. , Oropeza M. , Carnwath J. W. , Niemann H.
Published: August 2010

The Oct4 gene is an essential transcription factor for maintenance of pluripotency in mammals. Here, we report the production of cloned transgenic pigs carrying a genomic construct encompassing murine Oct4 regulatory regions and driving an enhanced green fluorescent protein (Oct4-EGFP) construct. We employed fetal porcine fibroblasts, stably co-transfected with neomycin and the mouse Oct4-EGFP construct, for somatic cell nuclear transfer to reconstruct transgenic embryos. The cloned embryos (811 embryos) were surgically transferred into the oviducts of 8 recipient animals. Two pregnancies were terminated at Day 25 for recovery of fetuses and the others delivered a total of 23 piglets, of which 11 survived the postpartum period. A detailed analysis showed that the Oct4-EGFP construct was active in cloned pig blastocysts from Days 5 to 6. EGFP fluorescence was found exclusively in the primordial germ cells of Day 25 fetuses, whereas somatic tissues did not express the transgene. We could also detect expression of Oct4-EGFP in individual cells of the postnatal testis. Testis-specific expression was confirmed by Northern blotting. We fused transgenic porcine fibroblasts with murine embryonic stem cells to analyze reactivation of the Oct4-EGFP transgene under experimental reprogramming conditions. The fused hybrids displayed stem cell morphology and a high proliferation rate and started to express EGFP fluorescence 72 h after fusion. In conclusion, we report the production of viable Oct4-EGFP transgenic piglets that express EGFP exclusively in germ line and pluripotent cells. This transgenic pig line is a valuable tool for derivation and maintenance of porcine embryonic stem cells and will be of utmost interest for reprogramming studies and for preclinical testing of stem cell therapies in a large animal model.

Funded by BMBF.
441 EFFECTS OF BYPASS LIPID SUPPLEMENTATION IN THE TRANSITION PERIOD ON REPRODUCTIVE PARAMETERS IN DAIRY GOATS AFTER PARTURITION
Authors: Carneiro C. , Souza J. M. G. , Torres C. A. A. , Silva W. J. , Denadai R. , Bruschi J. H. , Fonseca J. F.
Published: August 2010

The use of lipids for nutrition supplementation during reproduction phases is called flushing and directly influences body weight and body condition score, which could alter ovulation and fertility rate. Studies have reported the effects of its use for dairy cattle, but for goats this kind of information is incipient. The aim of this study was to evaluate the use of bypass lipid enriched in polyunsaturated fatty acids (Megalac® Arm and Hammer, Church & Dwight Company, Princeton, NJ, USA) in the transition period (i.e. 3 weeks before and after parturition) on the return of ovarian activity. This study was conducted from March to May 2009, in Piau/MG (21°35′S latitude and43°15′W longitude), Brazil. Nineteen Toggenburg (n = 16) and Saanen (n = 3) goats were equally assigned according to breed, body weight, and condition score into 4 treatments: animals received 2% dry matter of fat supplementation 21 days before and after parturition (T1), only before parturition (T2), or only after parturition (T3); the control group received no supplemental fat (T4). Goats were fed a complete mixture of napier grass and corn silage in a 50:50 forage/concentration ratio 4 times daily. Transrectal ultrasonography (5-MHz transducer; Aloka SSD 500®, Tokyo, Japan) was performed daily from 10 days after parturition until detection of ovulation. Estrous onset and its duration were detected daily with a fertile buck. Statistical analysis were performed using all tests at the 95% confidence interval with a SAEG® program (Funarbe, Viçosa, Brazil). The results are presented as mean ± SD. The interval (days) from parturition to first estrus was 20.5 ± 2.2 (T1), 30.0 ± 17.4 (T2), 20.2 ± 2.1 (T3), and 19.0 ± 2.5 (T4), and to first ovulation was 26.3 ± 4.0 (T1), 22.4 ± 3.3 (T2), 24.4 ± 1.1 (T3), and 24.2 ± 3.6 (T4) (P > 0.05). The diameter of ovulatory follicles (mm) was similar (P > 0.05) for T1 (7.21 ± 0.30), T2 (6.86 ± 0.31),T3 (6.66 ± 0.27), and T4 (7.32 ± 0.64). The number of ovulations was also not different (P > 0.05) for T1 (1.5 ± 0.3),T2 (1.2 ± 0.2), T3 (1.4 ± 0.2), and T4 (1.0 ± 0.0). A negative correlation (r = -0.68; P < 0.005) was detected between body condition score at the parturition and the interval from parturition to the first estrus, as well as to the first ovulation (r = -0.48; P < 0.05). A positive correlation (r = 0.47; P < 0.05) was found between body weight on the day of ovulation and the number of ovulations. These data show the importance of body weight and condition score to reproductive performance after parturition. No significant differences were registered among all treatments on reproductive parameters for goats after this amount of lipid supplementation. There is a need for more studies to be done using different supplement concentrations in order to achieve better reproductive performances after parturition.

Financial support: Embrapa Goats and Sheep, Fapemig, CNPq.
447 INDUCTION OF ESTRUS IN ANGLO-NUBIAN GOATS IN THE TRANSITION SEASON WITH SHORT-TERM PROGESTAGEN PROTOCOLS WITH OR WITHOUT eCG ADMINISTRATION
Authors: Souza J. M. G. , Torres C. A. A. , Ribeiro S. D. A. , Ribeiro A. C. , Becalete L. , Rigo A. G. , Silva W. J. , Facó O. , Fonseca J. F.
Published: August 2010

Synchronized estrus can be induced by different hormonal protocols including prostaglandin, progestagens, and gonadotrophins. Equine chorionic gonadotrophin is a FSH-like hormone produced by pregnant mares that can evoke antibody formation in goats. Thus, if possible, restriction of its use should be considered. The aim of this study was to investigate the possibility to omit the administration of eCG to induce estrus in Anglo- Nubian goats during the transition season. This study was done in February of 2009 in Espírito Santo do Pinhal/SP (22°57′S latitude and 46°58′W longitude), Brazil. The local breeding season extends from March to June. A total of 10 nulliparous and 10 pluriparous Anglo-Nubian goats were equally assigned into 2 treatments with (T1) or without (T2) 200 IU eCG (Novormon 5000®, Intervet Schering Plough Animal Health, São Paulo, Brazil) i.m. administration 24 h before sponge removal. All goats received (Day 0) intravaginal sponges (60 mg of MAP; Progespon®, Intervet Schering Plough Animal Health) and 50 mg d-cloprostenol (Ciosin®, Intervet Schering Plough Animal Health) i.m. Estrous behavior and transrectal ultrasonography (5-MHz transducer; Aloka SSD 500®, Tokyo, Japan) were performed at 12-h intervals (0600 and 1800h) until 96 h after sponge removal. Statistical analysis was performed using all tests at the 95% confidence interval with a SAEG® program (Funarbe, Viçosa, Brazil). Estrous response was 100% (10/10) for T1 and 70% (7/10) for T2 (P > 0.05). All animals that were detected in estrus ovulated, and none of the 3 that did not show estrus ovulated. The interval from sponge removal to estrous onset (h) was 36.0 ± 9.8 (T1) and 34.8 ± 10.5 (T2), whereas its duration (h) was 27.4 ± 14.5 (T1) and 32.6 ± 12.7 (P > 0.05). The interval from sponge removal to ovulation (h) was 67.0 ± 8.0 (T1) and 73.9 ± 6.4 (T2). Also, the interval from estrous onset to ovulation (h) was 25.2 ± 8.85 (T1) and 30.9 ± 6.4 (T2). No difference (P > 0.05) was observed in the number of ovulations for T1 (1.2 ± 0.4) and T2 (1.4 ± 0.5). The diameter of the ovulatory follicle (mm) was similar (P > 0.05) for T1 (6.7 ± 0.6) andT2 (6.5 ± 0.6) (P > 0.05). Further studies should be done to consider if the use of eCG is necessary in the transition season for Anglo-Nubian goats.

Financial support: Embrapa Goats and Sheep; Capritec; CNPq; Intervet Schering Plough Animal Health; Fapemig.
46 RECLONING USING TRANSGENIC FETAL FIBROBLASTS AS NUCLEI DONORS INCREASES DEVELOPMENTAL POTENTIAL OF RECONSTRUCTED EMBRYOS IN CATTLE
Authors: Bressan F. F. , Miranda M. S. , Perecin F. , De Bem T. H. C. , Bajgelman M. , Strauss B. , Krieger J. E. , Binelli M. , Meirelles F. V.
Published: August 2010

Genetically modified animals have numerous applications ranging from basic research to agriculture production. Cloning by nuclear transfer (NT) has made possible the production of transgenic animals using previously genetically modified cell lineages. Gene expression studies and adequate selection of the nuclei donor cell for NT guarantees the presence of the gene construction in the offspring and the absence of deleterious mutations caused by the random insertion of transgenes in functional areas of the genome. Embryonic development after NT requires a change in the transcriptome of the donor cell from a somatic to an embryonic pattern, causing cloning efficiencies to be low because of incomplete or defective nuclear reprogramming. Therefore, the establishment of methodologies able to increase cloning success is highly desirable. The experiment was designed to test if recloning of transgenic fetal fibroblasts increases cloned blastocyst production and the pregnancy rates of transgenic cloned embryos produced by NT. This study compared the developmental potential of cloned embryos reconstructed with fetal fibroblasts genetically modified by lentivirus random integration (control group) expressing the green fluorescent protein gene (eGFP), with a transgenic fetal fibroblast cell line established from a 30-day transgenic pregnancy (recloning group). Fusion, cleavage (72 h post-activation, hpa), blastocyst production (168 hpa), and 30-day pregnancy rates were analyzed. A total of 1213 embryos were reconstructed; 884 (10 replicates) with random transgene insertion fibroblasts and 329 (4 replicates) with cells derived from the cloned fetus. Results were analyzed by chi-square test at 5% significance. No difference was observed (P > 0.05) between control and recloned groups regarding fusion rate (n = 550, 62.22% and n = 189, 57.25%; respectively) or cleavage rate (n = 383, 69.45% and n = 132, 69.84%, respectively). The recloned group, however, showed a higher blastocyst development rate (P < 0.01) compared with the control group (n = 51, 26.98%, and n = 79, 14.36%, respectively) and a higher 30-day pregnancy rate (n = 6, 15.38% and n = 3, 5.56%, respectively). In conclusion, recloning of transgenic fibroblasts from a successfully established pregnancy augments the efficiency in the production of embryos and pregnancy establishment compared with the control group. We speculate that a second round of NT enhances the nuclear reprogramming of donor cells, and moreover, the use of a transgenic cell lineage already proven to be successfully reprogrammed may indicate that transgene integration is not deleterious in that specific cell lineage, resulting in a good source of donor cells to be used in order to produce a homogeneous bioreactor herd.

Financial support: FAPESP, Brazil.
47 ACTIVATION WITH IONOMYCIN FOLLOWED BY DEHYDROLEUCODINE AND CYTOCHALASIN B OF CLONED BOVINE EMBRYOS
Authors: Canel N. , Bevacqua R. , Salamone D.
Published: August 2010

A combined treatment of dehydroleucodine (DhL) and cytochalasin B (CB) was previously demonstrated to induce pronuclear formation of bovine oocytes (Canel and Salamone 2008 Reprod. Fertil. Dev. 21, 214-215). The aim of this study was to evaluate the potential of DhL combined with CB to induce diploid activation of parthenogenetic embryos and to employ this treatment to assist cloning by intracytoplasmic injection of whole cumulus cells. To do that, COCs were collected from cow ovaries obtained from a slaughterhouse and in vitro-matured in TCM-199, at 39°C under 6% CO2 in air for 24 h. After removal of cumulus cells, metaphase II (MII) oocytes were treated with 5 μM ionomycin (Io) for 4 min and randomly assigned to the following activation groups: a) DhL/CB (incubation with 1 μM DhL and 5 μg mL-1 CB, for 3 h); b) DhL/long CB (treatment DhL/CB for 3 h, followed by exposure to 5 μg mL-1 CB alone, for 3 additional hours); and c) DMAP (incubation with 2 mM 6-DMAP for 3 h). In experiment 1, activated oocytes underwent IVC for 48 h and cleaved embryos were treated with 1 μg mL-1 colchicine for 6 h, fixed on glass slides, and stained with 5% vol/vol Giemsa solution to assess chromosomal complements. In experiment 2, MII oocytes were mechanically enucleated and injected with whole cumulus cells obtained from IVM COCs. After 2 h, reconstructed eggs were treated with 5 μM Io for 4 min and randomly exposed to the activation treatments a, b, or c. Parthenogenetic control groups were also included. All embryos were cultured in SOF medium and rates of cleavage, morulae, and blastocysts were evaluated on Days 2, 5, and 8 (Table 1). Results showed that DhL/long CB diploidy rates were significantly higher than those of DhL/CB and DMAP (63.8, 40. and 31.6%, respectively; Fisher’s test, P < 0.05). Both DhL treatments induced polyploidy rates lower than DMAP (5.2, 10.6, and 31.6%, respectively; P < 0.05). Finally, Io followed by DhL/CB or DhL/long CB was able to induce cloned blastocyst rates not statistically different from Io plus DMAP (P > 0.05), but presumably with a higher degree of normal embryo ploidy.

Table 1.In vitro development of bovine cloned embryos activated with DhL and CB
5 CLONING AND CHARACTERIZATION OF NEWBORN OVARY HOMEOBOX GENE (NOBOX) IN CATTLE
Authors: Tripurani S. K. , Lee K. B. , Wang L. , Smith G. W. , Yao J.
Published: August 2010

Newborn ovary homeobox (NOBOX) is a homeobox gene that is preferentially expressed in the oocytes and is essential for folliculogenesis in mice. NOBOX knockout mice are infertile, and lack of NOBOX perturbs the expression of many germ-cell-specific genes and microRNAs. Furthermore, mutations in the NOBOX gene associated with premature ovarian failure have been described in humans. However, the temporal and cell-specific expression of NOBOX in bovine oocytes and the potential function of NOBOX in early embryogenesis have not been described previously. The objectives of this study were to clone the complementary (c)DNA encoding for bovine NOBOX, analyze the expression of NOBOX mRNA in bovine tissues including fetal ovaries of different developmental stages, and characterize the temporal expression patterns of NOBOX mRNA during oocyte maturation and early embryogenesis. Based on the sequence of a predicted cDNA for bovine NOBOX, we successfully amplified, using RT-PCR, a cDNA fragment representing the coding region of bovine NOBOX from bovine fetal ovary cDNA. Additional 5′ and 3′ sequences were obtained using rapid amplification of cDNA ends (RACE) procedures. The assembled full-length NOBOX cDNA is 2275 bp with an open reading frame encoding a protein of 500 amino acids with a conserved homeodomain and typical nuclear localization signal. The predicted NOBOX protein shares 61% and 49% amino acid sequence identity with its human and mouse counterparts, respectively. A BLAST search of the bovine genome database at the National Center for Biotechnology Information (NCBI) revealed that the bovine NOBOX gene is located on chromosome 4, spans approximately 5.5 kb, and is encoded by 7 exons. Northern blot analysis revealed an approximately 2.3-kb bovine NOBOX RNA transcript. RT-PCR analysis of RNA samples from a panel of 14 different bovine tissues revealed that expression of NOBOX mRNA is restricted to ovarian samples and can be detected in fetal ovaries harvested as early as 105 days of gestation, when primary follicles start to form. Further RT-PCR analysis using RNA isolated from oocytes and granulosa and cumulus cells of antral follicles indicates that bovine NOBOX is expressed in oocytes but not in other follicular cells. Real-time PCR analysis demonstrated that NOBOX mRNA is abundant in germinal vesicle and metaphase II stage oocytes, as well as from pronuclear to 8-cell stage embryos, but barely detectable in embryos collected at the morula and blastocyst stages, suggesting that NOBOX might be a maternal effect gene. Collectively, our results demonstrate that bovine NOBOX is specifically expressed in oocytes and may play a role in early embryonic development in addition to its known function in folliculogenesis.
51 DELAYED NUCLEOLOGENESIS IN PORCINE PREIMPLANTATION EMBRYOS DEVELOPED IN VIVO AND PRODUCED BY SOMATIC CELL NUCLEAR TRANSFER
Authors: Deshmukh R. S. , Østrup O. , Lemme E. , Peterson B. , Lucas-Hahn A. , Niemann H. , Hyttel P.
Published: August 2010

Nucleolus is known to be a well-suited morphological marker for embryo technologies. Failure in de novo nucleolar formation during embryonic genome activation (EGA) has been observed in many species. The aim of the present study was to investigate nuclear changes and nucleolar formation during EGA in the porcine preimplantation embryos developed in vivo and produced by somatic cell nuclear transfer (SCNT). Embryos were collected at early and late 1-cell stage, 2-, 4-, and 8-cell stage, early and late blastocyst stage, fixed in 3% glutaraldehyde for 1 h, and processed for transmission electron microscopy. In vivo embryos from 1- and 2-cell stages showed electron dense, spherical nucleolar precursor bodies (NPB) in centrally located nuclei with well-developed nuclear envelope and condensed chromatin. Two 1-cell-stage embryos, however, had represented metaphase chromosomes in the periphery. At the 4-cell stage, in vivo embryos displayed fibrillo-granular nucleoli containing all 3 functional nucleolar compartments: fibrillar centers (FC), dense fibrillar component (DFC), and granular component (GC). The nuclei were centrally located, round, and had complete nuclear envelopes. The same types of nuclei and nucleoli were observed for all following stages. On the other hand, embryos produced by SCNT at early 1-cell stage showed centrally located, irregular-shaped nuclei with incomplete nuclear envelopes and condensed chromatin with large intact NPB. Exceptionally, 1 out of the 5 embryos presented a peripheral nucleus with partially condensed chromatin lacking nuclear envelope and fibrillo-granular nucleolus probably persisting from donor fibroblast. Only 2 out of 5 late-1-cell SCNT embryos showed nuclear structures. The nuclei had irregular shapes, complete nuclear membranes, and contained large NPB. At the 2- and 4-cell stages, the embryos presented central nuclei with complete nuclear envelopes. Some of the embryos showed more than one nucleus of varying shapes and sizes. The fibrillo-granular nucleoli were first observed toward the 8-cell stage. The embryos from this stage contained irregularly shaped nuclei with well-developed nuclear envelopes. The nucleoli displayed fibrillar and granular compartments in SCNT 8-cell stage embryos, but lacked the typically structured functional nucleoli observed in in vivo embryos. The absence of formation of functional nucleoli at the 4-cell stage and altered nuclear ultrastructure during the EGA in SCNT embryos, thus, may be one of the main reasons for decreased developmental competence of SCNT embryos.
58 DEVELOPMENT OF α1,3-GALACTOSYLTRANSFERASE KNOCK-OUT CLONED PIG EMBRYOS
Authors: Hwang S. S. , Park M. R. , Shim J. H. , Yang B. C. , Ko Y. G. , Oh K. B. , Yang B. S. , Woo J. S. , Park E. W. , Park S. B.
Published: August 2010

This study was performed to increase the developmental rate of cloned embryos with the 1,3-Galactosyltransferase (GalT) gene knocked out (KO). Ovaries were collected from local slaughterhouse and immature oocytes were cultured in TCM-199 + 0.1% PVA + FSH + LH (0.5 μg mL-1) + EGF (10 ng mL-1) + 10% porcine follicular fluid (pFF) at 38.5°C in 5% CO2 humidified chamber for 40 h (1-step) or 20 h (with hormone) +20 h (without hormone; 2-step). After IVM, the oocytes with 1st polar body were enucleated and transferred the GalT KO donor cell originated from miniature pig. The embryos transferred with normal mini-pig ear fibroblast cell were used as control. The reconstructed embryos were fused with 2 electric pulses (DC) of 1.2 kV cm-1 for 30 μs. For the development of cloned embryos, the embryos were cultured in PZM-3 under 5% CO2 in air at 38.5°C for 6 days. The embryos were transferred to a surrogate (Landrace) at an earlier stage of the estrus cycle, and pregnancy diagnosis was determined at 28 days after embryo transfer using ultrasonography. Differences among treatment means were determined by a chi-square test. A probability of P < 0.05 was considered statistically significant. The maturation rate was significantly higher in the 2-step method (89.8 ± 2.75) compared with single maturation method (79.6 ± 8.95; P < 0.05). The blastocyst development of cloned embryos reconstructed with GalT KO donor cell (28.4 ± 2.14) was not different from cloned embryos by normal donor cell (27.4 ± 0.01). The cell number of GalT blastocyst (36.1 ± 11.1) was not different statistically from control (26.9 ± 9.3). The apoptosis rate was also not different in both groups (2.9 to 4.9%). Five surrogates were pregnant and the GalT KO fetuses were still ongoing pregnancy at 45 days after embryo transfer.

This work received grant support from the Agenda Program (No. 200901FHT010305146 and No. 200901FHT010305535), Rural Development Administration, Republic of Korea.
64 VALPROIC ACID ENHANCES IN VITRO DEVELOPMENT OF SOMATIC CELL NUCLEAR TRANSFER EMBRYOS IN PIGS
Authors: Kim Y. J. , Ahn K. S. , Kim M. J. , Shim H.
Published: August 2010

Epigenetic modification influences reprogramming and subsequent development of somatic cell nuclear transfer embryos. Such modification includes an increase of histone acetylation and a decrease of DNA methylation in transferred donor nuclei. Histone deacetylase inhibitors (HDACi) such as trichostatin A (TSA) and valproic acid (VPA) have been known to maintain high cellular levels of histone acetylation. Hence, the treatment of HDACi to NT embryos may increase efficiency of cloning. Indeed, TSA treatment has significantly enhanced the developmental competence of nuclear transfer embryos in several species including pigs (Zhang et al. 2007 Cloning Stem Cells 9, 357-363; Li et al. 2008 Theriogenology 70, 800-808). Valproic acid, another type of HDACi, has often been used to assist reprogramming of somatic cells into induced pluripotent stem cells in mice. In the present study, we tested the potency of VPA compared with TSA on the enhancement of in vitro development in porcine nuclear transfer embryos. Reconstructed embryos were produced by transferring nuclei of adult ear skin fibroblasts into enucleated oocytes. After electrical activation, these embryos were cultured in PZM-3 containing no HDACi (control), 5 mM VPA, or 50 nM TSA for 24 h, and another 5 days thereafter without HDACi. At least 3 replicates were conducted for the following experiments. The rates of cleavage were not different among the VPA, TSA, and control groups. However, the rate of blastocyst development was significantly higher (P < 0.05) in embryos treated with VPA than in those treated with TSA and without HDACi (125/306, 40.8% v. 94/313, 30.0% v. 80/329, 24.3%). Differential staining of inner cell mass (ICM) and trophectoderm (TE) also supported the beneficial effect of VPA treatment in NT embryos. Compared with the control group, the number of TE cells was significantly increased (P < 0.05) in the VPA and TSA treatment groups (79.3 ± 7.4 v. 74.6 ± 9.2 v. 40.0 ± 6.7). Moreover, VPA treatment significantly increased (P < 0.05) the number of ICM cells compared with the control (15.6 ± 1.7 v. 10.8 ± 2.6), whereas no differences were observed between the TSA treatment and control group (12.9 ± 3.0 v. 10.8 ± 2.6). The present study demonstrates that VPA enhances in vitro development of nuclear transfer embryos, in particular by an increase of blastocyst formation and the number of ICM cells, suggesting that VPA may be more potent than TSA in supporting developmental competence of cloned embryos. However, long-term effects of different HDACi in the development of nuclear transfer embryos, including any adverse outcome from destabilizing epigenetic condition, remain to be determined by further in vivo embryo transfer studies.
67 PRODUCTION OF CLONED TRANSGENIC RABBITS FROM MESENCHYMAL STEM CELLS
Authors: Li S. , Flisikowska T. , Kessler B. , Güngör T. , Kind R. , Wolf E. , Schnieke A. , Zakhartchenko V.
Published: August 2010

Mesenchymal stem cells (MSC) are adult stem cells with fibroblast-like morphology, which can be easily isolated from bone marrow and expanded in culture. Mesenchymal stem cells are able to grow from a single cell into a cell clone, which makes them potentially useful for gene targeting. In our recent study we investigated the dynamics of epigenetic reprogramming following nuclear transfer (NT) with MSC and found that these cells can support development of cloned embryos as good as genetically identical fibroblasts (Brero et al. 2009 Cloning Stem Cells 11, 319-329). In the present study we tested whether live cloned rabbits can be produced from MSC. Nuclear donor cells were isolated from a 6-week-old transgenic Ali/Bas rabbit, expanded in culture, and assessed for their differentiation potential. Mesenchymal stem cells were transfected with a green fluorescent protein (GFP) reporter gene construct and stable cell clones were selected (GFP-MSC). The MSC and GFP-MSC were used for NT at passage 3 to 7 after serum starvation for 2 to 4 days. Nuclear transfer was performed essentially as described previously (Yang et al. 2007 Reproduction 133, 219-320). To assess the development to blastocyst, reconstructed embryos were cultured in B2 medium for 5 to 6 days, whereas for in vivo development embryos were cultured only overnight and then transferred into recipients at the 4- to 8-cell stage. In the MSC group, 844 oocytes were used, 793 (94%) of them fused, 698/786 (89%) cleaved, and 48/128 (38%) developed to blastocyst. After transfer of 483 cloned embryos into 13 recipients, 2 from 8 pregnant recipients gave birth to 10 (2.4%) rabbits, from which 2 and 1 survived for more than 7 days and 3 months, respectively. In the GFP-MSC group, 444 oocytes were used, 412 (93%) of them fused, 377/409 (92%) cleaved, and 97/178 (55%) developed to blastocyst. Transfer of 216 cloned embryos into 8 recipients resulted in 4 pregnancies. One recipient gave birth to 6 (3.7%) live and 2 stillborn rabbits, from which 2 and 1 rabbits survived for more than 3 days and 2 weeks, respectively. All cloned rabbits carried a GFP gene, and green fluorescence could be detected in the follicles of the skin under a fluorescence microscope (Zeiss Axiovert200, Carl Zeiss, Germany). Our study demonstrates that live cloned rabbits can be produced from genetically modified MSC, thus paving the way to generate gene targeted animals.

This work is supported by Roche Diagnostic GmbH.
79 PSEUDOPHYSIOLOGICAL TRANSCOMPLEMENTARY ACTIVATION OF GOAT OOCYTES RECEIVING ADULT EAR CUTANEOUS FIBROBLAST CELL NUCLEI
Authors: Skrzyszowska M. , Samiec M.
Published: August 2010

The aim of the study was to determine the in vitro developmental potential of caprine cloned embryos following pseudophysiological (transcytoplasmic) transcomplementary activation of oocytes reconstructed with ear skin-derived fibroblast cell nuclei. The source of nuclear recipient cells were IVM doe oocytes. The reconstruction of the previously enucleated oocytes (i.e. ooplasts) was performed by microinjection of either the somatic cell-derived karyoplasts or intact whole tiny nuclear donor cells directly into the cytoplasm. The reconstructed oocytes were incubated in Upgraded B2 INRA medium for 30 min to 1 h before their pseudophysiological activation. The activation was achieved by electrofusion of clonal cybrids with the allogeneic cytoplasts isolated from caprine IVF-created zygotes, which led to the formation of triple allocytoplasmic hybrids (allocybrids). These originate from 3 sources: (1) homologous whole nuclear donor fibroblast cells or their karyoplasts; (2) enucleated oocytes (ooplasts), and (3) zygote-derived cytoplasts. Single zygote-descended cytoplasts (the so-called zygoplasts) were inserted into the perivitelline space of previously reconstituted oocytes. The resulting zygoplast-clonal cybrid couplets were subsequently subjected to electrofusion, which was induced by application of a single DC pulse of 2.4 kV cm-1 for 15 μs. The electrofusion of zygoplast and reconstructed oocyte plasma membranes occurred in an isotonic dielectric solution deprived of Ca2+ ions. The transcytoplasmically activated clonal cybrids were cultured in vitro in Upgraded B2 INRA medium for 48 h at 38.5°C in a 100% water-saturated atmosphere of 5% CO2 and 95% air. Afterward, cleaved embryos were co-cultured with Vero cells in medium supplemented with 10% fetal bovine serum for an additional 96 to 144 h up to morula and blastocyst stages under the same thermal and atmospheric conditions. A total of 53/78 (67.9%) oocytes reconstructed with fibroblast cell nuclei were successfully fused with zygoplasts. From among 53 cultured cloned embryos, 34 (64.2%) cleaved. The rates of embryos that reached the morula and blastocyst stages were 21/53 (39.6%) and 11/53 (20.8%), respectively. In conclusion, the relatively high percentages of morulae and blastocysts were noticed among in vitro-cultured caprine cloned embryos produced by the strategy of pseudophysiological transcytoplasmic activation of oocytes reconstructed with adult dermal fibroblast cell nuclei. Therefore, the use of cytoplasmic components originating from zygotes as the stimuli for activation of nuclear-transferred oocytes appeared to be an effective procedure in the generation of goat blastocysts by somatic cell cloning.
8 EFFECT OF SEMEN TYPES (SEXED v. NONSEXED) ON CONCEPTION RATES OF GIROLANDO HEIFERS INSEMINATED IN FIXED-TIME AI
Authors: Almeida J. , Resende O. A. , Silveira D. L.
Published: August 2010

With the goal of optimizing protocols, fixed-time artificial insemination (FTAI) was performed under field conditions in a study to evaluate conception rates in Girolando (5/8) heifers (18 to 24 months of age) treated with estradiol benzoate (BE) to induce ovulation. Forty heifers were allocated to 2 groups with 20 animals each: GI-sexed semen (2 million spermatozoa/dose) and GII-nonsexed semen (20 million spermatozoa/dose), with body condition score (BCS) of 3.0 ± 0.14 (scale of 1 to 5), middleweight of 335 ± 38 kg, so that it was made for reutilization of progesterone device. On Day 0 (morning), the animals received 2.0 mg of BE (RIC-BE®,Tecnopec, São Paulo, Brazil), 0.075 mg of D-cloprostenol (Prolise®, ARSA S.R.L., Buenos Aires, Argentina), and an intravaginal device of 1 g of progesterone (Primer®, Tecnopec). On Day 8 (morning) the device was removed and 0.075 mg of D-cloprostenol was managed in the GI animals, then these animals received 1.0 mg of BE 24 h after device removal. The insemination was in the late afternoon of Day 10 with a 6-hour delay (4 to 6 pm). The GII animals received Primer previously used for 8 days (being used in the GI animals under the same protocol). After pregnancy diagnosis at 60 days, a third group was created (GIII) using the not-pregnant animals of GI and GII. In GIII, Primer from third use [disinfected in solution Kilol®-L 1:250 (Quinabra, São Paulo, Brazil) and rinsed with water, dried, and packed in plastic sacks and kept for 60 days] was used following the GI protocol with sexed semen. There were no significant differences (P > 0.05) in the conception rates of 40, 58, and 65% among groups GI (8/20), GII (11/20), and GIII (13/20). These results indicate very good conception rates for heifers, contrary to recommendations of commercial companies of sexed semen that alert for the conception rate for mated animals to be lower when using FTAI and mating protocols in mass, compared with that of AI after estrus observation.
81 THE EFFECT OF FOLLICULAR AND OVIDUCT OOCYTES ON THE DEVELOPMENT OF RABBIT NUCLEAR TRANSFERRED EMBRYOS IN VITRO
Authors: Sung L.-Y. , Chen C.-H. , Lin T.-A. , Sung L.-J. , Su H.-Y. , Sung Y.-S. , Cheng W. T. K. , Ju J.-C. , Chen E. , Xu J. , Du F.
Published: August 2010

This study was designed to examine the effect of rabbit oocytes collected from oviducts v. follicles on the developmental potential of nuclear transplant (NT) embryos. Rabbit oocytes were flushed from the oviducts (oviduct oocytes) or collected from the ovarian Graafian follicles(follicular oocytes) of superovulated does at 12 h post-hCG injection (hpi). Cumulus cells were then removed from the oocytes by incubation in 0.5% hyaluronidase and pipetting. Oocyte enucleation was conducted in TCM-199 +10% fetal bovine serum (FBS) and confirmed under fluorescent microscopy. Skin fibroblasts from an adult rabbit were prepared and cultured to passage 8 to 10 before use as nuclear donors. A donor cell with a diameter of approximately 15 to 19 μm was transferred into the perivitelline space of an enucleated oocyte and subsequently fused with the recipient oocyte by applying 3 direct current pulses at 3.2 kV cm-1 for 20 μs per pulse. Fused oocytes were activated by the same electrical stimulation described above, and then cultured in TCM-199 + 10% FBS containing 2.0 mM 6-DMAP and 5 μg mL-1 cycloheximide for 1 h. Cloned embryos were cultured in 2.5% FBS B2 medium in 5% CO2 and 95% humidified air at 38.5°C for 3 d. Embryo development to cleavage (2- to 4-cell), 8-cell, and morula/blastocyst (Mor/BL) stages was evaluated. The data were analyzed by the General Linear Model procedure (SPSS 11.0, SPSS Inc., Chicago, IL, USA).The total number of oocytes collected per animal was 27.6 ± 1.3, with 47.8% from oviducts, and 52.2% from follicles. The percentage of oviduct oocytes that showed the first polar body was 98.3% (n = 150) at the time of collection, whereas follicular oocytes only had 54.8% at collection (n = 93), but it reached 92.4% when immature follicular oocytes were cultured for 3 h in vitro. The enucleation rates were similar between the follicular (82.7%) and the oviduct (79.1%) groups. Table 1 shows that a significantly higher fusion rate was found in follicular oocytes compared with that in the oviduct group (90.8 v. 63.4%; P < 0.05). There was no difference in the cleavage rate and Mor/BL development between the 2 groups, although the 8-cell(78.4 v. 63.9%; P = 0.11) and the overall efficiencies (30.6% v. 17.9%; P = 0.14) appeared higher in the follicular group. These results demonstrated that rabbit follicular oocytes at 12 hpi have potential equivalent or maybe better (fusion) than that with oviduct oocytes for promoting the preimplantational development of NT embryos.

Table 1.The effect of follicular and oviduct oocytes on the development of rabbit NT embryos

Supported by NIH1R43 RR023774-01A1 and 5R44HL091605-03.
83 THE ENDOMETRIUM AND EXTRAEMBRYONIC TISSUES RESPOND DIFFERENTLY TO CLONED v. NATURAL MATING EMBRYOS ON EARLY PREGNANCY
Authors: Woo J.-S. , Ko Y.-G. , Kim H.-N. , Yoo J.-G. , Yang B.-S. , Lee H.-C. , Lee J.-Y. , Hyung N.-W. , Park E.-W. , Hwang S.-S. , Park S.-B. , Park M.-R.
Published: August 2010

Porcine cloning by somatic cell nuclear transfer (SCNT) has limitations because of the high incidence of fetal failure after embryo transfer to recipient. The fetal failure may originate from disturbed embryo-uterine interactions in the early implantation period. In this study, we compared apoptotic-related gene expression and cytokines using real-time RT-PCR and protein chip array. Ovaries were obtained from prepubertal crossbred gilts at a local slaughterhouse. Oocytes were matured for 40 to 44 h at 38.5°C under 5% CO2 in air. As donor cells, fibroblast cells were cultured from ear skin tissue of 8-month-old MHC inbred miniature pig. After the enucleation and injection process, eggs were held in TCM-199. For fusion, two DC pulses of 1.2 kV cm-1 were applied for 30 μs. SCNT embryos were cultured under PZM-3 medium to the 1- to 4-cell stage. For embryo transfer, approximately 200 SCNT eggs were transferred into the oviduct of each synchronized recipients. We obtained the extraembryonic (embryo origin) and endometrium (maternal origin) tissues from 3 cloned fetus with that from normal fetus by natural mating at Day 30. Each experiment was repeated at least 3 times and the data were presented as mean ± SE. Values of P < 0.05 were considered statistically significant. Data from real-time RT-PCR are expressed as log (fold change) ± SE. Expression of Bax-α, p53, and caspase-3 mRNA was significantly higher in the SCNT group than in the control group (P < 0.05). Five cytokines were analyzed: Interleukin (IL)4, IL8, tumor necrosis factor-α, and epidermal growth factor were lower in the control group than in the cloned group (P < 0.05). However, the level of MCP1 was higher in the SCNT group (P < 0.05). Numbers of trophoblast cells in the cloned group were also lower than in the control group (P < 0.05). These findings indicate that failure of endometrium and extraembryonic tissues in cloned pregnancies may originate from abnormal embryo-maternal communication that develops during the implantation period.

This work received grant support from the Agenda Program (no. 200901FHT010305535), Rural Development Administration, Republic of Korea.