bluuebunny/crossref_metadata_2025 huggingface dataset:
22 VASCULAR ENDOTHELIAL GROWTH FACTOR mRNA EXPRESSION IN FETAL MEMBRANES DURING EARLY PREGNANCY AS A BIOMARKER FOR DEVELOPMENT OF HYDROPS PLACENTA LATER IN PREGNANCY IN BOVINE EMBRYOS CREATED THROUGH SOMATIC CELL NUCLEAR TRANSFER (SCNT)
Authors: Borowicz P. P. , Johnson M. L. , Beraldi R. , Edwards A. , Grazul-Bilska A. T. , Thorson C. , Kasinathan P. , Wang Z. , Robl J. , Reynolds L. P. , Redmer D. A.
Published: December 2008

Pregnancies obtained via somatic cell nuclear transfer (SCNT) show an increased incidence of pregnancy complications, including poor placental vascularization, hydrops placenta, and abnormal placental morphology. In a preliminary study of hydrops placentas from SCNT clones v. control cow placentas close to term, we observed a thickening of the endometrial epithelium, which appeared pseudostratified; in addition, the subepithelial endometrial vascular bed consisted of large, rare arterioles with thick basement membranes compared with the normal capillary bed observed in control animals. The fetal membranes of hydrops placenta from bovine SCNT clones also exhibited macroscopic morphological differences, which included edema, increased membrane thickness, and a gelatinous appearance. All of these morphological differences were similar to those we have reported for sheep SCNT clones (Palmieri et al. 2007 Placenta 28, 577–584). In the search for biomarkers that may indicate abnormal placental development early in pregnancy, placental tissues were collected on day 40 from pregnancies established after transfer of embryos created through SCNT (n = 17) or IVF (n = 6). Nuclear donor cells used to obtain SCNT embryos were those we have previously found to be highly susceptible to hydrops (from 5 to 19%), whereas the incidence of hydrops in IVF and controls in our laboratories did not exceed 0.5%. A sample of fetal membranes was fixed in 10% buffered formalin for histological analysis; another sample was snap-frozen in liquid nitrogen for analysis of mRNA levels for vascular endothelial growth factor (VEGF) and its receptors (KDR and FLT) by quantitative, real-time RT-PCR. Histological analysis of cell proliferation rate was performed using immunohistochemical localization of proliferating cell nuclear antigen and image analysis software (Image-Pro Plus). There were no differences (P = 0.39) in the rate of cell proliferation of the trophoblast in SCNT v. IVF (29.4 ± 4.9% v. 22.1 ± 3%). However, VEGF mRNA expression was less (P < 0.04) in the fetal membranes of SCNT than IVF on day 40 of pregnancy (0.30 ± 0.04 v. 0.53 ± 0.12, normalized to α-actin mRNA levels). The mRNA expression for KDR and FLT were similar in SCNT and IVF. These data suggest that the lower expression of VEGF, a major placental angiogenic factor, in fetal membranes of early pregnant SCNT fetuses may be an early biomarker for the development of hydrops placenta later in pregnancy.

NDAES Proj. ND01727.
220 SYNERGISTIC EFFECT ON EMBRYO DEVELOPMENT BY INCLUSION OF SUPPLEMENTAL EMBRYOS IN AGAR CHIPS
Authors: Senatore E. M. , Mannino M. E. , Suarez Novoa M. V. , Xu J. , Chaubal S. , Yang X. , Tian X. , Du F. , Presicce G. A.
Published: December 2008

The main scope of this study was to evaluate the likelihood of a helper effect of agar-embedded cleaved embryos on a low number of free embryos at a similar stage of development within the same culture droplet. Such an improved system could be beneficial within ovum pickup/in vitro embryo production (OPU/IVEP) combined protocols whenever a low number of OPU-derived cleaved embryos are produced per donor. Oocytes were recovered from abattoir ovaries, and after in vitro maturation (IVM) and in vitro fertilization (IVF), presumptive zygotes were deprived of cumulus investment and allocated into culture droplets for 24 h. At 48 h from IVF, 4- to 8-cell cleaved embryos were randomly allocated into a control and a treatment group. Control groups consisted of 1, 3, 5, and 10 embryos, respectively, in 50-μL droplets. Treatment groups consisted of 1, 3, and 5 free embryos with the addition of 9, 7, and 5 embryos, respectively, at a similar stage of development embedded in agar chips, so as to reach a total number of 10 cleaved embryos in each culture droplet. Culture was performed for both the control and treatment groups in SOF medium droplets covered with mineral oil, with the supplementation of essential and nonessential amino acids in a controlled gas atmosphere consisting of 5% CO2, 7% O2, and 88% N2 at 39°C. Final embryo output was checked at Day 7 from IVF. When considering only free embryos, the difference in progression to blastocyst development was highly significant between the control and treatment groups: 1) group 1 v. 1 + 9: 6.6 v. 84.3% (P = 0.00000); 2) group 3 v. 3 + 7: 11.1 v. 41.3% (P = 0.00001); 3) group 5 v. 5 + 5: 24.4 v. 42.2% (P = 0.00001). Rate of blastocyst development in the control group containing 10 cleaved embryos was not significantly different from free cleaved embryos in the 3 + 7 (39.2 v. 41.3%, P = 0.71) and 5 + 5 treatment groups (39.2 v. 42.2%, P = 0.54), but was significantly lower when compared with the 1 + 9 treatment group (39.2 v. 84.3%, P = 0.000). For 1, 3, 5, and 10 control group embryos, the numbers of replicates and total cleaved embryos used (n) were 30 (n = 30), 27, (n = 81), 27 (n = 135), and 39 (n = 390), respectively. For the 1 + 9, 3 + 7, and 5 + 5 treatment group embryos, the numbers of replicates and total cleaved embryos used were 32 (n = 32), 29 (n = 87), and 27 (n = 135), respectively. In conclusion, a beneficial effect of agar-embedded embryos on the development of free embryos within the same culture droplet was shown. A striking improvement in late-stage embryo development was particularly evident when considering the 1 v. 1 + 9 control and treatment groups. These results may foster a different strategic approach in in vitro culture to enhance embryo development from highly valuable donors.
23 EFFECT OF HISTONE ACETYLATION LEVELS ON THE IN VITRO DEVELOPMENT OF CLONED BOVINE EMBRYOS
Authors: Chacón L. , Jenkins J. A. , Leibo S. P. , Wirtu G. , Dresser B. L. , Pope C. E. , Gómez M. C.
Published: December 2008

The epigenetic status of donor cells is an important factor for their successful reprogramming during somatic cell nuclear transfer (SCNT). Environmental factors partly influence DNA methylation and histone modifications (Fraga et al. 2005 PNAS USA 102, 10 604–10 609; Ke et al. 2006 Carcinogenesis 27, 1481–1488; Rodenhiser and Mann 2006 CMAJ 174, 341–348); low temperatures have altered epigenetic events in plants (Amasino 2004 Plant Cell; Hao et al. 2002 Cryo Letters 23, 37–46). Because cryopreservation alters histone acetylation levels in donor cells and subsequent viability of cloned embryos (Gómez et al. 2008 Cloning Stem Cells, in press), similar alterations may occur in bovine cloned embryos reconstructed with donor cells thawed immediately before SCNT. The objectives of the present study were (1) to measure the relative levels of nuclear histone acetylation in bovine fibroblasts immediately after thawing (frozen/thawed) or following a period of culturing (cultured) and (2) to determine the influence of the epigenetic status of donor cells on the in vitro development of reconstructed, cloned bovine embryos by gauging blastocyst development. Cell cultures lines were derived from the skin of 3 adult cows and analyzed at passage 1 (P1), 2 (P2), and 10 (P10). For each of 3 passages, cells were cultured until reaching 100% confluence, followed by an additional 3 days of culture during which time acetylation levels were measured in cultured and frozen/thawed cells. For cryopreservation, cells at P1, P2, and P10 were disaggregated and resuspended in CryoStor™ (CS10; BioLife Solutions, Bothell, WA, USA) and cooled at 1.0°C min–1 to –80°C prior to storage in liquid nitrogen. Cells were fixed with ethanol for 12 h and incubated for 30 min with antibody directed against acetylated lysine 9 on histone 3 (H3K9). The cells were then incubated with a fluorescein isothiocyanate conjugated secondary antibody and DNA stain and evaluated by flow cytometry. Cloned embryos were reconstructed with cultured or frozen/thawed cells at P1, P2, and P10 as described by Vajta et al. 2005 (Reprod. Fertil. Dev. 17, 791–797). Derived embryos were cultured until Day 8, and cleavage and development to the blastocyst stage were evaluated. Histone acetylation levels for all 3 cell lines, either fresh or frozen/thawed, were significantly higher at P1 than at P2 and P10 (Table 1), and cryopreservation reduced histone acetylation levels only in cell culture line 2 at P1. Higher development to the blastocyst stage (25%) was observed when embryos were reconstructed with cultured cells at P2 and with cells that had lower histone acetylation levels (Pearson correlation, r = –0.55; P = 0.01)

Table 1.Relative levels of histone acetylation in bovine fibroblast culture and percentages of development to blastocyst stage after cloning
230 COMPARISON OF SEMEN SAMPLES FROM NORMAL AND MUSCULAR DYSTROPHY AFFECTED ANIMAL MODELS BY THIOBARBITURIC ACID REACTIVE SUBSTANCES TEST AND FLUORESCENCE-ACTIVATED CELL SORTING ANALYSIS
Authors: Peres M. A. , Nichi M. , Rocha A. M. , Mendes C. M. , Cavalcanti P. B. , Gonçalves J. S. A. , Visintin J. A. , Assumpção M. E. O. A.
Published: December 2008

Duchenne’s muscular dystrophy (DMD) is the most common human muscular affliction with high frequency in male individuals. The condition is characterized by progressive muscle degeneration, weakness, and loss of motion capacity. These individuals exhibit longer lifespans resulting from improvements in technical patient care and now raise new questions ranging from ethical to physiological issues never observed before, such as the possibility of reproduction. Thus, this study used Golden Retriever muscular dystrophy (GRMD)-affected dogs, a natural experimental model of DMD, to evaluate semen quality and oxidative stress by thiobarbituric acid reactive substances (TBARS) test. Thirty-seven ejaculates from 4 non-affected Golden Retriever and 5 GRMD-affected dogs (from 1.5 to 4.5 years old) were collected monthly and evaluated as 5 different replicates. Semen samples were processed, and initial analysis comprised of sperm concentration, percent of straightforward motility and morphology. Samples were then diluted to a final concentration of 106 spermatozoa mL–1 of prior incubation with each fluorescent probe. Acrosome integrity assessment was conducted with PSAFITC (100 g mL–1), and mitochondrial activity was assessed by JC1 (50 g mL–1). Fluorescence-activated cell sorting was performed in 103 spermatozoa, from a pre-selected gate that contained only these cells. Seminal plasma was submitted to TBARS quantification by spectrophotometer under normal and oxidative conditions (stress). Parametric data were compared by Student’s t-test. There were no significant differences in ejaculate volume, sperm concentration, acrosomal integrity, mithocondrial activity, and ejaculate TBARS tests between GRMD and normal dogs. Data are summarized in the table. GRMD does not affect the production and quality of semen of dogs. This animal model suggests that ejaculates from DMD men can be obtained by the proper stimulation and that conventional in vitro fertilization techniques combined with preimplantation genetic diagnosis for single gene disorders may be able to satisfy the desire of paternity of DMD patients with the birth of non-affected children.

Table 1.Sperm analysis and TBARS tests of ejaculates from GRMD and normal dogs

FAPESP for the financial support (06/50272-3); Surgery Department and Reproduction Department of FMVZ-USP; Canil GRMD Brazil.
231 SPERM SUBPOPULATIONS IN THE KOALA (PHASCOLARCTOS CINEREUS) EJACULATE
Authors: Satake N. , Johnston S. D. , Holt W. V.
Published: December 2008

Koala semen contains a heterogeneous mixture of sperm morphotypes, mainly attributable to extreme degree of shape variability displayed by the hooked sperm head. By analogy with other species, we anticipate that the morphotypes may exhibit correspondingly different sperm-motility behaviors, largely caused by the differences in hydrodynamic interactions with the suspending media. This trend has been shown in human spermatozoa where motility behavior was demonstrably correlated with the sperm head morphology (Overstreet et al. 1981). In this study, we have investigated the heterogeneity of koala sperm motility profiles in semen in an effort to determine whether distinct sperm subpopulations within ejaculates are recognizable by the use of computer-assisted sperm motility analysis. Ejaculates from 5 males were collected by electroejaculation, then diluted and transported in Tris-citrate-glucose (TCG) diluent. Spermatozoa were washed through a 35–60% Percoll gradient to separate seminal plasma and the majority of the prostatic bodies from spermatozoa. Spermatozoa from the washed pellet were then diluted in TCG at 35°C, incubated for 10 min, and video recorded using a negative phase ×10 objective. Sperm motion parameters were then analyzed using the Hobson sperm tracker (Hobson Vision Systems, UK: Holt et al. 1996 J. Androl. 17, 587–596). Multivariate pattern analysis (PATN; CSIRO Australia; Abaigar 1999 Biol. Reprod. 60, 32–41) was used to distinguish 3 sperm subgroups, consistently shown in each ejaculate, within the data (1936 tracks × 6 kinetic parameters; VCL, VAP, MAD, BCF, ALH, LIN). After group allocation by PATN, all parameters showed significant differences between each of the groups (P < 0.0001). Group 1, approximately 25% of the sperm tracks, showed profiles of spermatozoa with fast, non-linear motility (VCL 106.88 ± 28.15; BCF 3.23 ± 3.81; LIN 14.08 ± 10.20). Group 2, approximately 27% of sperm tracks, showed profiles of fast, linear motility (VCL 63.92 ± 13.50; BCF 7.90 ± 3.42; LIN 28.10 ± 12.15). Group 3, 48% of sperm tracks, showed profiles of slow, non-linear or circular patterns of motility (VCL 39.05 ± 11.92; BCF 0.02 ± 0.35; LIN 5.15 ± 4.88). The recognition of 3 clearly identifiable subgroups supports our hypothesis that heterogeneity of sperm motility patterns exists within koala ejaculates. These may be a reflection of the heterogeneity in sperm-head morphotypes in koala semen, but that remains to be investigated in more detail. The clear distinctions between these groups, and the observation that all 3 subpopulations exist in each of the ejaculates, also suggest that the spermatozoa exhibit functional differences, possibly related to biochemical or maturational status.

Many thanks to Dr. Michael Pyne and Dr. Vere Nicholson and their teams and animals at Currumbin Wildlife Sanctutary and Dreamwolrd QLD for all their help and support for the collection of samples.
234 MECHANISMS OF SPONTANEOUS ACTIVATION IN RAT OOCYTES
Authors: Chebotareva T. , Mullins J. , Wilmut I.
Published: December 2008

MII-arrested rat oocytes spontaneously resume meiotic maturation soon after recovery from oviducts. Time elapsed post oocyte retrieval, environmental factors, certain genetic background have been implied to promote spontaneous release from MII arrest in rat oocytes. The precise mechanism behind this process is unknown. This study was undertaken to explore signaling pathways, which may be involved in spontaneous activation in rat oocytes. Using triple immuno-staining and epifluorescence microscopy we have described morphological changes in the meiotic spindle of rat oocytes during 6h of in vitro culture (immature CD female rats from Charles River Laboratories). An ELISA-based method was used to evaluate cdc2 activity in rat oocytes subjected to in vitro culture. SDS-PAGE and western blotting were performed to study levels of phosphorylated and total p42/p44 MAPK, phosphorylated on Tyr15 and total cdc2, cyclin B1 and β actin. Expression of Emi2 was analysed by RT-PCR. Data were analyzed using GLMM or ANOVA followed by t-test. Freshly collected oocytes contained well-preserved spindle with chromosomes aligned in metaphase plate. After being in culture for 2 h oocytes demonstrated signs of activation, such as spindle rotation, preparing to extrude second polar body, and some oocytes had entered anaphase. In the majority of oocytes cultured for 6 h spindles had disintegrated and chromosomes were scattered in the oocyte cytoplasm; microtubules were found around condensed chromosomes. Significant drop in cdc2 activity was detected in oocytes after 2 h of in vitro culture. In oocytes cultured for additional 4h of cdc2 activity returned to the level observed in freshly collected oocytes. Inhibitory phosphorylation of cdc2 on Tyr 15 was not associated with cdc2 inactivation. We were not able to detect changes in the level of cyclin B1 during the MII to MIII transition. Phosphorylated forms of activated p42/p44 MAPK in eggs were present throughout in vitro culture. Emi2 is a novel candidate cytostatic factor in vertebrate eggs. Expression of Emi2 was detected at mRNA level in rat eggs and zygotes with no expression at later stages of preimplantation development. Taken together, the onset of spontaneous activation in rat oocytes is correlated with a sharp decline in cdc2 activity and stable level of phosphorylated p42/p44 MAPK. Dynamics of cdc2 and p42/p44 MAPK activity during spontaneous activation resembles that during the MI to MII transition, although spontaneously activated rat oocytes do not form MIII spindle. Molecular factors involved in spindle assembly may be missing during the MII to MIII transition. Persistence of p42/p44 MAPK in spontaneously activated rat oocytes could account for the absence of pronuclear formation. The role of Emi2 remains to be investigated.

This work is supported by the CMVM, ORS, Mary Orr Paterson studentships and the Framework 6 activity EURATOOLS.
236 CREATION OF PARTHENOGENETIC SHEEP EMBRYOS: EFFECTS OF SERUM AND BREEDING SEASON
Authors: Grazul-Bilska A. T. , Borowicz P. P. , Redmer D. A. , Bilski J. J. , Reynolds L. P.
Published: December 2008

The monoparental embryo (with only the maternal genome, termed a parthenogenote, or with only the paternal genome, termed an androgenote) is a powerful model to study the epigenetic status of developmentally regulated genes, including imprinted genes (those expressed only when inherited from one parent). Therefore, to use monoparental embryos for future study of placental development in normal and compromised pregnancies, the objective of this study was to test, validate and optimize the methodologies necessary to create parthenogenetic sheep embryos. In Exp. 1, cumulus–oocyte complexes (COC) were collected during the breeding season from nonpregnant and early pregnant ewes (n = 18) and matured overnight in vitro. The oocytes were then activated using ionomycin (a calcium ionophore) and 6-dimethylaminopurine (DMAP; a protein kinase inhibitor) in medium with (n = 47 COC) or without (n = 112 COC) 2% serum. In Exp. 2, COC were collected from nonpregnant, seasonally anestrous (n = 7; 79 COC) and late pregnant (n = 4; 44 COC) ewes, matured in vitro and activated as in Exp. 1 in the presence of serum. In Exp. 1, the rates of activation and blastocyst formation were not affected by reproductive status (nonpregnant v. pregnant). Activation of oocytes in serum-containing v. serum-free medium resulted in 76.3% v. 4.35% (P < 0.0001) cleavage rates and 21.9% v. 8.3% (P < 0.006) blastocyst formation rates, respectively. In Exp. 2, cleavage rates were similar for nonpregnant and pregnant ewes (31.9 v. 37.5%), but blastocyst formation was 13% in nonpregnant and 0% in pregnant ewes. These data demonstrate that (1) during the breeding season, the presence of serum in activation medium enhances cleavage and blastocyst formation rates; (2) in out-of-season ewes blastocyst formation but not cleavage rates are greater for nonpregnant than pregnant ewes, and (3) cleavage and blastocyst formation rates are greater (P < 0.0001 and P < 0.04, respectively) during the breeding season than out-of-season. Thus, oocytes obtained from ewes during the breeding season and activation medium containing serum should be used for creating parthenogenetic embryos in sheep. This study also demonstrated that creation of parthenogenotes in sheep, which can be used in the future to study parentally imprinted genes, is feasible but requires specific season and culture conditions.

Supported by USDA-NRI grant 2007-01215 to LPR and ATGB, and NIH grant P20 RR016741 (INBRE program of the NCRR, NIH).
238 CAFFEINE PREVENTS A LOSS OF CALCIUM OSCILLATORY RESPONSE ASSOCIATED WITH POSTOVULATORY AGING OF MOUSE OOCYTES
Authors: Wakai T. , Zhang N. , Fissore R. A.
Published: December 2008

Numerous studies have demonstrated that postovulatory aging of oocytes prior to fertilization has detrimental effects on oocyte quality and developmental competence. Oocyte aging is accompanied by abnormal oocyte activation and subsequent development, suggesting a disruption of Ca2+ oscillations after fertilization. The inositol 1,4,5-trisphosphate receptor type 1 (IP3R1) in mammals is responsible for the majority of Ca2+ release during fertilization (Miyazaki S et al. 1993 Dev. Biol.). Previously, we reported that phosphorylation of IP3R1 at an MPM-2 epitope may play an important role in facilitating the induction of Ca2+ oscillations at the MII stage (Lee B et al. 2006 Development), indicating that IP3R1 phosphorylation may be a good indicator of the health of the oocyte. However, few studies have investigated the alteration of the Ca2+ signaling and IP3R1 function associated with oocyte aging. On the other hand, a previous report showed that caffeine increased MPF activity and suppressed fragmentation after parthenogenetic activation of aged oocytes (Kikuchi K et al. 2000 Biol. Reprod.). Therefore, the purpose of the present study was to examine whether and how Ca2+ oscillatory activity changes during oocyte aging and to test if caffeine prevents the negative effects of oocyte aging. MII mouse oocytes were collected 14 h after hCG injection and cultured in vitro for 8, 24 or 48 h with or without caffeine (5 or 10 mm). Oocyte quality was assessed by the occurrence of spontaneous fragmentation, monitoring of Ca2+ oscillations after exposure to 10 mm strontium chloride, Western blot analysis of IP3R1 phosphorylation and immunostaining of IP3R1. In oocytes in vitro aged for 8 h, the duration of the first Ca2+ rise was significantly decreased compared with fresh MII oocytes, although this reduction was not observed in MII oocytes treated with 5 mm caffeine. The phosphorylation of IP3R1 at the MPM-2 epitope was slightly decreased during oocyte aging in both caffeine and noncaffeine treatment. Importantly, whereas IP3R1 in MII oocytes treated for 8 h with 5 mm caffeine displayed the typical cortical cluster organization, IP3R1 in aged oocytes without caffeine became dispersed in the cytoplasm. In addition, caffeine significantly suppressed the spontaneous fragmentation that is normally observed by 48 h of in vitro culture. These results suggest that the Ca2+ oscillatory activity is compromised during oocyte aging and caffeine prevents the loss of integrity of Ca2+ signaling possibly by keeping the cortical distribution of IP3R1.
239 CANINE OOCYTES CAN BE SPONTANEOUSLY PARTHENOGENERATED AND DEVELOPED TO 2-CELL TO 8-CELL STAGE PARTHENOGENERATED EMBRYOS IN COCULTURE SYSTEM WHICH THEY WERE COCULTURED WITH OVIDUCT CELLS OR OVIDUCT EXTRACTS
Authors: Wang L. X. , Wang S. , Hou J. , Chen Y. F. , Hu R. L. , An X. R.
Published: December 2008

The objective of this study was to coculture canine oocytes with oviduct cells or oviduct extracts in modified M199 culture medium to improve the MII rate. The canine oocytes were collected from the local slaughterhouse and were cultured in three culture systems while in the same culture medium. In culture system A, canine oocytes were cocultured with ‘small-round’ oviduct cells and ‘big-round’ oviductal cells which based on cell volume and morphology for 96 h. In culture system B, canine oocytes were cultured with in the modified M199 medium with 10% canine follicle fluid (in the CEF group) or without canine follicle fluid (in the compared group) for the first 6 h; following this, they were cocultured with ‘small-round’ oviductal cells for another 90 h, which were preblanced at least 2 h. In the culture system C, the canine oocytes were cocultured with canine estrous oviduct extracts for 96 h (in the compared group) or were cocultured with granulosa cells together with canine estrous oviduct extracts (in the G + CEOE group) for 96 h. These results demonstrated that, in culture system A, one 2-cell and one 8-cell stage parthenogenerated embryo in the small-round group and one 2-cell stage parthenogenerated embryo in the compared group were achieved after in vitro culture for 96 h. Meanwhile, the MII rates were greater in small-round group (47.1%) than that in big-round group (36.1%) and the compared group (32.4%). In the culture system B, one 3-cell stage parthenogenerated embryo was detected in CEF group. The MII rates were almost equal in CEF group (28.0%) and in the compared group (26.1%). In culture system C, one 2-cell stage parthenogenerated embryo was found in the compared group. The MII rate were almost equal in CEOE group (25.8%) and in the G + CEOE group (23.5%). It is concluded that some unidentified factors secreted by the oviductal cells or oviduct extracts may promote the development of canine oocytes in vitro and parthenogenerate the canine oocytes beyond 2-cell stage parthenogenerated embryos. The culture system in which the canine oocytes were cocultured with oviducts or oviductal cells has been improved to date.
258 FACTORS AFFECTING COMMERCIAL SEXING PROGRAM AND MULTIPLE GENETIC ANALYSIS PERSPECTIVES OF IN VITRO-PRODUCED BOVINE EMBRYOS
Authors: Alonso R. V. , Hellú J. A. A. , Perri S. H. V. , Visintin J. A. , Garcia J. F.
Published: December 2008

The present study aimed to evaluate the interactions among different factors on the viability and sex ratio of in vitro-produced (IVP) bovine embryos, submitted to a large scale sexing program. Additionally, whole genome amplification (WGA) technology was used to amplify genomic DNA from IVP bovine embryo biopsies, in order to perform multiple genetic analyses. The survey was performed in a 4650 IVP bovine sexed embryo database. Embryos were biopsied by a microaspiration technique and sex was determined by PCR of DNA from the biopsy. Only female embryos were transferred to synchronized recipients. Pregnancy diagnosis and fetal sex determination were carried out by ultrasound. The variables were classified in accordance with embryo sex (male, female, and indeterminate), five laboratories (A, B, C, D, and E), six bovine breeds (Nellore, Brahman, Girolando, Simmental, Holstein, and Jersey), embryo stage (MO, EB, BL, XB, and HB), embryo quality (1, 2, and 3) and biopsy quality (“standard” and “nonstandard”). The statistical analysis was carried out by association chi-square test, chi-square for a 1:1 ratio, and logistic regression analysis (PROC LOGISTIC) of SAS. PCR showed 93.3% efficiency, 93.2% accuracy, and male and female rates of 52.9% and 47.1%, respectively. Mortality rate of biopsied embryos was 10.3% and pregnancy rate was 31.7%. Significant differences were not observed between male and female viability, although indeterminate embryos resulted in more death after micromanipulation. For quality 2 and 3 embryos, the mortality rate after biopsy was 3.19 and 11.37 fold higher, respectively, than for quality 1 embryos. For embryos whose biopsies were classified as nonstandard, the embryonic mortality rate was 3.6-fold higher than standard ones. Mortality rate was not affected by embryo stage at biopsy (P > 0.05). Although sex ratio was significantly skewed to male embryos, differences were not observed among laboratories (P > 0.05) and breeds (P > 0.05) on the sex ratio of IVP bovine embryos. To test the feasibility of using WGA method for multiple genetic analysis, biopsies from 28 IVP embryos were submitted to the GenomePlex Single Cell System (Sigma-Aldrich, St. Louis, MO, USA). Aliquots from each DNA sample were purified using column chromatography and submitted to PCR using sexing primers BRY4a, SRY, UMN0920, and S4B. PCR was successful and in agreement among tested DNA aliquots from each single biopsy. The WGA strategy used herein was a useful tool for applications involving restricted amounts of starting genetic material (DNA), such as in preimplantation genetic diagnosis using IVP bovine embryos.

To FAPESP and UNESP.
275 DERIVATION OF PLURIPOTENT CELL LINES FROM PIG EMBRYOS: IN VITRO-FERTILIZED V. PARTHENOGENTIC ACTIVATION
Authors: Gandolfi F. , Pennarossa G. , Attanasio L. , Antonini S. , Gasparrini B. , Brevini T. A. L.
Published: December 2008

The establishment of porcine pluripotent ES cell lines would be an exciting and novel tool for animal biotechnology, such as cloning and transgenesis. Furthermore, it would represent a useful model for biomedical research, cell therapy, xenotransplantation as well as developmental biology research. However, in spite of several studies, no conclusive results have been obtained and a number of technical questions are still to be answered in order to derive genuine ES cells in the pig. Here we report the results obtained in our laboratory aimed at comparing IVF v. parthenogenetic embryos as a source for the establishment of putative ES cells. Oocytes were divided in two groups and subjected to IVF or parthenogenetically activated with ionomicyn and 6-DMAP. They were cultured in NCSU for 7 days and then subjected to immuno-surgery. Inner cell mass were plated onto inactivated STO feeder cells and outgrowth formation was monitored. Cells were passaged to a new STO monolayer every 7 days. Assessment of pluripotency markers was carried out both by RT-PCR and immunocytochemical analysis at every passage for up to 22 passages. Telomerase activity was measured every 5 passages. The results indicate that parthenogenetic embryos, although less resilient than IVF embryos to immuno-surgery, have a significantly greater ability to generate outgrowths and stable cell lines. Moreover, 77% of the 39 parthenogenetic lines derived v. only 33% of the IVF ones expressed pluripotency markers and displayed high telomerase activity. Altogether our findings are consistent with data obtained in the human where the efficiency to derive hES cell lines from parthenogenetic blastocysts appears greater as compared with regular blastocysts from IVF embryos (Cheng L 2008 Cell Research 18, 215–217).

Table 1.

Supported by: Prin 2005, 2006.
28 SIMPLIFIED ACTIVATION METHOD TO IMPROVE THE IN VITRO DEVELOPMENT OF HANDMADE CLONED (HMC) PORCINE EMBRYOS
Authors: Du Y. , Yang Z. , Lv B. , Lin L. , Kragh P. M. , Zhang X. , Vajta G. , Yang H. , Bolund L.
Published: December 2008

Delayed activation is commonly used in pig somatic cell nuclear transfer (SCNT) where electrical activation is followed by chemical activation. However, chemical incubation of several hours (up to 4 or 6) is logistically not very convenient even though handmade cloning (HMC) could improve the overall efficiency of pig cloning (Du et al. 2007 Theriogenology 68, 1104–1110). It was reported that a brief exposure of cycloheximide (CX) before electrical activation could significantly increase developmental rate and total blastocyst cell number when simultaneous activation was performed in micromanipulator-based pig cloning (Naruse et al. 2007 Theriogenology 68, 709–716). The purpose of our present work is to investigate whether such activation method is also applicable for pig HMC. Data were analyzed by t-test using SPSS (11.0, SPSS Inc., Chicago, IL, USA). After 42 h in vitro maturation, cumulus cells were removed. In vitro-cultured porcine fetal fibroblasts were used as donor cells. Cytoplast-fibroblast pairing, electrical fusion and activation of fused cytoplast-fibroblast pairs were performed as described previously (Kragh et al. 2005 Theriogenology 64, 1536–1545; Du et al. 2005 Cloning Stem Cells 7, 199–205). Three groups were compared due to different activation protocol. In Group 1 (control), reconstructed embryos were cultured in porcine zygote medium 3 (PZM3) supplemented with 4 mg mL–1 BSA, 5 μg mL–1 cytochalasin B (CB), and 10 μg mL–1 CX for 4 h. In Group 2 (CX priming), fused pairs and the other halves of cytoplasts were incubated in HEPES-buffered TCM-199 medium supplemented with 10% calf serum, 10 μg mL–1 CX for 10 min just before the second fusion or electrical activation. In Group 3 (CB + CX priming), treatment similar to Group 2 was performed except that additional 5 μg mL–1 CB was added for the 10-min incubation. Reconstructed embryos were in vitro cultured in the well of the well (WOW) system for 6 days. Blastocyst rates and total cell numbers of Day 6 blastocysts were evaluated. As illustrated in Table 1, embryos pretreated with both CB and CX gave the best results, with better blastocyst formation (53.8 ± 4.8%; mean ± SEM) and higher cell number (77.2 ± 5.4) compared to the other 2 groups. Our data suggested that CX and CB priming could be used as a solution to the long chemical incubation in porcine SCNT by HMC, making the embryos more receptive to electrical activation.

Table 1.In vitro development of HMC reconstructed embryos with different activation protocols
294 EFFECT OF SPERMATOZOA MOTILITY ON TRANSFERABLE EMBRYO RATE OF SUPEROVULATED JAPANESE BLACK CATTLE
Authors: Hayama K. , Takeuchi M. , Ideta A. , Urakawa M. , Sasatani M. , Nagamune Y. , Aoyagi Y.
Published: December 2008

Sperm motility is known to affect fertilization; however, little is known about the relationship between frozen–thawed sperm motility and in vivo fertilization following superovulatory treatment. The objective of this study was to evaluate a sperm function test as potential predictors of embryo production following superovulatory treatment in cattle. Two to five batches of semen (Japanese black bull, n = 4, A to D) were diluted with egg york-citrate-glycerol in 0.5 mL plastic straws, and they were stored in liquid nitrogen until analyzed. Frozen–thawed spermatozoa were evaluated for motility {motile sperm concentration (MSC, million mL–1), progressive MSC (PMSC, million mL–1) and velocity (μm s–1)} using a sperm quality analyzer for bulls (SQA-Vb, Medical Electronic Systems, Caesarea, Israel). Each sample of 20 μL aspirated into the disposable capillary, was inserted into SQA-Vb. Measurements were displayed within 75 s. Intra-assay CVs of MSC, PMSC, and velocity were 14.2, 7.3 and 7.5%, respectively. Inter-assey CVs of them were 13.5, 3.9 and 4.3% respectively. Superstimulated donors (Japanese black cows, n = 161) were artificially inseminated with one dose of frozen–thawed semen (bull A = 74, B = 46, C = 21 and D = 20). The proportion of transferable embryo (IETS grade 1 to 3) was examined on day 7 (day 0 = estrus). Data were analyzed using ANOVA followed by Scheffe multiple comparison test, and Fisher’s z-transformation. MSC, PMSC and velocity values differed significantly among each bull. The values of bull A were much lower than those of the other bulls. The proportion of transferable embryos produced by bull A was significantly lower than that of other bulls (P < 0.05, Table 1). Correlations showed significant association between MSC and proportion of transferable embryos (r = 0.99, P < 0.01). We conclude that bovine sperm motility using a SQA-Vb is a useful predictor of embryo production following superovulatory treatment.

Table 1.Relationship between sperm motility and proportion of transferable embryo
297 LACK OF IMPROVEMENT ON EMBRYO PRODUCTION BY THE REPLACEMENT OF THE LAST TWO DOSES OF pFSH BY eCG IN SUPERSTIMULATED NELORE HEIFERS
Authors: Sartori R. , Guardieiro M. M. , Barros C. M. , Bastos M. R. , Machado G. M. , Leme L. O. , Rumpf R.
Published: December 2008

Results from a previous experiment have shown that the replacement of pFSH by eCG on the last day of the superstimulatory treatment in Nelore (Bos indicus) cows resulted in a greater superovulatory (SOV) response as compared to the treatment exclusively with pFSH (Barros CM et al. 2008 Repr. Fertil. Dev. 20, 152 abst). The aim of this study was to investigate if a similar approach would be beneficial for embryo production in nulliparous Nelore heifers. Forty heifers were randomly divided into two SOV groups: FSH Group: eight pFSH injections or FSH-eCG Group: six pFSH injections followed by two eCG injections. Each female received both treatments 65 days apart in a cross-over design. The SOV protocols consisted of an IM injection of 2 mg estradiol benzoate (Estrogin, Farmavet, São Paulo, Brazil) and insertion of an intravaginal progesterone releasing device (1.9 g progesterone, CIDR, Pfizer, Hamilton, New Zealand) on Day 0. On Day 4.5, the superstimulatory treatments (70 mg pFSH; Folltropin-V, Bioniche Animal Health; Belleville, ON, Canada) were initiated and given in decreasing doses of 28, 21, 14, and 7 mg twice a day, over a 4-day period. The FSH-eCG Group had the last two doses of pFSH replaced by two doses of 150 IU eCG (Folligon, Bioniche). At the time of the fifth and sixth injections of FSH, 25 mg dinoprost tromethamine (Lutalyse, Pfizer, Paulinia, Brazil) was injected IM. The CIDR was removed at the time of the seventh superstimulatory injection. Ovulation was induced with an IM injection of 0.05 mg GnRH (gonadorelin acetate; Gestran Plus; ARSA S.R.L., Buenos Aires, Argentina) 12 h after the last superstimulatory injection. All heifers were artificially inseminated with frozen/thawed semen from the same bull 12 and 24 h after GnRH. Seven days after the first AI, embryos/ova were recovered using a nonsurgical uterine flushing technique and classified according to IETS standards. To determine the superstimulatory (number of follicles ≥6 mm 12 h prior to GnRH) and SOV (number of ovulated follicles 48 h after GnRH, confirmed by CL number at the time of embryo collection) responses, transrectal ultrasonography was performed. Data were analyzed by paired t test and are presented as mean ± SEM. There was no difference between FSH and FSH-eCG groups regarding superstimulatory (23.2 ± 1.9 v. 22.3 ± 1.6 follicles ≥6 mm, P = 0.56) or SOV (15.2 ± 1.1 v. 17.5 ± 1.4 CL, P = 0.21) responses. Treatments were also similar for number of total embryos/ova (9.6 ± 0.9 v. 9.5 ± 1.0, P = 0.91), viable embryos (4.9 ± 0.7 v. 3.7 ± 0.5, P = 0.17), or degenerate embryos (3.0 ± 0.6 v. 4.3 ± 0.7, P = 0.10) recovered. Contrasting with the results using Nelore cows, the present study did not observe improvement on embryo production by replacing of the last two doses of pFSH by eCG in superstimulated heifers.

Financial support from CNPq, FAPESP, EMBRAPA and Pfizer of Brazil.
303 EFFECT OF THE PRESENCE OF EXOGENOUS DNA AND RECOMBINASE-A PROTEIN ON THE BOAR SPERM FUNCTIONALITY
Authors: Gadea J. , Gutierrez-Adan A. , Garcia-Vazquez F. A.
Published: December 2008

Since 1989, a new method for the production of transgenic animals has been available, namely sperm-mediated gene transfer based on the intrinsic ability of sperm cells to bind exogenous DNA molecules and to transfer them into the oocyte. It has been reported that coating exogenous DNA with recombinase-A protein (RecA) may improve the efficiency of transgenic embryo production (Garcia-Vazquez FA et al. 2006 Reprod. Domest. Anim. 41, 338). The objective of this study was to investigate the effect of the presence of DNA and DNA-RecA complex on the sperm functionality. Spermatozoa were incubated with linealized plasmid 5.7 kb enhanced green fluorescent protein (DNA) or RecA-DNA complexes (40:1 or 40:3 protein:DNA w/w) in a relation of 108 cells mL–1 and 5 to 15 μg of DNA mL–1. Sperm viability, membrane lipid disorder, reactive oxygen species (ROS) generation, and motion parameters were evaluated by flow cytometry and computer-assisted semen analysis (CASA) for control (no DNA), DNA (5 μg), RecA 5 (5 μg), and RecA 15 (15 μg). A total of 5 replicates were used. Data were analyzed by ANOVA. The sperm viability measured by propidium iodide staining was significantly reduced by the presence of RecA DNA complexes (viable cells 77.5a v. 69.4ab v. 55.6b v. 35.8c%; P < 0.01). Lipid membrane disorder (measured by merocyanine 540 and Yopro 1 staining) were significantly reduced in the group with high exogenous DNA concentration (RecA 15 group), with a reduction of percentage of viable cells and low lipid disorder (67.4a v. 60.0a v. 51.5a v. 24.3b; P < 0.01). However, the ROS generation (measured by 2′,5′-dichlorofluorescein diacetate) was not different in the different experimental groups (fluorescent units 20.3 v. 25.5 v. 26.2 v. 13.9; P = 0.36). The motion parameters measured by CASA were affected by the presence of DNA-RecA complexes with a significant reduction in percentage of motile and progressive motile cells in the group RecA 15 (% progressive motility 46.7a v. 43.9a v. 46.8a v. 20.3b; P < 0.01). Also, it was detected a change in the pattern of motility with an increase in the sperm velocities (velocity average path, velocity straight line, and VCL) in groups DNA and RecA 5 and a reduction of these values when the concentration of DNA was 15 μg mL–1 (VCL μm s–1 97.5a v. 106.4b v. 109.1b v. 58.1c; P < 0.01). The motions parameters (LIN, STR, WOB, ALH, BCF) showed significant lower values for the group RecA 15. These results shown that the presence of DNA and RecA-DNA complex modified the pattern of motility and reduced the viability and functionality of the boar sperm in a concentration dose way. It is necessary to select an adequate DNA and RecA concentration that would protect the sperm functionality and optimize the binding process.

Supported by BIOCARM 10BIO2005/01-6463 and AGL2006-03495.
304 PRODUCTION OF TRANSGENIC CHICKENS EXPRESSING HUMAN ERYTHROPOIETIN IN A TETRACYCLINE-INDUCIBLE MANNER
Authors: Koo B. , Kwon M. , Roh J. , Lee H. , Kim N. , Kim T.
Published: December 2008

The use of livestock animals as bioreactors to address the growing demand for large quantities and increasing numbers of biopharmaceuticals is of prime strategic relevance to agricultural improvement and medical advancement. We report here the production of transgenic chickens that produce human erythropoietin (hEPO) using replication-defective Moloney murine leukemia virus-based vectors. Because it is well known that constitutive overexpression of some cytokine genes in the transgenic animals may cause serious physiological disturbances, the vectors were designed to express in the presence of tetracycline. In addition, we introduced woodchuck hepatitis virus posttranscriptional regulatory element sequence at 3′ end of hEPO gene to boost the gene expression under the inducible condition. Approximately 5 μL of vector virus solution concentrated as much as 109 cfu mL–1 was injected beneath the blastoderm of non-incubated chicken embryo (stage X). Out of 596 injected eggs, 36 chicks hatched after 21 days of incubation and 23 hatched chicks were found to express vector-encoded hG-GSF gene when fed with doxycycline. Quantitative analysis of the blood samples taken from some Go transgenic chickens resulted in more than 300 IU mL–1 of hEPO in the blood. These transgenic chickens have not exhibited any physiological abnormalities; therefore, it is possible that this controllable gene expression system may be useful in minimizing detrimental side effects when used to produce other transgenic animals. The biological activity of the recombinant hEPO was comparable to its commercially derived Escherichia coli counterpart. The significance of this work stems from the fact that it is the first successful report on the production of transgenic chickens expressing the hEPO gene. This approach can be employed to create a useful transgenic model system for further studies on the chicken embryo development and the efficient production of transgenic chickens as bioreactors.

This work was supported by: The BioGreen 21 Program of the Rural Development Administration, Republic of Korea; The SRC/ERC program of MOST/KOSEF (grant no. R11-2002-100-04005-0); The 2006–2011 Technology Development Program for Agriculture and Forestry (TDPAF), Ministry of Agriculture and Forestry, Republic of Korea.
34 COMPARISON OF IN VIVO DEVELOPMENT DURING EARLY PREGNANCY OF CLONED FETUSES DERIVED FROM BOVINE FETAL FIBROBLASTS AT THE EARLY G1 AND G0 PHASES
Authors: Ideta A. , Hayama K. , Urakawa M. , Tsuchiya K. , Nakamura Y. , Aoyagi Y. , Saeki K.
Published: December 2008

Enhanced development of bovine somatic cell nuclear transfer (NT) embryos to full term has been achieved using fibroblasts at the early G1 (eG1) phase instead of cells at the quiescent (G0) phase (Urakawa et al. 2004 Theriogenology 62, 714–728). The high abortion rate and abnormal placental development of NT embryos using G0 phase cells is related to the low formation rate of embryonic disks and the aberrant development of the trophectoderm in utero until Day 14 of gestation (Ideta et al. 2007 Cloning Stem Cells 9, 571–580). The purpose of this study was to examine the morphological development of conceptuses such as fetuses and fetal membranes in the early pregnancy of NT embryos using eG1 phase cells (eG1-NT embryos) and G0 phase cells (G0-NT embryos). Blastocysts derived from eG1-NT and G0-NT embryos were transferred to recipient heifers, and the conceptuses at Day 50 of gestation were retrieved nonsurgically using prostaglandin F2α (PGF2α) and oxytocin (Lavoir and Betteridge 1996 J. Reprod. Fertil. 106, 95–100). In vitro-fertilized (IVF), parthenogenetic and artificially inseminated (AI) embryos were used as controls. Data were analyzed using chi-square test and Student’s t-test. Pregnancy rates at Day 30 of recipient heifers carrying eG1-NT, G0-NT, IVF, parthenogenetic, and AI embryos were similar (57 to 100%; 4/7 to 8/8). Two recipient heifers carrying parthenogenetic embryos returned to estrus between Day 30 and 50 of gestation, whereas all other pregnancies remained viable. Most fetuses at Day 50 of gestation of all experiment groups (20/24) were recovered nonsurgically by several PGF2α and oxytocin treatments. The recovery rates of normal fetuses derived from eG1-NT embryos (83%, 5/6), IVF embryos (80%, 4/5), and AI embryos (88%, 7/8) were greater than those of G0-NT embryos (33%, 2/6) and parthenogenetic embryos (0%, 0/7). The amniotic fluid volume of G0-NT embryos was significantly greater than that of AI embryos (P < 0.05). But the amniotic fluid volume of eG1-NT embryos was the same as that of AI embryos (P > 0.05). The fetal weights of eG1-NT and IVF embryos were significantly greater than the fetal weight of AI embryos (P < 0.05). Our results suggest that efficient production of cloned offspring is possible by NT using donor cells that are in the early G1 phase.