bluuebunny/crossref_metadata_2025 huggingface dataset:
151 EFFECTS OF HEXOSES SUPPLEMENTED IN THE MATURATION AND PRE IMPLANTATION MEDIUM ON THE IN VITRO DEVELOPMENT OF PORCINE OOCYTES
Authors: Wongsrikeao P. , Budiyanto A. , Ooki R. , Teniguchi M. , Otoi T.
Published: December 2004

The present study was conducted to determine the effects of hexoses on in vitro maturation, fertilization, and development of porcine oocytes. In the first experiment, porcine oocytes were matured in modified North Carolina State University (NCSU)-37 medium supplemented with 0.6 mM cysteine, 1 mM dibutyryl cyclic AMP (dbc AMP), 10 IU/mL equine chorionic gonadotrophin, 10 IU/mL human chorionic gonadotrophin, 50 μ/mL gentamycin (Sigma-Aldrich, Tokyo, Japan), 10% (v/v) porcine follicular fluid, and various hexoses (glucose, fructose, and galactose) at various concentrations of 0 (control), 2.5, 5.5, and 10 mM. They were subsequently cultured in the maturation medium without hormones and dbcAMP for an additional 22 h. Fertilization was performed according to Kikuchi et al. (2002 Biol. Reprod. 66, 1033–1041); 15 oocytes were co-incubated with 1 million frozen thawed sperm/mL in fertilization medium for 5 h. Supplementation of either glucose (2.5 or 5.5 mM) or fructose (5.5 mM) in the maturation medium significantly increased the percentages of maturation to metaphase II (68.5%, 79.4%, and 70.2%, respectively) and monospermic fertilization of oocytes (55.0%, 64.5%, and 58.9%, respectively), as compared with control group (metaphase II: 52.8%; monospermic: 42.7%; P < 0.05). Supplementation of galactose had no effect on the meiotic maturation and monospermic fertilization of oocytes. In the second experiment, presumptive zygotes were cultured in modified NCSU-37 supplemented with 4 mg/mL BSA, 0.17 mM sodium pyruvate, 2.73 mM sodium lactate, and 50 μg/mL gentamycin. The cleaved embryos were collected at Day 3 after in vitro fertilization and then cultured for a further 4 days in modified NCSU-37 medium supplemented with 5.5 mM of glucose, fructose, or galactose. The percentages of blastocyst formation, calculated from cleaved embryos, were significantly higher in the glucose and fructose groups (20.4% and 18.4%, respectively) than in the galactose group (12.9%) and the non-supplemented control group (9.2%). Although fructose supplementation did not accelerate blastocyst formation, it did significantly increase the mean cell number of blastocysts (48.0 ± 2.9 vs. 38.6 ± 1.8) and reduced the index of DNA-fragmented nuclei in the blastocysts stained by the TUNEL method (7.6 ± 0.9 vs. 11.8 ± 0.9), as compared with glucose supplementation. In the present study, although all hexoses were used through the glycolysis pathway, supplementation of galactose in the maturation and embryo culture medium had no beneficial effect on development. In contrast, both glucose and fructose enhanced development with an optimal concentration of 5.5 mM. Replacement of glucose with fructose did not accelerate blastocyst formation but did enhance embryo quality in terms of increasing cell number and decreasing the number of fragmented nuclei.
155 DEVELOPMENT OF BOVINE AGGREGATE EMBRYOS CONSTRUCTED FROM NUCLEAR TRANSFER EMBRYOS AND ELECTROFUSED IVF-DERIVED EMBRYOS
Authors: Landry A.M. , Murakami M. , Denniston R.S. , Williams J.L. , Echelard Y. , Godke R.A.
Published: December 2004

The production of animals by nuclear transfer can be hindered by placental and developmental abnormalities in the fetus. Studies in mice have indicated that tetraploid embryo complementation can be used to rescue embryos with lethal placental deficiencies and produce live offspring. The objectives of this experiment were to produce bovine electrofused embryos by blastomere fusion and to utilize those embryos for aggregation with nuclear transfer (NT) embryos. Oocytes were obtained from a commercial source (BoMed, Madison, WI, USA) and were allocated for use in either NT or in vitro fertilization (IVF). NT embryos were produced using standard procedures. Bovine transgenic fibroblast cells maintained in active culture were used as the nuclear donor. Oocytes were fertilized with frozen semen using standard IVF procedures. Zygotes were observed for cleavage and selected at the 2-cell stage for electrofusion at 28, 30, 32, and 34 h post-insemination in 0.3 M mannitol fusion buffer. Embryos were aligned using a 5 s, 7.5 V AC pulse and were fused using a 1.4 kV cm−1 100 μs DC pulse. Treated embryos were observed after ∼1 h for fusion of cell membranes between the two blastomeres and were returned to culture (IVF-Fused). Good quality 8-cell embryos produced from the NT and IVF-Fused groups were selected for aggregation at 72 h post-insemination. Aggregate embryos were constructed by removing 3–4 blastomeres from an 8-cell NT embryo. The zona pellucida of an 8-cell IVF-Fused embryo was then removed by placing the embryo in a 0.25% pronase solution for approximately 1 min. After the zona pellucida was removed, 3–4 blastomeres were aspirated from the IVF-Fused embryo and were injected into the NT embryo using a glass pipette. Embryos were returned to culture in CR1aa media and were examined at 168 and 192 h post-insemination for blastocyst (BLST) development (Table 1). This study demonstrates that NT/IVF-Fused aggregate embryos can be constructed and develop at the same rate as controls. An attempt was made to determine the nuclear status of the electrofused embryos but technique limitations did not permit differentiation between tetraploid and multinucleate cells. Further research is needed to determine nuclear status of IVF-Fused embryos and the allocation of NT and IVF-Fused cell lineages within the developing embryo.

Table 1.
Blastocyst rates of NT/IVF-Fused aggregate embryos constructed at the 8-cell stage
166 EFFECT OF TREATMENT WITH hCG OR GnRH AT THE TIME OF EMBRYO TRANSFER ON PREGNANCY RATES IN COWS SYNCHRONIZED WITH PROGESTERONE VAGINAL DEVICES, ESTRADIOL BENZOATE, AND eCG
Authors: Tribulo R. , Balla E. , Cutaia L. , Bo G.A. , Baruselli P.S. , Peres L.
Published: December 2004

Although several studies have investigated the relationship between circulating progesterone and pregnancy rates in cattle, the beneficial effect of treatments that increase progesterone concentrations, by insertion of a progesterone (P4) releasing device or induction of an accessory CL with hCG, GnRH, or LH treatment, has resulted in inconsistent effects on pregnancy rates in embryo recipients. An experiment was designed to evaluate the effect of hCG or GnRH treatment, given at the time of embryo transfer without estrus detection, on pregnancy rates in recipients treated with intrauterine P4-releasing devices, estradiol benzoate (EB), and eCG. The experiment was performed in two replicates; non-lactating Bos taurus × Bos indicus crossbred beef cows with a body condition score between 2.5 to 3.5 (1-to-5 scale) were used (replicate 1, n = 180; replicate 2, n = 140). All cows received 1 g of P4 via a P4-releasing device (DIB, Syntex, Argentina) and 2 mg EB i.m. (Syntex) on Day 0, and 400 IU of eCG i.m. (Novormon 5000, Syntex) plus 150 μg d(+)cloprostenol i.m. (Ciclase, Syntex) on Day 5. DIBs were removed on Day 8 and all cows received 1 mg EB i.m. on Day 9. Recipients were not observed for signs of estrus, and those >1 CL, or a single CL with an area >256 mm2, received 195 Grade 1 and 46 Grade 2 frozen/thawed “direct transfer” embryos on Day 17. At the time of embryo transfer, recipients were randomly allocated to 1 of 3 treatment groups to receive 1500 IU hCG (Ovusyn, Syntex), 50 μg Lecirelina (GnRH, Gonasyn, Syntex), or no treatment (control) at that time. Ovarian ultrasonography was performed on Day 0 to determine ovarian status (only cows with a CL or a follicle >10 mm and uterine tone were used), on Day 17 to measure CL area, and 40 days after embryo transfer to determine pregnancy status. Data were analyzed by logistic regression and the effects of replication, technician, treatment, and embryo quality were considered in the model. From the 320 recipients treated with a DIB plus EB and eCG, 241 (75.3%) were selected to receive an embryo. Nine (3.7%) and 1 (0.4%) of the selected recipients had 2 and 3 CL, respectively. Pregnancy rates did not differ between replicates (replicate 1: 80/140, 57.1%; and replicate 2: 57/101, 56.4%; P = 0.84), technicians (technician 1: 65/118, 55.1%; and technician 2: 72/123, 58.5%; P = 0.64), or treatments (hCG: 43/80, 53.8%; GnRH: 45/83, 54.2%; and control: 49/78, 62.8% P = 0.99). However, pregnancy rates were higher (P = 0.001) in recipients receiving Grade 1 embryos (121/195, 62.1%) than in those receiving Grade 2 embryos (16/46, 34.8%). GnRH or hCG treatment at the time of embryo transfer did not increase pregnancy rates in recipients synchronized with P4 releasing devices, EB, and eCG.
Research was supported by Syntex S.A., Estancia El Mangrullo S.A., and Agencia Cordoba Ciencia S.E.
170 ASSESSING THE POTENTIAL OF STEM CELLS TO GENERATE CHIMERIC RATS
Authors: Guo J. , Fida S. , Gou K. , Zhang C. , Morrison J. , Du Z.
Published: December 2004

Embryonic stem (ES) cells are pluripotent cells derived from inner cell masses (ICMs) of blastocysts. The capacity of pluripotency in differentiation is assumed to contribute to embryonic development to form a chimeric individual when these cells are reintroduced into embryos. Chimeric mice can be routinely generated by aggregation of ES cells with morulae or injection into blastocysts, which are then implanted in pseudopregnant foster mothers. Furthermore, recent studies have demonstrated that bone marrow-derived stem cells and neural stem cells can integrate into the embryonic development in mouse (Geiger et al. 1998 Cell 93, 1055–1065; Clarke et al. 2000 Science 288, 1660–1663). We therefore tried to assess the ability of rat ICMs and neural stem cells to form chimeras by injecting these cells into rat blastocysts. Forty-two rat ICMs from Day 6 blastocysts of Dark Agouti (DA) inbred rat were injected into Day 5 blastocysts of Sprague-Dawleyd (SD) outbred rats; 14 pups were born following embryo transfer of these blastocysts injected into Hooded Wistar (HW) recipients. One male of the 14 pups was coat color-patched and displayed germline transmission. Following embryo transfer of 22 SD blastocysts injected by Day 5 DA ICMs, 7 pups were born and 2 of them were coat color-patched. Nine pups were obtained from 23 DA blastocysts injected by Day 5 SD ICMs; 4 of them were coat color-patched. The ICM cells were isolated and cultured for 6 days. No chimeras were generated by injection of the cultured ICM cells, as assessed by coat color patching. These results suggest that rat embryonic ICMs have potential to develop into chimeras, but the chimeric potential of ICMs was rapidly lost in our culture system. Investigation of potential chimeric development of rat fetal neural stem (rFNS) cells transfected with Lac Z was carried out. Staining was observed in tissues from 2 of 41 E14 fetuses. These results demonstrated that rFNS cells can integrate into the early embryonic environment although the ability of these cells to contribute to chimeric formation was marginal. No coat color chimerism was observed in any of the 88 pups generated from the LacZ-rFNS cell experiments.
181 Oct-4: A POTENTIAL MARKER FOR PLURIPOTENCY IN CATTLE
Authors: Vejlsted M. , Avery B. , Gjoerret J. , Schmidt M. , Greve T. , Maddox-Hyttel P.
Published: December 2004

The POU (Pit-Oct-Unc) domain transcription factor Oct-4 is one of the most acknowledged markers for pluripotency in murine and primate embryonic cells. At the blastocyst stage, expression of Oct-4 has been shown to remain high in the inner cell mass (ICM) while being rapidly down regulated in the trophectoderm (TE). Furthermore, in these species, expression of Oct-4 is maintained in pluripotent derivatives of the ICM and in embryonic stem (ES) cells, but lost upon differentiation. In the bovine embryo, a marker with similar qualities has long been sought. The aim of this study was to investigate, using a commercially available antibody for immunohistochemistry (IHC), whether Oct-4 might serve this role. In vitro produced (IVP) embryos were transferred to synchronized recipients at Day 6 post insemination (p.i) and flushed at Day 12; in vivo-derived embryos were flushed at Day 14. Day 8 IVP embryos (n = 20) were fixed and processed for IHC in paraffin sections together with Day 12 (n = 18) and Day 14 (n = 3) embryos. From Day 8 IVP embryos, outgrowth colonies (OCs) were formed by intact blastocyst culture on mouse SNL feeder cells. OCs were photographed using a stereomicroscope on Days 12, 14, and 16 p.i., and were examined for Oct-4 expression by in situ IHC at Day 16 p.i. (n = 94). From isolated embryonic discs of Day 12 embryos, OCs were derived by similar culture and were either processed for IHC on paraffin sections at Days 16, 18, and 20 p.i. (n = 9) or used for establishment of ES-like cell lines. Of colonies formed, representative specimens from each of the initial 5 passages (n = 18) were examined for Oct-4 expression either in paraffin sections or in situ. In Day 8 embryos, Oct-4 expression was demonstrated in all nuclei of both ICM and TE cells except for presumptive apoptotic ones. Approximately one-fifth of the OCs presented a substantial amount of Oct-4 positive cells of putative ICM, but also of TE origin. Apparently, the formation of Oct-4 positive OCs was favored by initial attachment of the embryonic pole to the feeder cells. In Day 12 and 14 embryos, specific and exclusive Oct-4 staining of nuclei of the complete epiblast, but not the hypoblast and the TE, was revealed. All OCs derived from Day 12 embryonic discs showed specific staining for Oct-4 in nuclei of putative epiblast origin only. On subsequent culture of these isolated epiblast derivatives, loss of Oct-4 staining from colonies was observed by passage 3. This study has, for the first time, shown expression of Oct-4 to be limited to pluripotent cells of bovine Day 12 and 14 embryos. Compared with murine and primate embryos, down-regulation of Oct-4 expression in bovine TE cells appears to be delayed. Findings indicate that Oct-4 may be used as a marker for pluripotency in bovine ES-like cells, although TE derivatives may maintain Oct-4 expression when isolated from Day 8 embryos.
185 A NOVEL AND EFFECTIVE PROCEDURE FOR REMOVING HIV-1 AND HEPATITIS B AND C VIRUSES FROM SPIKED HUMAN SEMEN
Authors: Loskutoff N.M. , Huyser C. , Singh R. , Morfeld K.A. , Walker D.L. , Thornhill A.R. , Smith M. , Morris L. , Webber L.
Published: December 2004

The objectives of this study were to determine the effectiveness of a novel, trypsinized density gradient treatment designed to remove viruses from semen and to evaluate sperm viability after treatment. Exp. 1: Cryopreserved human semen (n = 6 donors) was layered on 2-mL columns of 45% Isolate (Irvine Scientific, Santa Ana, CA, USA) with or without 0.25% trypsin (trypsin-exposed and control, respectively), which overlaid 2-mL columns of 90% Isolate with or without 10 μg/mL soy-based trypsin inactivator (Sigma, St. Louis, MO, USA) and centrifuged (700g for 30 min). The layering of multiple density gradients is facilitated by a novel polypropylene tube insert, which also prevents contamination when extracting treated sperm (USA and international patents pending). Pellets were washed and then incubated at room temperature. Sperm were examined (motility and supravital staining) at 0, 2, 24, and 48 h post-treatment and the results evaluated using Wilcoxon Signed Rank and Rank Sum tests. Exp. 2: A cytopathic cell (MT-2) assay was conducted (6 replicates) to determine the effect of trypsin (1-min exposure) on HIV-1 RNA infectivity. Viral replication was assessed by syncytium formation and p24 antigen production. Exp. 3: Two pools of fresh human semen (N1 = 3 and N2 = 8 donors) were inoculated (1:1) with 1 × 108 copies/mL of cultured HIV-1 RNA, and one pool (N2) was inoculated (1:1) with plasma collected from patients infected with either Hepatitis B DNA (HBV) or C (HCV) RNA viruses; spiked and non-spiked aliquots were processed as in Exp. 1. Treated sperm pellets were analyzed for HIV-1 or HBV and HCV concentrations by the Bayer Versant branched DNA (bDNA; version 3.0) and/or the Roche Amplicor quantitative RT-PCR (1.5 ultrasensitive) assays at Toga Laboratories (Pty), Ltd. (Edenvale, South Africa). As a result of Exp. 1, there was significantly (P < 0.05) lower motility (but not supravital staining) between trypsin-treated and control sperm at 0 h (58.0 vs. 69.3%) and 2 h (54.7 vs. 62.9%) post-washing; however, no differences were noted after 24 h (P > 0.05). In Exp. 2, trypsin exposure affected HIV-1 RNA infectivity negatively as compared to controls in terms of MT-2 cell syncytium formation and p24 antigen production. Results of the bDNA and/or RT-PCR assays in Exp. 3 indicated that the procedure effectively reduced HIV-1, HBV, and HCV viral copies in the spiked semen samples to undetectable levels or levels below clinical relevance. In conclusion, the novel trypsin density gradient procedure was effective for removing HIV-1, HBV and HCV from spiked semen without markedly affecting sperm survival. Extrapolation of these results to natural infections may be unfounded for viruses (e.g. HBV) that are thought to integrate into sperm chromatin.
2 CORRELATION BETWEEN OXYGEN RESPIRATION RATES AND MORPHOLOGY, SEX, DIAMETER AND DEVELOPMENTAL STAGE OF SINGLE BOVINE IVP-EMBRYOS
Authors: Lopes A.S. , Ramsing N. , Larsen L.H. , Räty M. , Peippo J. , Greve T. , Callesen H.
Published: December 2004

A simple, non-invasive, rapid and sensitive oxygen microsensor system was developed to investigate correlations between oxygen respiration rates of individual bovine embryos and their morphology, sex, diameter and developmental stage. Bovine IVP-embryos (n = 78; Holm et al. Theriogenology 52, 683–700) were analysed around the 8-cell stage (Day 3; n = 18) and at various blastocyst stages (Day 7; n = 60). Each embryo was morphologically evaluated, its outer diameter measured and was then loaded into a glass tube (i.d. 0.68 mm, length 3 mm). After 1 h, oxygen concentration gradients generated by the embryo’s respiration were measured over app. 8 min with an oxygen microelectrode (www.unisense.com). Five embryos were measured in one round together with an empty tube as control. The procedure was repeated twice for each embryo with app. 1 h interval. Individual respiration rates in nL O2/embryo/h (nL/h) were calculated from these gradients. The measurements were performed at 38.5°C under constant flow of humidified 5% CO2 in air (app. 19% O2). After this, 64 embryos (14 Day 3; 50 Day 7) were lysed for sex diagnosis by PCR. Values are given as mean ± SD. The sensitivity of the oxygen measurement system was high (controls: 0.034 ± 0.035 nL/h, n = 15) and its repeatability from 1st to 2nd measurement was 99.7 ± 9.8% (n = 71). The average embryo respiration rate was 0.39 ± 0.05 nLl/h on Day 3 (n = 18) and 1.31 ± 0.52 nLl/h on Day 7 (n = 60). For Day 7 embryos, the respiration rates varied according to their morphological quality, being 1.87 ± 0.46a (n = 18), 1.17 ± 0.32b (n = 23), 0.95 ± 0.27b,c (n = 14) and 0.72 ± 0.24c (n = 4) nL/h for quality 1, 2, 3, and 4 embryos, respectively (Proc Mixed,a,b,c: P < 0.05; values with different superscripts differ significantly). The sex ratio (male:female) was 9:5 (Day 3) and 32:18 (Day 7), and on Day 7 this ratio varied between qualities: 11:2, 12:8, 8:4, and 1:3 for quality 1, 2, 3, and 4, respectively. The average respiration rate on day 3 was the same for males and females, as it was on day 7 (1.22 ± 0.43 nL/h (females) and 1.31 ± 0.58 nL/h (males), P > 0.05). There was a correlation between embryo diameter and respiration rate (r2 = 0.65, n = 74), which was even stronger for Day 7 male embryos (r2 = 0.72, n = 32). In conclusion, a highly reliable, repeatable and sensitive system was established for measuring respiration rates in single bovine embryos, even at early developmental stages. The respiration rate was lower on day 3 compared to Day 7 embryos, and it was correlated with the morphological embryo quality on Day 7. Oxygen consumption could be a valuable supplementary indicator of embryo viability, especially in difficult evaluations (e.g. quality 2 and 3 after IVP). It remains to be demonstrated if such measurements can also reveal quality differences already at Day 3, which would be of interest in, e.g. the human field.
ASL is supported by FCT, Portugal.
209 NORGESTOMET IMPLANTS REDUCE LH RELEASE PATTERN IN ZEBU COWS UNDERGOING REPEATED OOCYTE PICK-UP
Authors: Viana J.H.M. , Camargo L.S.A. , Ferreira A.M. , Sa W.F. , Fernandes C.A.C. , Marques Junior A.P.
Published: December 2004

Ultrasound-guided follicular puncture (OPU) has become the most used technique to recover cumulus-oocyte complexes (COCs) from valuable donors for in vitro embryo production, because of the low risk and the possibility of collecting COCs at intervals as short as twice-a-week. However, repeated aspiration of ovarian follicles may induce endocrine abnormalities due to partial luteinization of punctured follicles and interference with follicular development. The use of exogenous progestagens is an alternative used to control these side effects, and is under evaluation. The aim of this study was to evaluate whether the effect of norgestomet treatment on intra-follicular and systemic steroid concentrations and on ovarian follicular dynamics is related to changes in LH releasing pattern. Pluriparous non-lactating Gir breed (Bos indicus) cows (n = 10) were randomly distributed between treatment (norgestomet ear implants, replaced weekly) and control (no hormone used) groups, and had their ovarian follicles larger than 3 mm in diameter aspirated twice a week, during the next two consecutive weeks. Follicular dynamics were evaluated every 12 h between OPU sessions, and the largest follicles present were used to recover samples of follicular fluid. Blood samples were collected daily for progesterone evaluation in all cows, and 3 times within a 4-h window interval, at 24, 48, 72, and 96 h after follicle puncture for LH evaluation, in 6 cows (3 from treated and 3 from control groups). LH was measured by a standardized RIA procedure. Data was analyzed by ANOVA, and means were compared by Tukey's test. Results are presented as means ± SEM. Treatment with norgestomet reduced mean progesterone plasma concentration during the evaluated period (36.3 ± 14.0 vs. 250.3 ± 49.3 pg/mL; P < 0.0001), the incidence of follicles growing above 9 mm (30% vs. 65%; P < 0.05) in the intervals between OPU sessions, and intrafollicular estradiol and progesterone concentrations in the largest follicles (n = 27) present (265.5 ± 47.4 and 34.9 ± 5.4 ng/mL vs. 765.2 ± 169.1 and 173.3 ± 43.4 ng/mL, respectively; P < 0.05). Plasma LH concentrations were consistently lower during the 3 session intervals in cows treated with norgestomet (0.16 ± 0.04, 0.22 ± 0.03, 0.22 ± 0.09 and 0.17 ± 0.01 vs. 0.44 ± 0.15, 0.53 ± 0.04, 0.42 ± 0.05 and 0.39 ± 0.11 for 24, 48, 72, and 96 h after OPU, respectively; P < 0.05). These results confirm the theory that norgestomet treatment is associated with a reduction in the LH-release pattern, as expected due to the reduction in both luteinization of punctured follicles and in the steroidogenic activity of growing follicles observed during the experiment. The use of norgestomet ear implants can be an alternative in the management of donor cows undergoing oocyte pickup.
This work was sponsored by FAPEMIG and CNPq. The authors thank Dr. Robert Douglas for aiding with LH RIA.
210 COMPARISON OF REAL-TIME PCR AND END-POINT PCR FOR ANALYSIS OF GENE EXPRESSION IN PREIMPLANTATION EMBRYOS
Authors: Baji Gal A. , Carnwath J.W. , Dinnyes A. , Herrmann D. , Wrenzycki C. , Niemann H.
Published: December 2004

The purpose of this study was to compare real-time PCR and end-point PCR with respect to their suitability for the analysis of gene expression in samples in which the number of cells is limited, for example, in studies of pre-implantation embryonic development. The real-time instrument was a LightCycler® from Roche Diagnostics (Budaors, Hungary) which is a capillary-based PCR system. Primers for histone H2A (housekeeping gene) were used for all PCR reactions. The end-point PCR system included an MJ Research PT-100 thermocycler, agarose gel electrophoresis, ethidium bromide staining, and image acquisition with a 12-bit CCD camera and densitometry. The sensitivity, dynamic range, and standard error of both PCR systems were compared using a single stock solution of cDNA. The more precise real-time PCR system was then used to determine the precision of a protocol for reverse transcription and the precision of a complete gene expression protocol including mRNA purification and reverse transcription. The real-time system was 100 times more sensitive than the end-point system and had a dynamic range of more than four orders of magnitude. The linear range for end-point PCR was extended for two orders of magnitude using a fixed end-point of 31 cycles. The standard error of the mean based on 30 replicates was 0.14% for the real-time system and 6.8% for the end-point system. The standard deviations for reverse transcription combined with real-time analysis and for the complete gene expression protocol were 0.6% and 1.4%, respectively. The t standard deviation was 1.8% for expression analysis of 6 bovine oocytes. In conclusion, real-time PCR system has advantages in sensitivity, dynamic range, and precision of measurement. New research areas which involve subtle changes in expression reprogramming or the analysis of low copy number transcripts (even from single cells and embryos) clearly benefit from the advent of real-time PCR analysis. However, when genes with high transcription levels are analyzed, the amount of cDNA taken from the reverse transcription reaction can be adjusted to lie within the operating range of end-point PCR. Pooling embryos is a valuable approach for both methods when the goal is to determine the behavior of the average embryo rather than variation between embryos. In many cases, the magnitude of biologically significant expression changes is so great that the higher levels of precision afforded by real-time PCR are not essential for the analysis.
This work was funded by the Bilateral Scientific and Technological Collaboration Agreement (TET) between Hungary and Germany (TET D-6/01) and by the National Office of Research and Technology (NKTH) (BIO-00017/2002).
214 DIFFERENT GENE EXPRESSION OF INDIVIDUAL BLASTOMERES IN EARLY MOUSE EMBRYO DETECTED BY REAL TIME PCR
Authors: Bodo Sz. , Baji Gal A. , Boonkusol D. , Gorhony B. , Balogh E. , Dinnyes A.
Published: December 2004

The objective of this study was to detect gene expression differences among blastomeres from 4- and 8-cell stage mouse embryos by a sensitive method. In earlier studies gene expression was determined in pooled or single embryos. With the higher levels of precision afforded by our method, we could quantify mRNA in single blastomeres. For comparison of gene expression, different inner cell mass specific (Oct-4, Nanog) and trophectoderm-specific (Glut-3) genes and the housekeeping beta-actin gene (for reference) were chosen. Late 4- and early 8-cell stage embryos were obtained from oviducts 58 and 66 h post-hCG, respectively. The embryos were collected in CZB-HEPES and the zonae pellucidae were dissolved by acid Tyrode solution. Blastomeres were separated mechanically from each other by gentle pipetting with a fire-polished glass capillary. Messenger RNA was isolated from the frozen blastomeres using the Dynabeads® mRNA DIRECTTM Micro Kit (Kvalitex Kft., 1136 Budapest, Hungary) following the manufacturer's protocol. The reverse transcription reactions were performed at 25°C for 10 min, and then at 42°C for 1 h followed by 99°C for 5 min. Real-time PCR was performed in an iCycler iQ Real-Time PCR Detection System (Bio-Rad Laboratories, 1094 Budapest, Hungary). Reaction mixtures, which were separated for each gene, consisted of QuantiTect iQ SYBR Green Supermix (Bio-Rad Laboratories), 500 nm of both primers, and a 1/8 aliquot of the blastomere cDNA. The cDNA template was denatured by heating to 95°C for 2 min and amplified by 50 cycles of 95°C for 20 s, 60°C for 20 s, and 72°C for 20 s, with a single fluorescence measurement at each cycle. When the reaction was finished, melting curves were plotted to confirm product purity. By this procedure we could measure the expression level of four genes in six 4- and 8-cell-stage embryos. Glut-3 expression was not detectable in the blastomeres of 4-cell stage embryos. In spite of the 1.3–1.5-fold technical error, Oct-4 and Nanog showed 10-fold and 9-fold differences, respectively. Among the blastomeres of the same embryo, one of the 4-cell-stage showed notably high expression of Oct-4. In the 8-cell stage blastomeres Oct-4 expression showed slight differences (2–8-fold), and Nanog and Glut-3 showed 28-fold and 12-fold differences, respectively. Two of the 8-cell blastomeres showed notably high expression of Nanog. In conclusion, this method was accurate and sensitive enough to measure the expression of different genes in a single blastomere. We found that the mRNA levels of Oct-4 and Nanog have remarkably discrete differences within the blastomeres of a 4-cell-stage embryo. Expression of Nanog gene showed notable differences within blastomeres of an 8-cell-stage embryo. These differences may show a very early sign of the cell fate determination.
This work was funded by the Wellcome Trust International Senior Fellowship and Bio-Rad Laboratories.
23 PREGNANCY RATE IN NELORE COWS AFTER TEMPORARY CALF REMOVAL, AND USE OF HORMONAL PROTOCOLS WITH eCG
Authors: Pinheiro V. , Souza A. , Ereno R. , Barros C.
Published: December 2004

Both temporary calf removal (TCR) and administration of eCG are potentially useful to improve pregnancy rates in animals treated with fixed-time artificial insemination (FTAI) protocols. In the present work, two experiments were performed to compare the efficiency of eCG and/or TCR in FTAI protocols, with or without exogenous progesterone. In experiment 1, lactating Nelore cows (40 to 70 days post-partum, n = 220) were allocated to two Groups. At a random stage of the estrous cycle (Day 0), animals from Group GPE (GnRH/PGF2α/Estradiol)/eCG were treated with GnRH (50 mg lecireline, i.m.; Gestran Plus®; Tecnopec, Sao Paulo, Brazil). Seven days later (Day 7) they received PGF2α (150 mg D-cloprostenol, i.m.; Prolise®, Tecnopec) and eCG (300 UI, i.m.; Novormon®, Syntex). On Day 8 estradiol benzoate (EB, 1 mg, Estrogin®, Tecnopec) was administered, and 30–36 h later the animals were inseminated (FTAI). In Group DIB (bovine intravaginal device)/eCG an intravaginal progesterone-releasing device (1.0 g, DIB®, Syntex, Buenos Aires, Agrentina; Day 0) was inserted into the vagina of cows and EB (2.5 mg, i.m.) was given in parallel. Eight days later (Day 8), eGC (300 UI, i.m.) and D-cloprostenol (150 mg) were administered. Then the DIB was removed. Twenty-four hour after DIB removal, cows were treated with EB (1.0 mg, i.m.), and 30–36 h later the animals were inseminated (FTAI). Ultrasonographic evaluation of ovaries was performed in all experiments 10 days before and at the beginning of the treatments or TCR, in order to detect the presence of CL. Cows from Group DIB/eCG showed higher pregnancy rates than those from Group GPE/eCG (58%, 72/124 vs. 39.5%, 38/96, respectively, P < 0.01). Furthermore, only in Group GPE/eCG were pregnancy rates higher in animals with CL (47.6%, 20/42) when compared to those without CL (33%, 18/54, P < 0.05). In a second experiment, a possibly beneficial effect of TCR on GPE/eCG protocol was tested in lactating Nelore cows (40 to 70 days postpartum, n = 140). Animals of Group GPE/eCG (control) were treated as described above, whereas calves were removed for 48 h from cows in Group RTB/GPE/eCG prior to hormonal treatments. Lactating Nelore cows having their calves removed showed a significant increase in pregnancy rates compared to those without TCR (51.2%, 34/66 v. 28.4%, 21/74, respectively, P < 0.01), in both situations: animals with CL (54.8%, 17/31 v. 33.3%, 11/33, respectively, RTB/GPE/eCG v. GPE/eCG) or without CL (48.5%, 17/35 v. 24.3%, 10/41, respectively, RTB/GPE/eCG v. GPE/eCG). In conclusion, these results indicate that addition of eCG to the GPE protocol was not efficient enough to produce comparable results to those obtained with DIB/eCG protocol. However, calf removal before the GPE/eCG treatment increased pregnancy rates in cycling or anestrous (without CL) lactating Nelore cows.
24 TRANSGENESIS AND NUCLEAR TRANSFER USING STEM CELLS FROM CULTURED PORCINE PRIMORDIAL GERM CELLS
Authors: Ahn K.S. , Yang H.S. , Heo S.Y. , Shim H.
Published: December 2004

Embryonic germ (EG) cells are undifferentiated stem cells isolated from cultured primordial germ cells (PGC). These cells share many characteristics with embryonic stem cells including their morphology and pluripotency. Undifferentiated porcine EG cell lines demonstrating capacities of both in vitro and in vivo differentiation have been established (Shim H et al. 1997 Biol. Reprod. 57, 1089–1095). Since EG cells can be cultured indefinitely in an undifferentiated state, whereas somatic cells in primary culture are often unstable and have limited lifespan, EG cells may provide an inexhaustible source of karyoplasts in nuclear transfer (NT). This would be particularly advantageous in maintaining nuclear donor cells carrying a transgene. In addition, genome-wide demethylation of DNA occurs in pre-implantation embryos as well as PGC. Nuclear transfer embryos using EG cells rather than somatic cells may be close to embryos from normal fertilization in their DNA methylation status. If combined with NT technique, EG cells may potentially be useful for genetic manipulation in pigs. In this study the efficiencies of transgenesis and NT using porcine fetal fibroblast and EG cells were compared. Two different techniques were used to perform NT. When conventional NT procedure (Roslin method) involving fusion of donor cells with enucleated oocytes was used, the rates of development to the blastocyst stage were 16.8% (59/351) and 14.1% (50/354) in EG and somatic cell NT, respectively. In piezo-driven micromanipulation (Honolulu method) involving direct injection of donor nuclei into enucleated oocytes, the rates of blastocyst formation in EG and somatic cell NT were 11.9% (15/126) and 7.5% (12/160), respectively. Although the differences between EG and somatic cell NT were statistically insignificant, the rates of blastocyst development in EG cell NT were comparable to the somatic cell counterpart regardless of NT methods used in the present study. To investigate if EG cells can be used for transgenesis in pigs, GFP gene was introduced into porcine EG cells. Nuclear transfer embryos using transfected EG cells gave rise to blastocysts (29/137, 21.2%), and all embryos that developed to the blastocyst stage expressed GFP, based on observation under fluorescence microscope. In this study, the possibility of using EG cells as karyoplast donors in NT procedure was tested. The results suggest that EG cell NT may be used as an alternative to somatic cell NT, and transgenic pig embryos may be produced using EG cells.
This research was supported by a grant (SC14033) from Stem Cell Research Center of the 21st Century Frontier Research Program funded by the Ministry of Science and Technology, Republic of Korea.
29 COMPARISON OF TERM PLACENTAS IN CLONED AND CONTROL PREGNANCIES IN CATTLE
Authors: Batchelder C.A. , Bertolini M. , Hoffert K.A. , Mason J.B. , Moyer A.L. , Petkov S.G. , Anderson G.B.
Published: December 2004

Somatic cell nuclear transfer is associated with high incidence of fetal loss, late-term pregnancy complications, perinatal mortality, and abnormal placental development. Several groups have described abnormalities of early and mid-gestation cloned placentas (Hill et al. 2000 Biol. Reprod. 63, 1787–1794; Lee et al. 2004 Biol. Reprod. 70, 1–11). The objective of our study was to characterize differences in the placentas of clones and control calves at term delivery. Clones were produced from ovarian cell lines from two donors (Holstein, n = 5; Hereford, n = 2). Breed-matched controls included AI (Holstein, n = 3) and embryo transfer (Holstein, n = 3; Hereford n = 3) calves. All calves were delivered alive with no visible birth defects between Days 273 and 280 of gestation, and placentas were recovered for measurement and morphological analysis. When possible, pregnancies were delivered via caesarian section, and the entire uterus was recovered for classification of anatomical shape of placentomes. Each placentome was measured, weighed, and classified by type as (A) engulfing mushroom-like; (B) sub-engulfing mushroom-like; (C) flattened, non-engulfing; and (D) convex (adapted from Penninga and Longo 1998 Placenta 19, 187–193, for sheep). Mean number of placentomes per placenta was significantly greater in controls than clones, while total mass of placentomes in the pregnant horn was significantly greater in clones than in controls (Table 1). Total surface area of placentomes in the pregnant horn tended to be larger and more variable in clones (range: 2710–7450 cm2) than in controls (range: 3120–5030 cm2; P < 0.10). A two-fold increase was observed in cloned placentas, as compared with control placentas, in mean surface area per placentome and mass per placentome. Anatomically, cloned placentas differed from controls in the percentage of placentomes classified Type A (controls > clones) and Type C (clones > controls). Other abnormalities noted in cloned placentas included moderate to severe edema, teratomas, enlarged vessels, and large areas devoid of placentation. All clones and 2/9 controls displayed enlarged umbilical vessels. Significant placental abnormalities were observed in all cloned pregnancies.

Table 1.
Placental characteristics of term cloned and control pregnancies
296 LEUKEMIA INHIBITORY FACTOR INFLUENCES SHEEP OOCYTE PARTHENOGENETIC DEVELOPMENT DURING THE TRANSITION FROM GERMINAL VESICLE TO EARLY PRONUCLEAR STAGE
Authors: Ptak G. , Lopes F. , Loi P.
Published: December 2004

Leukemia inhibitory factor (LIF) is an indispensable cytokine for female fertility. The influence of LIF on embryo development and particularly implantation has been recently confirmed; however, the effect of this cytokine on the oocyte has not been studied. The presence of LIF in human follicular fluid implies its possible role in the acquisition of oocyte competence. Furthermore, the up-regulation of LIF by steroid hormones in sheep makes entirely feasible the hypothesis that ovulatory estradiol peak plays a role in the preparation of female gamete for fertilization. With this in mind, we studied the effect of LIF during in vitro development of sheep oocytes mimicking the physiological expression of LIF induced by the ovulatory peak of estradiol in mice. GV stage oocytes matured and chemically activated in the presence of LIF and anti-LIF antibody were cultured to the blastocyst stage in our standard media. To eliminate the effect of the putative presence of LIF in heat inactivated fetal calf serum used for oocyte maturation, aliquots of LIF were treated at 56°C for 30 min and added to the maturation medium. The proportion of embryos that reached the blastocyst stage in vitro was significantly higher (P < 0.001) for oocytes matured and activated with LIF (36/93; 39%) than for the group incubated with antibody against LIF (6/68; 9%). The significant effect of anti-LIF antibody (P < 0.001) was also observed when compared with blastocysts developed from the control group of oocytes matured without LIF addition (31/106; 29%). Although the beneficial influence of LIF treatment on embryo development demonstrated with those preliminary data was not confirmed statistically, due to low number of oocytes involved, the proportion of embryos reaching the blastocyst stage in vitro was about 10% higher for those incubated with LIF than for either those cultured without the cytokine or those, matured in the presence of heat-treated LIF (15/55; 27%); however, the rate of blastocyst development appeared very similar to that of the control group. This study revealed for the first time a role of LIF in determining oocyte competence. Further investigation to determine how LIF achieves its effects on the oocyte are ongoing in our laboratory.
This work was supported by FIRB RBNE01HPMX, COFIN 2002074357, COFIN 2003073943 002, and British Council 2004.
321 DEVELOPMENT IN VIVO AND IN VITRO OF PORCINE OOCYTES FERTILIZED BY INTRACYTOPLASMIC INJECTION OF A FREEZE - DRIED SPERM HEAD
Authors: Nakai M. , Kikuchi K. , Takizawa A. , Ozawa M. , Noguchi J. , Kaneko H. , Shino M. , Kashiwazaki N.
Published: December 2004

The present study investigated the development in vivo and in vitro of in vitro matured porcine oocytes injected with a freeze-dried (FD) boar sperm head. In mice, DNA damage was induced during the holding period after rehydration and before sperm injection (Wakayama, T. and Yanagimachi, R. 1998, Nat. Biotechnol., 16, 639–641). Here, we examined the relationship between duration of rehydration of FD sperm and in vitro development of FD sperm-injected porcine oocytes. We also assessed the in vivo developmental competence of the injected oocytes after embryo transfer. Ejaculated boar spermatozoa were suspended in Pig-FM (Suzuki, K. et al. 2002, Int. J. Androl. 25, 84–93) and sonicated for 1 min to separate sperm heads from the tails. An aliquot (100 μL) of the sperm suspension was put into a glass tube and then pre-cooled at −40°C for 6 h. Each tube was attached to a freeze-dry system (DuraDry μP, FTS Systems, Stone Ridge, NY, USA) for 12 h. The ampules were closed and stored at 4°C for more than 7 days before use. For rehydration, 100 μL of distilled water was added into the ampules. In Experiment I, we injected FD sperm heads which were kept for 0–60, 60–120, or 120–180 min after rehydration. At 1 h after the injection, the injected oocytes were stimulated with a DC pulse and cultured for 6 days. The rate of blastocyst formation and the number of cells in the blastocysts were examined. Embryos after in vitro fertilization (IVF) were evaluated as a control. As shown in Table 1, the rates of blastocyst formation were not different (by χ2 test) for duration of rehydration and the control. However, the cell numbers of FD groups were lower (P < 0.05; by Student's t-test) than that in the control. In Experiment II, oocytes injected with a single FD sperm head and stimulated were transferred to both oviducts of a total of ten recipient gilts. Two recipients were diagnosed as pregnant at Day 30 of gestation. At Day 39, one of the pregnant recipients had an abortion, and two fetuses were recovered. The other pregnancy was not maintained. The results suggest that oocytes fertilized with a single FD sperm head have competence to be implanted and to develop to the early fetal stage, and also that the duration for rehydration does not influence in vitro developmental ability in pigs.

Table 1.
Effects of the duration from rehydration of freeze-dried sperm heads to the injection of the heads into in vitro matured oocytes on in vitro development of the oocytes in pigs
35 PRODUCTION OF CLONED MINIATURE CALVES USING CYTOPLASTS FROM COWS OF STANDARD SIZE
Authors: Givens M. , Carson R. , Riddell K. , Stringfellow D. , Waldrop J. , Whitley E. , Kasinathan P. , Sullivan E.
Published: December 2004

Numerous genetically similar cattle for use in research or teaching can be produced with chromatin transfer technology (Sullivan et al. 2004 Biol. Reprod. 70, 146–153). Miniature cattle can provide advantageous biotechnological models for teaching and study of human and animal diseases. Miniature cattle are approximately one fourth the size of standard cattle and, therefore, represent a potentially less expensive, safer, and more easily managed animal model. Our limited attempts to reproduce miniature cattle via in vivo and in vitro production of embryos resulted in poor response to superovulation and logistical challenges in recovering embryos or oocytes due to the small size of the animals. Thus, the purpose of this study was to evaluate the potential of embryos derived by chromatin transfer from fibroblasts of a miniature cow and cytoplasts from cows of standard size to produce viable offspring after transfer into recipient cows of standard size. The donor of somatic cells was a heifer that weighed 7.7 kg at birth. Chromatin transfer resulted in 19% (82/428) blastocyst formation. A total of 66 cloned blastocysts (65 excellent/good quality, 1 fair quality) were transferred into 26 synchronized recipients. While ultrasound revealed 13 pregnancies prior to 67 days of gestation, only 4 pregnancies of 5 fetuses were maintained beyond 100 days. Parturition was induced with dexamethasone and prostaglandin on Day 286 of gestation. One singleton (12.3 kg) and a set of twins (10.2 and 11.1 kg) were healthy at birth and normal at 1 week of age. Two fully developed singletons, weighing 21.4 and 13.6 kg, died in utero. The latter fetus exhibited a fixed dorsolateral deviation of the neck that complicated delivery despite a caudal obstetrical presentation. No abnormalities were noted in the size or structure of any placenta. Our results indicate that healthy miniature calves can be gestated by recipient cows of standard size after transfer of embryos derived by chromatin transfer. Unfortunately, the fetal wastage, fetal anomalies, and stillbirths observed with standard sized cattle also may occur.