bluuebunny/crossref_metadata_2025 huggingface dataset:
118 TRANSFER OF IRON FROM MOTHER TO FETUS IN WATER BUFFALO: ERYTROPHAGOCYTOSIS AND UTEROFERRIN
Authors: Pereira F. , Braga F. , Kfoury Jr J.R. , Oliveira L. , Papa P. , Carvalho A.F. , Kohayagawa A. , Hamlett W. , Oloris S. , Dagli M.L. , Bazer F. , Miglino M.A.
Published: December 2004

The transplacental transport of iron by uteroferrin (Uf), and the hemophagous areas in the water buffalo placenta were analyzed to clarify the mechanism of blood extravasation in the materno-fetal interface with consequent transfer of iron to the fetus through the trophoblastic erythrophagocytosis (Murai and Yamauchi 1986 Nippon Juigaki Zasshi 48, 75–88) and in the endometrial glands (Bazer et al. 1991 Exp. Hematol. 19 910–19 915). In the water buffalo placenta this mechanism remains unclear; uteroferrin is very important in this process because it is both an iron transporter and a progesterone-induced hematopoietic growth factor. Our objective was to characterize these hemophagous areas and the endometrium of the water buffalo, focusing in the materno-fetal transfer of iron. Small pieces of the placentomes and interplacentomal region of water buffaloes (Bubalus bubalis bubalis, n = 51) in all of the gestation periods were fixed in 4% paraformoldehyde, and 2.5% glutaraldehyde in PBS, processed, and stained for light and transmission electron microscopy to characterize the hemophagous areas and endometrial glands morphologically, histochemically (Perls, acid phosphatase reaction), and immunohistochemically with rabbit anti-pig uteroferrin antibody to confirm the iron transfer. The hemophagous areas were present in the placentome from 4–10-months-pregnant placentae. The Perls reaction for ferric iron staining was negative in the placentome and positive in the endometrial glands, and the acid phosphatase reaction to detect phagocytic activity was positive in the placentome as well as in the interplacentomal region. The uteroferrin immunohistochemical reaction was positive in the trophoblast, mainly in determined regions of the materno-fetal interface and in other points deep in the placentome, and the endometrial glands showed a strong reaction in the epithelium and in the lumina. The ultrastructure of the hemophagous areas revealed ingested erythrocytes inside the epithelial cells of trophoblasts, endocytic vesicles, and caveolae. The endometrial gland epithelium is of the columnar type with microvilli and basal nuclei. The results obtained mainly by histochemistry and immunohistochemistry indicated that the hemophagous areas and endometrial glands are very important sites for iron transfer in water buffalo, and are thus involved in the regulation of fetal hematopoiesis.
This work was funded by FAPESP.
124 THE MAJOR GENE EXPRESSION PATTERNS IN ENDOMETRIAL TISSUE OF PIGS DURING EARLY GESTATION
Authors: Kim B.-K. , Chung H.J. , Ko Y.G. , Kim Y.M. , Seong H.-H. , Jung J.K. , Kim N.-H. , Chang W.K.
Published: December 2004

Although the expression of important genes in the embryo at pre-implantation stage, which encompasses the period from fertilization to implantation, have been reported for mice and cows, little information relevant to this subject is known in pigs. The objective of this study was to investigate the changes of importantly expressed genes and proteins in endometrial tissue of pigs from fertilization to implantation. Six genes, including estrogen receptor-α, estrogen receptor-β, LIF, LR (LIF receptor), TGFβ1, and TGFβ2, that may play important roles in regulating uterine receptivity and successful implantation and that show different expression patterns by the stages of pregnancy were selected. As a step toward understanding the role of gene and protein expression changes in endometrial tissue of pigs during the preimplantation stage (Day 2, Day 6, Day 8, Day 12, and Day 17, n = 3/group) and the post-implantation stage (Day 21 and Day 33, n = 3/group) and Day 0 (estrous), real-time PCR methods for quantitative analysis of genes and immunohistochemistry methods to localize protein expression were utilized. Data from quantitative real-time PCR were analyzed by ANOVA. The results of this experiment indicated that estrogen receptor-β mRNA level was sharply increased to Day 12 of pregnancy, while estrogen receptor-α mRNA did not change drastically during early pregnancy stage. In contrast, levels of LIF and LR mRNA were increased from Day 2 to Day 33. Although TGFβ1 mRNA reached peak on Day 17 and TGFβ2 mRNA showed the highest level on Day 17, TGFβ2 did not appear to change drastically. For the protein expression patterns, estrogen receptor-α and estrogen receptor-β proteins were expressed in both luminal epithelium and glandular epithelium, but they were only partially expressed in some tissues of stroma cells. LIF protein was expressed in all cell types, while TGF β1 protein was high expressed in glandular epithelium. Also, ERs, LIF, and TGF β1 mRNA and protein expression showed stage- and cell-specific expression patterns. We also investigated the gene expression of TGF β1 mRNA and TGF β2 mRNA in early conceptus (Day 12 and Day 17). TGF β1 mRNA expression was low in Day 12 embryos, and increased progressively to Day 17. This indicated that both the maternal uterus and the conceptus represent the same gene expression pattern. These results suggest that estrogen receptor-β could be an important factor in estrogen action in endometrial tissue during early gestation in pigs, and TGF βs function in both autocrine and paracrine interactions. Progressive increase in TGF β1 mRNA expression in conceptus and uterine tissues suggest important roles of TGF β1 in conceptus development and establishment of the uterine receptivity during the peri-implantation period.
136 EVIDENCE OF A DIRECT EFFECT OF P4 ON IVF-DERIVED BOVINE 8-CELL EMBRYOS
Authors: Ferguson C.E. , Davidson T.R. , Mello M.R.B. , Lima A.S. , Kesler D.J. , Wheeler M.B. , Godke R.A.
Published: December 2004

There has been much debate over a direct role for progesterone (P4) in early bovine embryo development. While previous attempts to supplement bovine embryos in vitro with P4 produced results that vary and are often contradictory, this may be a response of administering P4 at inappropriate times. Therefore, the objective of these experiments was to determine if P4 could exert a direct effect on developing IVF-derived bovine embryos when administered at an appropriate time of embryo development. In Exp. I, IVF-derived bovine 8-cell embryos were randomly allotted to treatments: (1) control, CR1aa medium (n = 168); (2) vehicle, CR1aa + ETOH (0.01%) (n = 170); and (3) P4, CR1aa + ETOH + P4 (20 ng/mL in 50-μL droplet) (n = 173). In Exp. II, IVF-derived bovine 8-cell embryos were randomly allotted to treatments: (1) control, CR1aa medium (n = 160); (2) vehicle, CR1aa + DMSO (0.01%) (n = 180); and (3) P4, CR1aa + DMSO (0.01%) + P4 (20 ng/mL in 50-μL droplet) (n = 170). All embryos were evaluated on Days 6 to 9 post-insemination and rates calculated from 8-cell embryos. In Exp. I, ETOH tended to have a detrimental effect with significantly fewer (P < 0.05) embryos (53%) developing to the blastocyst stage on Day 7 compared with the control (62%) and P4 (71%) groups. At Day 7, significantly more embryos cultured in P4 (71%) developed to the blastocyst stage compared with the control group (62%). P4 treatment significantly increased the number of Grade 1 blastocysts (25%) on Day 7 compared with vehicle (15%) and control (17%) groups. At the end of culture, there were also significantly more Day 9 hatched blastocysts in the P4 group (33%) compared with vehicle (22%) and control (21%) groups. Supplementing P4 in the culture medium increased the rate of development, resulting in significantly more blastocysts (8%) on Day 6 and hatched blastocysts (21%) on Day 8 compared with vehicle (3% and 12%) and control (0% and 8%) groups, respectively. In Exp. II, there were no significant differences between treatment groups for Day 7 blastocysts (control 54%, DMSO 61%, P4 57%) and Day 9 hatched blastocysts (control 46%, DMSO 51%, P4 46%). However, there were significantly more Grade 1 blastocysts in the P4 group (22% and 36%) on Days 6 and 8 compared with vehicle (11% and 23%) and control (13% and 23%) groups, respectively. The lack of improvement in Day 7 blastocysts and Day 9 hatched blastocysts rates leads to further uncertainty in understanding the P4 vehicle interactions. In conclusion, the results of these two experiments indicate that P4 can exert a direct effect on the developing IVF-derived bovine embryo; however, due to P4 vehicle interactions; other inert vehicles need to be explored to further evaluate the direct effects of P4 on the developing bovine embryo.
149 INTRAUTERINE CULTURE OF IN VITRO PRODUCED BOVINE EMBRYOS AND RECOVERY OF THE EMBRYOS AT DAYS 12 - 14
Authors: Schmidt M. , Avery B. , Greve T.
Published: December 2004

For stem cell production and detailed morphological analysis 12–14-day-old bovine embryos are suitable. However, it has been proven to be difficult to extend the in vitro culture period beyond Days 8–9, and it was the aim of the present experiment to examine whether it might be possible to culture 6–7-day-old in vitro-produced (IVP) embryos for a period of 5–7 days in the uterine horns of heifers. The IVP embryos were produced by standard procedures. Briefly, IVM took place in DMEM medium supplemented with 5% serum, EGF, and eCG/hCG, and IVF was carried out in TALP medium under 5% CO2 in humidified air and at 38.5°C. IVC took place in SOFaaci supplemented with 10% serum under 5% CO2, 5% O2 and 90% N2 at 38.5°C .The embryos were cultured in vitro to Days 6–7 post insemination, when morulas and blastocysts of excellent quality were placed in HEPES-buffered TCM199 with 10% serum, loaded in numbers of 10–30 into 0.25 mL straws, and then transported to the place of transfer in a portable incubator at 38.5°C. The embryos were transferred nonsurgically to the mid or distal part of the uterine horns of 28 dairy heifers which were heat synchronized with injections of cloprostenol (Estrumat Vet, Schering-Plough, Farum, Denmark) to a cycle stage of embryo age +1 day. In 16 heifers, embryos were transferred into both sides and for the remaining ones only into the horn ipsilateral to the ovary bearing the corpus luteum. After 5–7 days, the heifers were flushed nonsurgically by standard method, using a flushing catheter of large caliber (Minitab® 18 G) and slow infusion and evacuation of the fluid. The differences in recovering rate among horns were identified by Fisher's Exact test. Data are given as LS means ± SEM values and statistical differences assigned at the P < 0.05 level. In 6 of the 28 heifers no embryos were obtained; in these 6 cases, the quality of the transferred embryos, the transfer procedure, the heifers, and the flushing procedures did not differ in any obvious way from those of the successful flushings, which numbered 22 (79%). The mean embryo recovery rate was 40 ± 3% with a variation from 7% to 93%. There was a minor but not statistically significant difference between the overall recovery rate of embryos from the ipsi- versus contralateral horn, respectively (44 ± 5% vs. 38 ± 6%). In only 4 of the 16 heifers where transfer occurred to both horns was the recovery rate higher in contralateral side, compared to 9 heifers where the highest recovery rate was seen in the ipsilateral side. The oldest elongated embryos were in one occasion damaged and in another tangled, making it difficult to isolate the individual embryo; apart from that, all of the embryos seemed of excellent quality making it possible to isolate the embryonic discs. It can be concluded that it is possible to culture in vitro produced Day 6–7 bovine blastocysts in the uterus of synchronized heifers and to achieve an acceptable recovery of Day 12–14 embryos.
161 PREGNANCY RATES OBTAINED AFTER EMBRYO TRANSFER AT FIXED TIME OF IN VIVO-, IVF- AND CLONED-DERIVED EMBRYOS
Authors: Lagioia J. , Panarace M. , Marfil M. , Basualdo M. , Gutierrez J. , Révora M. , Medina M.
Published: December 2004

The most important factor in bovine embryo transfer programs is the low efficiency in the utilization of the recipients; this low efficiency is associated with low response to synchronization protocols and failures in estrus detection. It has been shown that cows transferred at fixed time with in vivo-derived embryos resulted in high rates of recipients selected for transfer and high overall pregnancy rates (recipients pregnant/recipients treated) (Tribulo et al. 2002 Theriogenology 57, 563). An experiment was designed to evaluate the pregnancy rate in recipients transferred with in vivo (fresh and frozen), IVF, and cloned-derived embryos without estrus detection. A total of 1555 non-lactating Bos Taurus crossbred beef cows was divided into two groups. Cows from group 1 (n = 421) were synchronized with a progesterone intravaginal releasing device (1 g P4; DIB, Syntex®, Buenos Aires, Argentina) plus 2 mg of estradiol benzoate (EB) i.m. (Syntex®) on Day 0. On Day 5, they received 400 IU of eCG (Novormon 5000, Syntex®) i.m. and 150 μg of D-Cloprostenol (PGF2α) (Bioprost-D, Biotay®, Buenos Aires, Argentina). The DIB devices were removed on Day 8 and on Day 9, 1 mg of EB was injected. Day 10 was arbitrarily considered as the day of estrus. Cows from group 2 (n = 1134) received 2 doses of PGF2α 14 days apart and were checked for heat during 5 days after the second PGF2α dose. Cows of both groups were examined 7 days after estrus by ultrasonography (Pie Medical Scanner 200®) and those with a corpus luteum >10 mm of diameter were transferred nonsurgically with in vivo (fresh and frozen), IVF, and cloned-derived embryos. In group 1, 360 cows were transferred, and in group 2, 726 cows were transferred (Table 1). Pregnancy was diagnosed 23 days later by ultrasonography (Pie Medical Scanner 200®). The pregnancy rates were compared statistically between groups 1 and 2 by analysis of variance (Infostat, LSD Fisher). There was no significant statistic difference (P > 0.05) between pregnancy rate in group 1 and 2 with in vivo (fresh), IVF, and cloned-derived embryos. However, pregnancy rate of frozen in vivo-derived embryos was lower in group 1 than in group 2 (P < 0.05). Results showed that treatment using DIB combined with EB, PGF2α, and eCG associated with embryo transfer without estrus detection (group 1) had no difference in pregnancy rate when compared with the treatment where synchronization with PGF2α and heat detection were used (group 2). Another important advantage is the use the group 1 treatment for increasing the flexibility and efficiency in the management of the recipients of in vivo, IVF, and cloned-derived embryo transfer programs.

Table 1.
Comparison of pregnancy rates between group 1 (embryo transfer at fixed time) and group 2 (embryo transfer 7 days after estrus detection)
164 EFFECT OF ACCUMULATION OF LIPIDS DURING IN VITRO CULTURE ON BOVINE BLASTOCYST YIELD AND FETAL DEVELOPMENT
Authors: Rooke J.A. , Ewen M. , McCallum G.J. , Watt R.G. , McEvoy T.G.
Published: December 2004

Previous research has shown that increased embryo lipid content, through culture in the presence of serum, is associated with reduced blastocyst yield and quality; provision of antioxidants ameliorates the effects of lipid accumulation (McEvoy et al. Reprod. Fertil. Dev. 16, 200). The present study extended these observations to assessment of fetal development from blastocysts which had accumulated lipid in vitro. Bovine oocytes, aspirated from abattoir-derived ovaries, were matured and fertilized using standard procedures. Cleaved zygotes were assigned to culture (5% CO2; 5% O2; 90% N2; 38.5°C) in four treatments: (1) synthetic oviductal fluid containing 0.3% bovine serum albumin and amino acids (SOF); (2) SOF plus supplementary bovine lipoproteins (2%, SOFLP); (3) SOF plus the antioxidant Trolox® (100 μM; 6-hydroxy-2,5,7,8-tetramethylchromane-2-carboxylic acid, Sigma-Aldrich Co., Inc., Irvine, UK; SOFT); and (4) SOF plus lipoproteins and Trolox (SOFLPT). Blastocysts were synchronously transferred to recipient cattle (day 7; 23/treatment) together with a control group (n = 21) of artificially inseminated (AI) cattle. Reproductive tracts were recovered on Day 70 and fetal total and organ weights recorded. Data were analyzed by ANOVA (with fetal sex as covariate) and chi-square analyses. Culture in the presence of lipoproteins increased blastocyst total fatty acids (mean ± SD) from 98 ± 9.7 to 124 ± 7.3 ng/blastocyst. Blastocyst yields (%; 23 batches of ovaries) were reduced (P = 0.002) by inclusion of lipoproteins in culture (SOF, 22.0 ± 8.20; SOFLP, 16.4 ± 8.57; SOFT, 22.8 ± 9.03; SOFLPT, 24.2 ± 7.30) unless Trolox was present. Blastocyst grade was poorer (P < 0.001) after culture in the presence of lipoproteins irrespective of the presence of Trolox (SOF, 2.4 ± 0.43; SOFLP, 2.6 ± 0.45; SOFT, 2.4 ± 0.47; SOFLPT, 2.6 ± 0.40). Pregnancy rates (Day 70) were greater (P < 0.001) for AI (91%) than culture (26%) but were not affected by culture treatment. Although there were no differences in fetal weights between AI and IVC fetuses, SOFLP fetuses (Table 1) were lighter, had smaller relative liver weights, and had greater crown-rump length-to-weight ratios than other IVC fetuses. Therefore, IVC in conditions that increased blastocyst lipid content without adequate antioxidant protection reduced blastocyst yield and influenced fetal development.

Table 1.
Fetal weights (g) and relative liver weights (g/kg) and crown-rump lengths (CRL, mm/g) at day 70

SAC receives financial support from SEERAD.
212 HETEROGENEITY OF RIBOSOMAL RNA GENE ACTIVATION AMONG CELLS OF IN VITRO-PRODUCED PORCINE EMBRYOS
Authors: Bjerregaard B. , Strejcek F. , Rasmussen Z. , Laurincik J. , Niemann H. , Maddox-Hyttel P. , Thomsen P.D.
Published: December 2004

In vitro production (IVP) of porcine embryos by in vitro maturation of oocytes followed by fertilization and culture in vitro is hampered by great deficiencies. Initiation of at least the major embryonic genome transcription, which includes activation of ribosomal RNA (rRNA) genes and the associated formation of a fibrillo-granular nuclealus, is normally seen during the 4-cell stage in pigs. We have investigated the activation of rRNA synthesis and the presence of silver staining nucleolar proteins in porcine IVP embryos as a marker of transcriptional activity and, thus, developmental competence. A total of 205 porcine IVP embryos from the 2-cell to the blastocyst stage were examined using sequential fluorescent in situ hybridization (FISH) to the rRNA genes and their transcripts and silver staining of nucleolar proteins as previously described (Viuff et al. 2002 Biol. Reprod. 66, 629–634). Briefly, cumulus-oocyte complexes with at least three cumulus cell layers and evenly granulated ooplasm were isolated from 2–5 mm ovarian follicles with stereomicroscopic evaluation. Subsequently, oocytes were matured in NCSU-37 and mechanically denuded followed by fertilization using frozen-thawed epididymal semen. Presumptive zygotes were then cultured in NCSU-23 at 39°C, 5% CO2. Around the time of expected cleavage, the embryos were examined every second hour to determine the time of cleavage. Embryos at the 2-cell stage were harvested at 5 h post-cleavage (hpc), 4-cell embryos late during the third cell cycle at 30 hpc, and tentative 8- and 16-cell embryos at 10 hpc. Blastocysts were harvested at Day 5 post-insemination. In general, nuclei of 2-cell embryos displayed 4 small foci of FITC labelling (presumably the rDNA), but no specific silver staining, and were consequently categorized as transcriptionally inactive. At the late 4-cell stage, 58% of the embryos resembled the 2-cell stage. However, in the remaining embryos (42%), some or all nuclei displayed large areas of FISH labelling (presumptive rDNA and rRNA) co-localized with silver staining, and were catagorized as transcriptionally active. Among the 8-cell embryos, 64% displayed a majority of transcriptionally active nuclei, whereas this was the case in 83% and 92% of the embryos in the 16-cell embryos and the blastocysts, respectively. In general, the majority of the embryos contained a mixture of transcriptionally active and inactive cells. These findings show that the porcine IVP embryos are often delayed and asynchronous with respect to activation of the rRNA genes.

Table 1.
Categorization of nuclei according to transcriptional activity

This work was supported by grants from “Disease models, disease prevention and animal welfare improvement: The pig embryo as a model.” Danish Research Agency (Grant: 9901178), NATO (Grant: 978658), and Deutsche Forschungsgemeinschaft (DFG).
221 TYPICAL HIF1-REGULATED GENES ARE UNALTERED BY OXYGEN IN BOVINE BLASTOCYSTS
Authors: Harvey A. , Kind K. , Thompson J.
Published: December 2004

Oxygen-regulated gene expression in the bovine embryo contrasts markedly with that observed in the mouse. Under low (2%) post-compaction oxygen conditions moderate changes in gene expression are observed in the bovine blastocyst (Harvey et al. 2004 Biol. Reprod. 71, in press), compared with 3–4 fold increases in the mouse (Kind et al. 2004 Mol. Reprod. Dev., in press). Specifically, GLUT-1 (Harvey et al. 2004), myotrophin, and anaphase-promoting complex 1 (Harvey et al., unpublished) mRNAs are increased in bovine blastocysts following 2% oxygen culture, compared with those cultured under 20% oxygen. These oxygen-mediated differences in gene expression in the bovine are most likely regulated by hypoxia-inducible factor (HIF)2 transcription factor activity, as we have previously observed that HIF1α protein is not detectable in bovine embryos whereas HIF2α is readily detectable (Harvey et al. 2004). The aim of this study was to determine the effect of post-compaction oxygen concentration on the expression of typically HIF1-regulated and potential HIF2-regulated (suggested from a mouse knockout study; Scortegagna et al. 2003 Nat. Genet. 35, 371) genes in bovine blastocysts. In vitro-produced bovine embryos were generated using standard protocols. Compact morulae were randomly allocated to treatments under 2%, 7%, or 20% oxygen for 72 h from Day 5. Blastocyst RNA was isolated using TriReagent (Molecular Research Center, Inc., Cincinnati, OH, USA) and samples were reverse-transcribed using Superscript II (Invitrogen, Melbourne, Australia). Amplification and analysis of cDNA was achieved by real-time PCR using specific primers and Sybr green PCR master mix (Applied BioSystems, Melbourne, Australia). Statistically significant differences in gene expression were analyzed by ANOVA, P < 0.05. Examination of expression of genes known to be regulated by HIF1 in somatic cells (reviewed by Semenza 2002 Biochem. Pharm. 64, 993) revealed no oxygen-mediated alteration in expression of aldose reductase, cyclooxygenase 2, or inducible nitric oxide synthase. However, the expression of lactate dehydrogenase A (LDHA) displayed a 4-fold increase under 2% oxygen, compared with 7% and 20% oxygen (P < 0.001). Expression of glutathione peroxidase, and CuZn- and Mn-superoxide dismutase (putative HIF2-regulated genes) was not influenced by oxygen concentration post-compaction. This study suggests that typical HIF1-regulated genes are not influenced by alterations in the external oxygen environment in the bovine embryo. These results complement previous observations that HIF1α protein is not detectable in blastocyst-stage bovine embryos, and suggest that LDHA may be an HIF2 target gene in the bovine embryo. As embryo development is influenced by oxygen concentration, levels of LDHA at the blastocyst stage may be used as a marker of oxygen responsiveness.
222 METHYLATION OF THE 52-UPSTREAM REGION OF THE H19 GENE IN MOUSE SOMATIC CELL, GAMETES, WILD TYPE AND ANDROGENETIC ES CELLS
Authors: Kato H. , Murakami H. , Kawasumi M. , Kunieda T. , Okuno M. , Kishimoto M. , Soma M. , Iwai D. , Anzai M. , Mitani T. , Matsumoto K. , Saeki K. , Hosoi Y. , Iritani A.
Published: December 2004

In mammals, several genes influenced by the phenomenon of genomic imprinting are critical during development. Recently, Kono et al. (2004 Nature 428, 860–864) reported the production of intact female mouse individuals that had only two haploid sets of maternal genome. They obtained these mice by combining a normal haploid maternal genome and a mutant haploid maternal genome with a 13 k base deletion in the H19 gene and its 5′-upstream region. This genomic combination resulted in the appropriate expression of the Igf2, H19, and other imprinted genes. In the mouse genome, there are four CTCF-binding sites in the 5′-upstream region of the H19 gene. The binding of CTCF to these binding sites regulates the expression of the Igf2 and H19 genes. The binding of CTCF to its binding sites is regulated by methylation of CpG sites in binding sites. In this study, as the first step to elucidate the role of the paternal genomic imprinting during development, we investigated the methylation of CpG sites in the 5′-upstream region of the H19 gene in mouse somatic cells, gametes, and two types of ES cells. Genomic DNA was isolated from BDF1 (C57BL/6N × DBA/2N) mouse's tail (male and female somatic tissue, mST and fST, respectively), spermatozoa (S), oocytes (O), and wild type and androgenetic embryonic stem cells (wtES and agES, respectively). The methylation of CpG sites was evaluated by using the bisulfite sequencing assay. There were 13 CpG sites and a CTCF-binding site in the region from −4413 to −3976 in the H19 gene relative to the transcription start site. The percentages of CpG sites in this region that were methylated were 88% (160/182), 79% (27/130), 93% (230/247), 8% (10/130), 77% (10/13) and 89% (314/351) for mST, fST, S, O, wtES, and agES, respectively. In the CTCF-binding site core motif (CCGCGTGGTGGCAG), the percentages of methylated CpG sites were 93% (26/28), 80% (16/20), 95% (36/38), 0% (0/20), 50% (1/2) and 96% (52/54) for mST, fST, S, O, wtES, and agES, respectively. The CpG sites in the sequence of agES were highly methylated similar to the finding in spermatozoa. However, an aberrant methylation pattern was observed in some clones of agES. From these results, it was concluded that the methylation of CpG sites in the genomic sequence of agES was well conserved and, therefore, agES is useful to elucidate the role of the paternal genomic imprinting during development.
This work was supported by Wakayama Prefecture Collaboration of Regional Entities for the Advanced of Technological Excellence, Japan, and by a Grant-in-Aid for the 21st Century COE Program of the Japan MEXT.
243 PREGNANCY RATES IN THE FIELD AFTER THE TRANSFER OF BOVINE IVP EMBRYOS VITRIFIED BY THE CRYOLOGIC VITRIFICATION METHOD
Authors: Fry R. , Earl C. , Fry K. , Lindemans W.
Published: December 2004

Although large numbers of IVP embryos can be produced from donor cattle in a short period of time, commercial acceptance of the technology depends on the ability to cryopreserve these embryos and achieve a 50% pregnancy rate in large-scale embryo transfer programs. Many studies have reported low pregnancy rates of about 20% after the transfer of cryopreserved IVP embryos. We have developed the user friendly CryoLogic Vitrification Method (CVM) that vitrifies embryos on a solid surface at −196°C and warms them rapidly in a one-step procedure prior to transfer (Lindemans et al. 2004 Reprod. Fertil. Dev. 16, 174). We present an overview of the pregnancy rates in the field after vitrification of bovine IVP embryos by the CVM. The bos taurus IVP embryos in southern Australia (bT) and the bos indicus-based IVP embryos in northern Australia (bI) were produced by our standard TVR and IVP methodology (Fry et al., 2003 Theriogenology 59, 446). Pregnancy was determined by rectal palpation between Day 40 and Day 90 and differences between treatments were analyzed by chi-square. The development of the CVM has enabled the successful cryopreservation of bovine IVP embryos. In the laboratory the typical survival (90% re-expansion) and development (80% hatching) of IVP embryos post-vitrification is high and, as demonstrated here, the pregnancy rates after transfer are approaching commercially acceptable levels. However, further research is required to identify factors that may influence success under full field conditions, for both the IVP and the vitrification technologies.

Table 1.
Pregnancy rates for fresh IVP, CVM-vitrified IVP, and traditionally flushed embryos cryopreserved in glycerol
27 MORPHOLOGICAL AND IMMUNOHISTOCHEMICAL CHARACTERIZATION OF DAY 21 IVP AND NT BOVINE EMBRYOS
Authors: Alexopoulos N.I. , Maddox-Hyttel P. , Tecirlioglu R.T. , Cooney M.A. , French A.J.
Published: December 2004

A major limitation of somatic cell nuclear transfer (NT) for the production of cloned calves is that only 1–5% of cloned embryos produce viable calves. The high rate of mortality is attributed to both pre- and post-natal losses and is primarily due to incomplete reprogramming of donor cells. Almost 50% of the pregnancy losses occur in the first trimester of pregnancy, indicating a major disruption in normal embryo development at NT. The aim of this study was to analyze germ layer formation by stereomicroscopy and immunohistochemical techniques for both NT embryos and their in vivo counterparts on Day 21 and to compare deviations from normal embryonic development as a measure of developmental capacity. Blastocyts derived by IVF (n = 20), conventional NT (n = 20), or hand made cloning (HMC; n = 20) were non surgically transferred to each synchronized recipient cow (n = 3). Each group of twenty embryos was transferred to one recipient. Cows were slaughtered on Day 21 and uterine tracts recovered and flushed with phosphate-buffer solution with 10% serum. Recovered Day 21 embryos were fixed in 4% paraformaldehyde, and embedded in paraffin; serial sections were stained with hematoxylin and eosin and evaluated by light microscopy. Immunohistochemical localization of cytokeratin 8 was used as a marker for potential ectoderm, alphafetoprotein for potential endoderm, and vimentin for potential mesoderm. Four IVF (20%; 4/20) embryos, six NT (30%; 6/20) embryos, and ten HMC (50%; 10/20) embryos were recovered following flushing. No obvious morphological differences were seen in the formation of a neural tube, differentiation of mesoderm, and number of somites among IVF (25%; 1/4), NT (33.3%; 2/6), and HMC (20%; 2/10) embryos. Delayed development with respect to the formation of neural groove and mesoderm differentiation was observed in 25% (1/4) of IVF, 16.7% (1/6) of NT, and 30% (3/10) of HMC embryos. In addition, 25% (1/4) of IVF, 16.7% (1/6) of NT, and 33.3% (2/6) of HMC embryos had not initiated gastrulation (i.e. displayed hypoblast and epiblast), suggesting a more substantial developmental delay. The remaining embryos showed gross abnormalities compared to their in vivo counterparts, including degenetration of epiblast and hypoblast cells. Cytokeratin was detected in the trophoblast, ectoderm, hypoblast, and endoderm. Alpha-fetoprotein was detected in the hypoblast while vimentin was seen in the mesoderm. In conclusion, although localization of staining in IVF and cloned embryos was consistent with that of in vivo embryos, the intensity was weaker, suggesting compromised or delayed development. It is also possible that the differences observed were due to the recipient and not to the treatment group.
This work was supported by Genetics Australia Cooperative Limited, Victoria, Australia.
293 TWO-STEP MATURATION OF BOVINE OOCYTES WITHOUT CDK INHIBITORS: AN ALTERNATIVE TO AFFECT THEIR SUBSEQUENT DEVELOPMENTAL COMPETENCE
Authors: Pavlok A. , Lapathitis G. , Cech S. , Kubelka M. , Lopatarova M. , Holy L. , Klima J. , Motlik J. , Havlicek V.
Published: December 2004

The acquisition of meiotic and developmental competence seems to correlate not only with the size of follicles and oocytes but also with the morphology and transcriptional activity of the oocyte nuclei and nucleoli. To secure or increase the fertilization and the developmental competence of bovine oocytes, we have developed a two-step culture system using the specific cyclin dependent kinase inhibitors (Butyrolactone I, Bohemine). However, these drugs have several side effects during the prolonged time of culture. To avoid this disadvantage, we have used in the present experiments modified culture conditions simulating the intrafollicular block of meiosis. In the first step of culture, bovine oocytes isolated from small, medium, and large follicles (2–3, 3–4, and 4–6 mm in diameter, respectively) were kept under conditions that secured for at least 48 h the intact germinal vesicle stage (GV) in more than 90% of oocytes. The second step represented the subsequent 20–22 h in conditions stimulating resumption of meiosis. The effectiveness of this model depended mainly on medium composition: reduced NaHCO3, substitution of serum with serum albumin, addition of antioxidants (curcumin), increased viscosity of a medium by agar (0.3%), and reduction of oxygen concentration (within 6–9%). The reduction of the proportion between the number of cumulus-oocyte complexes (COC) and the amount of medium (within 6–7 mL per COC) should amplify the GVBD-inhibiting effect of oocyte-surrounding granulosa cells. The COC were situated in clots of 6–7 COC per clot. The effectiveness and reversibility of GVBD inhibition depends also on the duration of COC isolation. The full reversibility of GVBD inhibition was controlled morphologically and also by measuring histone H1 and MAP kinase activities. The two-step versus one-step (24 h) maturation technique was evaluated by the percentage of total and hatched Day 9 blastocysts. When compared with one-step maturation, the two-step culture showed a slightly increased proportion of total and hatched blastocysts developed from the smallest follicular category (13.9 vs. 7.1% and 9.2 vs. 3.3% for total and hatched blastocysts, respectively). No significant difference was noticed between between one- and two-step culture when oocytes from large healthy follicles were used. However, the two-step maturation of oocytes from regressing follicles substantially reduced the blastocyst yield (9.7 vs. 39.1% and 4.9 vs. 26.7% for total and hatched blastocysts, respectively).
This study was supported by grant of GA CR No. 524/02/0674.
294 EFFECT OF CYSTEAMINE ADMINISTRATION DURING EQUINE OOCYTE MATURATION ON GLUTATHIONE CONTENT, NUCLEAR MATURATION, AND DEVELOPMENTAL CAPABILITY AFTER INTRACYTOPLASMIC SPERM INJECTION
Authors: Perazzoli F. , Galbusera C. , Modina S. , Goudet G. , Gerard N. , Luciano A.M.
Published: December 2004

In the recent years, assisted reproduction methods have produced only limited success in equine species in comparison with other domestic mammals. A major factor affecting oocyte viability during in vitro culture is oxidative stress. Oxidative modifications could be responsible for oocyte-defective in vitro maturation and consequently compromise subsequent fertilization and embryonic development. Low-molecular-weight thiol compounds such as cysteamine, added during in vitro culture of bovine, porcine, and ovine oocytes, increase intracellular glutathione (GSH) synthesis, which prevents oxidative damages and consequently improves in vitro maturation and embryo development. The present study was aimed at investigating whether equine oocyte maturation efficiency and embryonic developmental capability following ICSI benefit from the addition of cysteamine during in vitro maturation (IVM). Cumulus oocyte complexes (COCs) were collected from slaughtered ovaries and cultured for 30 h at 38.5°C in 500 μL of control medium (TCM199 + 0.4% BSA + 0.1 IU/mL rhFSH + 50 ng/mL EGF) either supplemented with 100 μM cysteamine or not. After culture, nuclear stage was assessed by Hoechst 33342 staining after cumulus cell removal, and MII oocytes were analyzed for GSH content (Baker MA et al. 1990 Anal. Biochem. 190, 360–365). Groups of COCs matured under the same conditions were denuded with hyaluronidase and only oocytes with a visible polar body were fertilized by ICSI. The number of embryos that reached the 2–4 cell stage was assessed by nuclear staining with propidium iodide after 72 h of culture in SOF supplemented with 5% calf serum at 38.5°C in a modified atmosphere (5% CO2, 5% O2, and 90% N2). Our data indicated that oocytes cultured in the presence of cysteamine had a nuclear maturation rate similar to those cultured in control medium (Table 1). Intraoocyte GSH content increased during IVM, and the addition of cysteamine induced a significant GSH accumulation in matured oocytes. After ICSI, a similar proportion of zygotes in each group developed beyond the two-cell stage after 72 h of culture. The results of this study demonstrate that the addition of cysteamine to the IVM medium increases GSH content in equine oocytes. However, this affects neither the maturation rate nor the capability to reach the early embryonic development after ICSI. We hypothesize that factor(s) other than GSH content are responsible for the limited in vitro developmental capability of equine oocyte.

Table 1.
Effect of cysteamine administration on maturation rate, oocyte GSH content (pmol/oocyte), and early embryonic development after ICSI

This work was supported by a 2003 UniMi Grant.
317 FACTORS AFFECTING PRODUCTION EFFICIENCY OF TRANSGENIC RATS	BY ICSI-MEDIATED DNA TRANSFER
Authors: Kato M. , Hochi S. , Hirabayashi M.
Published: December 2004

Successful DNA transfer via intracytoplasmic sperm injection (ICSI) was first reported in mice (Perry et al. 1999, Science 284, 1180), and was recently extended to rats (Kato et al. 2004, Mol. Reprod. Dev. 69, 153). In the present study, factors affecting the production efficiency of transgenic rats by the ICSI-mediated DNA transfer were investigated. Cauda epididymal spermatozoa from Sprague-Dawley rats were sonicated (SO) and/or frozen-thawed (FT) for tail-cutting and membrane-disrupting. The sperm heads were exposed for 1 min to different concentrations (0.02–2.5 ng/μL) of 3.0 kb EGFP DNA solution, and then microinjected into denuded F1 (Donryu × LEW) rat oocytes. The optimal concentration of EGFP DNA was 0.1 ng/μL, as determined by the in vitro developmental competence into morulae/blastocysts and the EGFP expression of the ICSI oocytes. The presumptive 1- or 2-cell stage zygotes were transferred into oviducts of pseudopregnant Wistar females, and the presence of EGFP DNA in the offspring was examined by fluorescence under the 480 nm UV light. The production efficiency of transgenic rat offspring was 2.8% (2/71 zygotes transferred), 1.6% (1/63), and 3.3% (2/61) in the oocytes into which SO-, FT-, and SO+ FT-treated sperm heads were injected, respectively. The founder transgenic rats carrying EGFP DNA transmitted the transgenes to their progeny according to the Mendelian fashion (43.8–54.8%), suggesting the stable incorporation of the transgenes into rat genomes. Four rat strains (F344, LEW, Donryu, and Sprague-Dawley) were compared for their suitability as sperm/oocyte donors in the production of transgenic rats by ICSI with SO + FT-treated and 0.1 ng/μL EGFP DNA-exposed sperm heads. The production efficiency of the transgenic rats in the Sprague-Dawley strain (8.2%, 8/98) was significantly higher than that in LEW strain (0.9%, 1/114), while those in F344 (4.3%, 4/92) and Donryu (4.4%, 5/114) strains were intermediate. Attempts were made to introduce three other DNA constructs (5.0 kb plasmid and 208 kb BAC, both with Fyn gene, and 186 kb BAC with Svet1/IRES-Cre gene) into rat genomes by ICSI with SO + FT-treated and 0.1 ng/μL DNA-exposed sperm heads. PCR analysis showed that the Fyn, Fyn/BAC, and Svet1/IRES-Cre DNA constructs were successfully introduced into Sprague-Dawley rat offspring via ICSI, with production efficiencies of 2.8% (3/109), 0.9% (1/109), and 2.4% (3/125), respectively. These results indicate that transgenic rats can be produced by ICSI-mediated DNA transfer using the various types of exogenous DNA and rat strains with different genetic backgrounds.
48 PREGNANCY AND FETAL CHARACTERISTICS AFTER TRANSFER OF VITRIFIED IN VIVO AND CLONED BOVINE EMBRYOS
Authors: Lonergan P. , Evans A.C.O. , Boland E. , Rizos D. , Sung L.-Y. , Du F. , Chaubal S. , Fair S. , Scraggs E. , Duffy P. , Xu J. , Yang X. , Tian X.C.
Published: December 2004

This study was conducted to examine pregnancy progression and fetal characteristics following transfer of vitrified bovine nuclear transfer (NT) vs. in vivo-derived (Vivo) embryos. Nuclear transfer was conducted using skin fibroblast cells derived from cultured ear explants taken from an elite Holstein-Friesian dairy cow. Expanding and hatching blastocysts on Day 7 were vitrified using liquid nitrogen surface vitrification (Shen et al. 2004 Reprod. Fertil. Dev. 16, 158). Day 7 in vivo embryos, produced using standard superovulation procedures applied to Holstein-Friesian heifers (n = 6), were vitrified in the same way. Following warming, embryos were transferred to synchronized recipients (2 per recipient; NT: n = 81 recipients; Vivo: n = 20 recipients). Pregnancies were monitored by ultrasound scanning on Days 25, 45, and 75, and a sample of animals was slaughtered at each time point to recover the fetus/placenta for further analyses. On Day 25, the entire conceptus was weighed together; on Day 45, the fetus and placenta were weighed separately; on Day 75, the fetus was dissected and the major organs were weighed. Significantly more animals remained pregnant after transfer of in vivo-derived embryos than NT embryos at all time points: Day 25 (95.0 vs. 61.7%, P < 0.01), Day 45 (93.3 vs. 39.7%, P < 0.001), and Day 75 (90.9 vs. 19.8%, P < 0.001). There was no significant difference (P = 0.10) in the weight of the conceptus on Day 25 from NT transfers (1.14 ± 0.23 g, n = 8) vs. Vivo transfers (0.75 ± 0.19 g, n = 8). On Day 45, there was no significant difference in the weight of either fetus (P = 0.393) or membranes (P = 0.167) between NT embryos (fetus: 2.76 ± 0.40 g, n = 12; membranes 59.0 ± 10.0 g, n = 11) or in vivo-derived embryos (fetus: 2.60 ± 0.15 g, n = 6; membranes 41.8 ± 5.2 g, n = 4). However, on Day 75, the weights of the fetus and several of the major organs were heavier from NT embryos (Table 1). These data suggest that the large calf syndrome is manifested after Day 45.

Table 1.
Fetal characteristics on Day 75 of gestation following transfer of vitrified in vivo-derived vs. NT blastocysts

This work was supported by the U.S. Department of Agriculture.
85 ADDITION OF REDUCED GLUTATHIONE TO FREEZING MEDIUM IMPROVED THE SPERM FUNCTIONALITY OF THAWED BOAR SPERMATOZOA
Authors: Gadea J. , Garcia-Vázquez F. , Gumbao D. , Rodríguez F.A. , Matás C.
Published: December 2004

The processes of cooling and freezing/thawing produce physical and chemical stresses on the sperm membrane that are associated with the oxidative stress and reactive oxygen species (ROS) generation that reduce sperm viability and fertilizing ability. It is known that the process of freezing is associated with a significant reduction of the GSH content in boar sperm (Gadea et al. 2004 Theriogenology 62, 690–701). The addition of antioxidants to freezing medium could prevent the formation of ROS and improve the seminal parameters. The aim of these experiments was to investigate the effects of the addition of reduced glutathione (GSH) to freezing extenders on capacitation status and changes in the sulfhydryl groups of proteins on the sperm surface. Ejaculate-rich fractions from three mature boars were frozen by classic methodology (Westendorf et al. 1975 Dtsch. Tierarztl Wochenschr. 82, 393–395) using lactose/egg-yolk extender with 0 mM (group 0), 1 mM (group 1), or 5 mM (group 5) GSH. To detect increase in plasma membrane lipid packing disorder, sperm samples were stained with merocyanine 540 (M540) and Yo-Pro 1 (Harrison et al. 1996 Mol. Reprod. Dev. 45, 378–391). Cells were classified as low M540 (viable, noncapacitated), high M540 (viable, capacitated), or Yo-Pro-1 positive (dead sperm) using flow cytometry. The sulfhydryl status of proteins from spermatozoa surface was evaluated with a fluorescent stain 5-iodoacetamidofluoresceine (5-IAF). The addition of GSH to the freezing medium had a positive influence on the parameters studied, increasing the proportion of viable noncapacitated spermatozoa and reducing the number of dead with a similar number of viable capacitated (Table 1). The proportion of spermatozoa stained by 5-IAF was significantly lower when GSH was added. In conclusion, we can assume that the addition of reduced glutathione to the freezing medium had a protective effect on spermatozoa functionality.

Table 1.
Capacitation status and changes in sulfhydryl groups of proteins (5-IAF staining) after thawing of frozen boar spermatozoa

This work was supported by AGL- 2003-03144.
Absorption of artificial piggery effluent by soil: A laboratory study
Authors: Smiles D. E. , Smith C. J.
Published: December 2004

Urine, faeces, and waste feed flushed from pens of intensive piggeries produce large volumes of nutrient-rich effluent, which is often most productively used for irrigation. These effluents tend to be similar, with pH values in the range 7.6–8.0 together with high water-soluble ammonium and potassium but lower concentrations of calcium, sodium, and magnesium. There is little experimental information on the behaviour of these mixed ion systems in soils, so the effects of artificial but chemically similar effluents were examined in absorption experiments on columns of ‘natural’ soils. In one series of experiments, NH4+ was excluded from the artificial effluent to assess the consequences of its presence or absence. Water content and solute concentration data scaled according to distance divided by the square root of time in experiments terminated at different times. This showed that basic flow and reaction equations were valid and that water-soluble and exchangeable cations appeared to be in equilibrium in the ‘time-scale’ of absorption. Systematic patterns of behaviour emerged: chloride, the dominant anion in the absorbed solution, moved with the water and its front coincided with the piston front that would exist if the absorbed water completely displaced the original soil solution; cation exchange was restricted to the inflow end of the columns in an environment of constant Cl– concentration and was retarded relative to the moving water. High concentrations of NH4+ appeared not materially to affect the exchange isotherms of other cation pairs, although they reduced retardation of other exchangeable cation fronts. The method provides a useful way to define exchange isotherms in an appropriate environment of competing cations.
Eye-safe lidar measurements for detection and investigation of forest-fire smoke
Authors: Utkin Andrei B. , Fernandes Armando , Lavrov Alexander , Vilar Rui
Published: December 2004

The problem of eye safety in lidar-assisted wildland fire detection and investigation is considered as a problem of reduction of the hazard range within which the laser beam is dangerous for direct eye exposure. The dependence of this hazard range on the lidar characteristics is examined and possible eye-safety measures discussed. The potential of one of the cheapest ways of providing eye safety, which is based on placing the lidar in an elevated position and using a 1064-nm laser beam with increased divergence, is also investigated experimentally. It is demonstrated that a lidar system operating with wider beams maintains its ability to detect smoke plumes efficiently. Providing eye-safe conditions allows scanning of the internal 3D structure of smoke plumes in the vicinity of fire plots. Examples are given as layer-by-layer smoke concentration plots on the topographic map.