bluuebunny/crossref_metadata_2025 huggingface dataset:
122 INTRACELLULAR ICE FORMATION IN VITRIFIED MOUSE MORULAE DURING WARMING
Authors: Jin B. , Kusanagi K. , Ueda M. , Edashige K. , Kasai M.
Published: December 2006

Vitrification is an ice-free cryopreservation method. However, ice can form during warming if the concentration of cryoprotectant is insufficient. For the best use of vitrified embryos, it is important to know the condition in which intracellular ice (ICI) is formed, since ICI is fatal to the cell. So, we tried to estimate the conditions in which ICI forms in vitrified embryos during warming. Compacted morulae were obtained from ICR mice. Vitrification solutions were prepared by diluting ethylene glycol (EG) with FS solution (PB1 medium containing 30% Ficoll and 0.5 M sucrose) at 10, 20, 30, 40, and 50% (EFS10, EFS20, EFS30, EFS40, and EFS50, respectively). Morulae were treated with the solutions for 30, 60, or 120 s at 25�C, loaded in a straw, and vitrified using liquid nitrogen. The sample was warmed by various methods. The survival of embryos was assessed by their ability to develop to expanded blastocysts in culture. The difference in survival was analyzed by the chi-square test. In addition, the morphology of damaged embryos was examined to estimate the type of injury. It was confirmed that 20% or more EG was necessary to prevent the formation of extracellular ice during cooling without embryos. In the rapid warming protocol (control), in which vitrified samples were directly immersed in 25�C water, high embryonic survival was obtained with EFS30 (79–88%) and EFS40 (96–99%), but not with EFS20 (12–45%) and EFS50 (0–14%). This suggests that embryos vitrified with EFS20 were injured by ICI, whereas those vitrified with EFS50 were injured by the chemical toxicity of the solution. When embryos vitrified with EFS30 and EFS40 were warmed slowly by holding the straw in air at 25�C for 120 s, the survival rate decreased significantly (44–64%), probably because the cytoplasm of the embryos that had vitrified in a state of supercooling devitrified during slow warming. To estimate the condition in which ICI forms, embryos vitrified with EFS30 and EFS40 were kept at intermediate temperatures for various times before being warmed in 25�C water. Compared with control, survival rates decreased significantly by keeping samples for 3 min at -80�C (27–65%), -60�C (16–39%), -40�C (6–26%), or -20�C (33–46%). These results suggest that ICI forms at a wide range of temperatures between -80�C and -20�C, but more likely forms at temperatures between -60�C and -40�C. When the holding time was prolonged to 1 h and 24 h, the survival decreased further (0–58% and 0–14%, respectively). This shows that ICI forms not only quickly but also slowly at an intermediate temperature. From the morphological examination, embryos that should be injured by ICI swelled at recovery in PB1 medium and collapsed in culture, whereas those that should be injured by the toxicity of solution underwent decompaction after culture. This observation reinforces our supposition that slowly warmed embryos are injured by ICI.
123 CRYOPROTECTANT EXPOSURE AFFECTS THE PROTEOME OF THE MOUSE METAPHASE II OOCYTE
Authors: Larman M. , Katz-Jaffe M. , Sheehan C. , Schoolcraft W. , Gardner D.
Published: December 2006

The ability to routinely cryopreserve oocytes will have a significant impact on human- and domestic animal-assisted reproduction, and on wildlife conservation. However, success with oocyte cryopreservation has been relatively limited. Previous studies have shown that slow freezing alters mouse oocyte physiology, including the proteome. To improve cryopreservation techniques, it is vital to investigate the effects of the cryopreservation procedure on oocyte physiology. It was therefore the aim of this study to determine at which stage of the slow freezing procedure the proteome was affected, or whether exposure to the cryoprotectant (1,2-propanediol; PrOH) alone was responsible for cellular perturbations. Metaphase II (MII) oocytes were collected from superovulated F1 (C57BL/6 x CBA) mice at 13 h post-hCG. Denuded MII oocytes were randomly allocated to treatments that mimic parts of the slow freezing protocol: (A) 37�C for 20 min (control), (B) room temperature for 20 min (temperature control), (C) 1.5 M PrOH exposure for 20 min (i.e. the equilibration and dehydration step), and (D) 1.5 M PrOH exposure and seeding (i.e. the equilibration, dehydration, and seeding step). Oocytes were then collected in groups of 5 (n = 12 replicates per group), extracted, processed, and analyzed by time-of-flight mass spectrometry. Statistical analysis was performed with the Mann-Whitney non-parametric test. Oocytes maintained at room temperature for 20 min displayed a protein profile similar to that of oocytes incubated at 37�C. The protein expression profile was altered when oocytes were exposed to PrOH for twenty min, with several proteins showing at least a two-fold change of expression level. A similar effect on protein expression was also observed with 20 min of PrOH exposure plus seeding. On comparison with control oocytes, those exposed to PrOH (with or without seeding) had a significantly (P < 0.05) lower abundance of 6 proteins and up-regulation of 4 proteins. Analysis of the mouse MII oocyte at different stages of the slow freezing protocol revealed that there is negligible impact on protein expression when oocytes are maintained at room temperature for 20 min. However, PrOH exposure at room temperature for the same amount of time induces significant perturbation of the oocytes' proteome. These data demonstrate that temperature changes per se (during cooling to room temperature and seeding) impact negligibly on the oocyte proteome, whereas exposure of oocytes to 1.5 M PrOH alone, which mimics the equilibration phase of slow freezing, significantly alters the proteome. The detrimental effect on the oocyte proteome suggests that chronic exposure to PrOH during slow freezing negatively impacts oocyte cryopreservation. To conclude, analyzing the effects of cryopreservation on cell physiology is pivotal for allowing the selection of appropriate techniques and implementing appropriate modifications.
This study was supported by a grant from Vitrolife.
125 IN VITRO-PRODUCED BOVINE EMBRYOS VITRIFIED IN GLASS CAPILLARIES USING TCM-199 OR DULBECCO'S PHOSPHATE-BUFFERED SALINE AS VITRIFICATION-WARMING MEDIA
Authors: Mucci N. , Kaiser G. G. , Rios G. , Alberio R. H. , Ferré L. B. , Aller J.
Published: December 2006

In previous studies (unpublished data) we observed that the replacement of open pulled straws (OPS) with glass capillaries (GC) did not affect the embryo survival rate after vitrification–warming. The aim of this work was to evaluate the post-Cryopreservation survival rate of in vitro-produced bovine blastocysts (B) and expanded blastocysts (eB) using Dulbecco's phosphate-buffered saline (PBS) or TCM-199 (TCM; Sigma-Aldrich, St Louis, MO, USA) as holding medium during vitrification and warming in glass capillaries. Cumulus–oocyte complexes were in vitro-matured and fertilized as previously described (Mucci et al. 2006 Theriogenology 65, 1551–1562), and cultured in 4 well plates in groups of 50 in 400-�L drops in serum-free CR1aa under low oxygen condition. Grade B1, B2, and eB were selected at Day 7 post-insemination and allocated to 3 groups: vitrification in TCM, vitrification in PBS, and control (without vitrification). Vitrification and warming were performed according to Vajta et al. (1998, Mol. Reprod. Dev. 51, 53–58), replacing OPS with GC (Tecnon Argentina S.A., Buenos Aires, Argentina); 75 mM length, 1.4 mM internal diameter, 1.6 mM external diameter). Briefly, B and eB were incubated in 1.78 M ethylene glycol (EG) and 1.3 M dimethyl sulfoxide (DMSO) in TCM or PBS supplemented with 20% estrous cow serum (ECS) for 3 min. Embryos were then transferred for 25 s to TCM or PBS supplemented with 3.56 M EG, 2.6 M DMSO, 0.5 M sucrose, and 20% ECS (vitrification solution: VS). Loading of embryos (2 per capillary) was performed by touching a 1-�L drop of VS with the capillary. After this, each capillary was immediately submerged into and stored in liquid nitrogen. Warming was performed by placing the capillary tip directly into TCM or PBS supplemented with 0.25 M sucrose for 5 min. Embryos were then transferred to TCM or PBS containing 0.15 M sucrose and 20% ECS for 1 min. After warming, embryos were cultured for 72 h in CR1aa + 5% ECS to evaluate embryo survival (hatching rate). Data was analyzed using the CATMOD procedure (SAS Institute, Inc., Cary, NC, USA). No interaction was found between holding media and embryo stage. Vitrified-warmed embryos had a significantly lower hatching rate compared with the control group (P < 0.05), whereas no differences were found between TCM and PBS. Expanded blastocysts had a higher hatching rate than blastocysts (P < 0.05). In conclusion, TCM can be replaced with PBS for its use in vitrification procedures. This protocol modification allows a simplified use of this technique in field conditions.
127 IN VITRO DEVELOPMENT OF IVF CRYOPRESERVED BOVINE EMBRYOS SUPPORTED BY TCM-199 OR SOFaa CULTURE MEDIUM
Authors: Nicacio A. C. , Feitosa W. B. , Rovegno M. , Simões R. , Gonçalves J. S. de A. , Assumpção M. E. O. D'Á. , Visintin J. A.
Published: December 2006

In vitro bovine embryo production is commercially applied around the world. However, the cryopreservation of these embryos is not yet possible, which raises difficulties for the expansion of this biotechnology. The aim of this work was to evaluate the influence of the culture media on embryo development after cryopreservation. Cumulus–oocyte complexes obtained from slaughterhouse bovine ovaries were in vitro-matured, fertilized, and cultured. A total of 600 expanded blastocysts (between 7 and 9 days of culture) were cryopreserved by slow freezing, quick freezing, or vitrification methods. For slow freezing (slow group), the embryos were exposed to 10% ethylene glycol (EG) for 10 min and cryopreserved at 1.2�C per minute. For quick freezing (quick group), the embryos were exposed to 10% EG for 10 min and to 20% EG + 20% glycerol (Gly) for 30 s. For vitrification (vitrification group), the embryos were exposed to 10% EG for 10 min and to 25% EG + 25% Gly for 30 s. The straws (quick and vitrification groups) were placed in nitrogen vapor (0.8 cm over the liquid nitrogen) for 2 min and then immersed in liquid nitrogen. The embryos were thawed in air for 10 s and in a water bath at 25�C for 20 s. For warming, embryos were washed in PBS + 0.2% BSA + 0.3 M sucrose for 3 min and in PBS + 0.2% BSA for 3 min. To evaluate development after thawing, the embryos were cultured on a granulosa cell monolayer with TCM-199 or SOFaa for 4 days. The embryos of the slow group showed re-expansion rates of 55.55% (55/99) and 29.00% (29/100), respectively, for TCM-199 and SOFaa. The quick group showed re-expansion rates of 4.85% (5/103) and 7.22% (7/97), and the vitrification group 10.89% (11/101) and 14.00% (14/100), respectively, for TCM-199 and SOFaa. The slow group showed hatching rates of 47.47% (47/99) and 11.00% (11/100), respectively; the quick group did not show hatching rates in either medium. The vitrification group showed hatching rates of 7.92% (8/101) and 6.00% (6/100), respectively, for TCM-199 and SOFaa. The results were analyzed by chi-square test, and all values were significant at P < 0.05. The slow group showed difference in re-expansion and hatching rates when the different media were compared. The quick and vitrification groups did not show differences in re-expansion and hatching rates when the different media were compared. The slow group showed higher re-expansion and hatching rates than the quick and vitrification groups. In conclusion, the culture medium influences embryo development after slow freezing, and the TCM-199 is more appropriate than SOFaa.
This work was supported by FAPESP 04/05335-1
13 MANAGEMENT SYSTEM FOR FROZEN BULL SEMEN USING A RADIO FREQUENCY IDENTIFICATION TAG
Authors: Kato S. , Mukojima K. , Otani T. , Sakai K. , Tamura K. , Miyazaki K. , Minami N. , Imai H.
Published: December 2006

An appropriate traceability system is essential for safety during production and distribution of food products. In Japan, the ear-tag system has been applied for this purpose. In this system, newborn calves are tagged individually with a bar code, and are registered to the database, so that everyone can know the information regarding individual cattle and meat. However, the management of frozen semen and the validation of frozen straws are out of the scope of the system. A traceability system for beef production totally covering the progression from semen to meat is necessary for food safety and validation. We applied radio frequency identification (RFID) for the identification of individual frozen semen straws. RFID is the management technology that uses a small tag with a radio transmitter (RFID tag) that has recently been used in the field of distribution management. We consider this technology as a tool to trace artificial insemination (AI) data before the birth of a calf and to establish a management system for production of the Japanese Black cattle breed. The microchip (�-chip) we used as an RFID tag was manufactured by Hitachi, Ltd., Tokyo, Japan. The �-chip is the smallest RFID chip in the world (0.4 mm square) and has a unique identification number (capable of corresponding to 128 bits, 1038 in decimal notation) readable by the RFID tag reader. The �-chip was attached to the frozen semen straw by ultrasonic sealing. We demonstrated that the �-chip retained its function after being stored in liquid nitrogen for at least several months. The information for bull semen, which was dispensed into frozen semen straws with the attached �-chips, was stored in the system database, and the straws were stored in liquid nitrogen. These straws with the �-chips were distributed to local farmers and inseminated into recipient cattle; the AI conditions, results of pregnancy diagnosis, and birth conditions were recorded in the system database. This system using the �-chips may be useful for confirmation and acquisition of trust for a commercially valuable bull and for the study of the improvement of AI for reproduction.
130 EFFECTIVENESS OF BUTYLATED HYDROXYTOLUENE AS EGG YOLK SUBSTITUTE FOR CRYOPRESERVATION OF BOAR SPERMATOZOA
Authors: Rodriguez-Vilar L. , Hernandez M. , Lopez-Sanchez C. , Vazquez J. M. , Martinez E. A. , Roca J.
Published: December 2006

Butylated hydroxytoluene (BHT) has proven to be efficient as a supplement for cryopreservation boar spermatozoa (Roca et al. 2004 J. Androl. 25, 397–405). Moreover, it has been successfully used as an egg yolk substitute to cryopreserve goat spermatozoa (Khalifa and El-Saidy 2006 Anim. Reprod. Sci. 93, 303–315). The objective of this study was to evaluate the effectiveness of BHT as an egg yolk substitute for freezing boar spermatozoa. Nine sperm-rich ejaculate fractions were collected from 3 boars (3 ejaculates per boar) using the gloved-hand method. After centrifugation (2400g for 3 min), the sperm pellet of each ejaculate was split into 5 aliquots. The aliquots were diluted (to a final concentration of 1 � 109 sperm/mL) in a Tris-citric-glucose extender with 3% glycerol and supplemented with 20% egg yolk (positive control, PC aliquot) or BHT at the final concentrations of 0 (negative control, NC aliquot), 0.2, 0.4, and 0.8 mM. Diluted semen samples were dispensed into 0.5-mL straws, and frozen in a programmable cell freezer at 20�C min. Thawing was carried out in a water bath at 70�C for 8 s. Post-thaw sperm survival was assessed according to total sperm motility (TSM, %) using a CASA system (SCA�; Microptic, Barcelona, Spain), and plasma membrane integrity (PMI, %) and acrosome membrane integrity (AMI, %) using a flow cytometric procedure (SYBR-14/propidioum iodide/FITC-phycoerythrin), at 30 and 150 min post-thawing in diluted Beltsville thawing solution with spermatozoa held in a waterbath at 37�C (3 straws per ejaculate). Data were analyzed using a ANOVA mixed model including the main effects of aliquot, boar, post-thaw assessment time, and their interactions, with ejaculate and straw as random effects. All main effects had significant influence (P ≤ 0.01) in all post-thaw sperm assessments. However, no interactions (P ≥ 0.05) among main effects were shown. Data were combined for the 2 post-thaw assessment times. The best (P ≤ 0.05) post-thaw sperm quality (mean � SEM) was achieved in PC aliquots (47.11 � 3.10, 58.98 � 2.78, and 51.35 � 3.42 for TSM, PMI, and AMI, respectively). In NC aliquots, the percentage of TSM, PMI, and AMI were always below 1% (P ≤ 0.05). BHT has a beneficial (P ≤ 0.05) effect on post-thaw sperm assessments, and no differences (P ≥ 0.05) among concentrations were shown. The mean post-thaw sperm quality in the BHT aliquots was 8.50 � 0.80, 20.29 � 0.53, and 16.03 � 0.55 for TSM, PMI, and AMI, respectively. On the basis of these data, we can conclude that BHT has a protective effect for boar spermatozoa during the cryopreservation process. However, BHT alone is insufficient to replace the protective effect of egg yolk.
This work was supported by CICYT (AGF2005-00706), Madrid, Spain.
132 SUCCESSFUL EMBRYO TRANSFER OF IN VIVO-PRODUCED RED DEER (CERVUS ELAPHUS) EMBRYOS AFTER CRYOPRESERVATION BY SLOW FREEZING OR VITRIFICATION
Authors: Soler J. P. , Kaiser G. G. , Mucci N. , Ferre L. B. , Alberio R. H.
Published: December 2006

Multiple ovulation and embryo transfer (MOET) programs for red deer (Cervus elaphus) have been established commercially over the last decade, with embryo cryopreservation being a related practice necessary to enhance the use of valuable genetic information. The aim of this work was to establish alternative methods for red deer embryo cryopreservation by using slow freezing with ethylene glycol (SF–EG) and vitrification by open pulled straw (OPS) methods. After surgical flushing of 18 superstimulated donors, 54 transferable embryos were recovered; 28 were transferred fresh to synchronized recipients and the others were cryopreserved by SF–EG (n = 11) or OPS (n = 15), respectively thawed or warmed, and transferred to recipients. Fresh embryos were maintained in Dulbecco's PBS + 20% cow serum (holding medium, HM) until transfer (maximum 3 h after collection). SF–EG cryopreserved embryos were suspended in HM + 1.78 M EG + 0.1 M sucrose + 4 mg mL−1 BSA. After a 10-min equilibration, embryos were loaded individually into 0.25-mL plastic straws and placed into a −7°C methanol bath chamber. After seeding (5 min later), the straws were cooled from −7 to −35°C at a rate of 0.5°C min. Straws were plunged into and stored in liquid nitrogen. Thawing was performed by placing the straws in a 30°C water bath for 30 s; their contents were drained into HM until transfer. Embryos were vitrified using the OPS method with minor modifications. They were first incubated in HM + 1.78 M EG + 1.3 M DMSO for 3 min and then transferred for 25 s into a vitrification solution of HM + 3.56 M EG + 2.6 M DMSO + 0.5 M sucrose. Each embryo was loaded by touching a 1-µL drop with the straw, which was immediately submerged into and stored in liquid nitrogen. Warming was done by placing the narrow end of the straws into HM + 0.25 M sucrose for 5 min. Embryos were then transferred into HM + 0.15 M sucrose for 5 min and finally to HM until transfer. Both types of cryopreserved embryos were transferred a few hours after collection, immediately after thawing or warming. Before embryo transfer, the presence of corpus luteum (CL) of recipients was confirmed by laparoscopic examination. Each embryo was surgically transferred into the apical extreme of the uterine horn ipsilateral to the CL of one recipient. Pregnancy was determined by ultrasonography 41 days after embryo transfer. The pregnancy rate between groups was compared with the chi-square test (P < 0.05). No statistical differences were found between groups (Table 1). Our results show that both vitrification and slow freezing methods with EG are suitable to cryopreserve red deer embryos.

Table 1.
Pregnancy rates in recipient hinds after transfer of fresh, vitrified, or frozen red deer embryos
135 SHORT-TERM CRYOPRESERVATION OF VITRIFIED MOUSE MORULAE AT - 79°C
Authors: Takagi Y. , Shimizu M. , Morimura M. , Yokomizo S. , Hara K. , Sakamoto M.
Published: December 2006

Embryos of various species are successfully vitrified and cryopreserved in liquid nitrogen (<−150°C). Like the preservation of frozen somatic cells cooled by dry ice (−79°C), the cryopreservation of embryos at −79°C is useful for a reduction in the shipping costs. The purpose of this study was to evaluate the effect of the cryopreservation period at −79°C on the in vitro embryo viability of vitrified mouse morulae after thawing. Morula-stage mouse embryos were collected from superovulated ICR donors 70 h after hCG injection. The embryos were exposed first to 5% DMSO + 5% ethylene glycol (EG) in Dulbecco's PBS + 20% FCS (mPBS) for 2 min, and then equilibrated for 20–30 s in a vitrification solution composed of 10% DMSO + 10% EG + 0.6 M sucrose in mPBS. The embryos were loaded onto cryoloops (Lane et al. 1999 Nat. Biotech. 17, 1234–1236) and plunged directly into liquid nitrogen. The cryoloops were placed in 1.2-mL cryotubes and stored in a −79°C freezer for 1–7 days. The embryos were warmed by passing through 4 dilution media and rinsed with mWM culture medium. They were then cultured at 37°C in 5% CO2 for 44 h. Non-cryopreserved embryos and embryos cryopreserved in liquid nitrogen served as controls. Data were analyzed by the chi-square test and the Student's t-test. Results are shown in Table 1. There was no significant difference (P > 0.01) in the developmental abilities to the blastocyst stage of the vitrified embryos that were cryopreserved at −79°C for 1 day, 3 days, and 5 days, the embryos cryopreserved in liquid nitrogen, and the non-vitrified control. The blastocyst rate of embryos was significantly lower (P < 0.01) for the Day 7 group than for the control group. The cell numbers of blastocysts were significantly lower (P < 0.01) for the Day 1, Day 3, Day 5, and Day 7 groups than for the control group. This study suggests that vitrified mouse morulae can be successfully cryopreserved at −79°C for 5 days.

Table 1.
Effect of the cryopreservation period on the viability of vitrified mouse morulae preserved at −79°C
140 LOCALIZATION OF PRIMORDIAL GERM CELLS IN DAY 21 BOVINE EMBRYOS
Authors: Alexopoulos N. I. , D'Cruz N. T. , Maddox-Hyttel P.
Published: December 2006

In most animal species, germ cell precursors, i.e., primordial germ cells (PGCs), arise from the epiblast and then migrate to the future gonadal ridge during development. At least in the mouse, PGCs may be cultured as embryonic germ cells that remain pluripotent. PGCs are the only cells in which OCT4 expression is maintained after gastrulation. The present study aimed at identifying the localization of PGCs in Day 21 in vivo-derived bovine embryos by immunohistochemical staining against OCT4. Six embryos were obtained after slaughter of superovulated heifers 21 days after insemination. The uterine tracts were flushed and embryos fixed, paraffin-embedded, and processed for immunohistochemistry. Embryos were sagitally sectioned, and selected serial sections were immunohistochemically stained for OCT4 to identify potential PGCs. Two embryos were at the neural groove stage. At this stage of development, the primitive gut had not yet been abstricted from the yolk sac and the allantois was not visible. A weak homogeneous OCT4 staining was localized to nuclei in a well-defined region of the epiblast, which was in the process of a gradual anterior to posterior differentiation into neural and surface ectoderm. Moreover, a strong OCT4 staining was localized to a few scattered cells found in the visceral mesoderm associated with the yolk sac in the region of the endoderm-hypoblast transition at some distance from the embryo proper. Four embryos were at the neural tube/somite stage. At this stage of development, the primitive gut had been defined and only the midgut was connected to the yolk sac. Furthermore, the allantois was visible as an anchor-shaped structure at the posterior end of the embryo. A strong OCT4 staining was found in nuclei of solitary cells in the endoderm and its associated visceral mesoderm of the ventral aspect of the mid and hindgut. The described OCT4 staining corresponds well with previous findings in the pig, in which presumptive PGCs are found in the endoderm epithelium during the neural groove stage. Later, during the early somite stages, they are localized in the endoderm and visceral mesoderm of the yolk sac and allantois, and in later somite stages, they are found in the developing genital ridge. This is, however, the first study to demonstrate the localization of these cells, at least by OCT4 staining, in bovine embryos at the neural groove and neural tube/somite stages.
148 EFFECT OF FOLLISTATIN TREATMENT POST-FERTILIZATION ON TIME TO FIRST CLEAVAGE, DEVELOPMENT TO THE BLASTOCYST STAGE, AND CELL ALLOCATION OF IN VITRO-PRODUCED BOVINE EMBRYOS
Authors: Lee K. B. , Bettegowda A. , Ireland J. J. , Smith G. W.
Published: December 2006

Previous studies from our laboratory have demonstrated a positive association of follistatin mRNA abundance with oocyte competence. Follistatin mRNA is greater in germinal vesicle stage oocytes collected from prepubertal (model of poor oocyte competence) vs. adult animals. Furthermore, follistatin mRNA abundance is also greater in early-cleaving 2-cell bovine embryos (collected prior to the maternal zygotic transition and initiation of significant transcription from the embryonic genome) than their late-cleaving counterparts. Given these results and the fact that early-cleaving embryos develop to the blastocyst stage at a greater rate, we hypothesized that follistatin has a stimulatory role in early embryonic development. To begin to test this hypothesis, we determined the effects of follistatin treatment of in vitro-produced bovine embryos (during the initial 72 h post-fertilization) on time to first cleavage, development to the blastocyst stage (Day 7), and blastocyst cell allocation (quality). Cumulus–oocyte complexes (COCs) were harvested from ovaries obtained from a local abattoir, matured, and fertilized in vitro. After 20 h of co-incubation with spermatozoa, presumptive zygotes were stripped of cumulus cells and cultured in KSOM medium supplemented with 0.3% BSA containing 0, 1, 10, or 100 ng mL-1 follistatin (n = 25 presumptive zygotes per treatment; n = 6 replicates). Proportions of embryos reaching the 2-cell stage within 30 h (early-cleaving), 30–36 h (late-cleaving), and within 48 h post-fertilization (total cleavage rate) were recorded. Embryos at the 8–16-cell stage were separated 72 h after fertilization and cultured in fresh KSOM medium supplemented with 0.3% BSA and 10% FBS until Day 7. The proportion of embryos reaching the blastocyst stage at Day 7 post-fertilization was recorded and the numbers of inner cell mass (ICM) and trophectoderm (TE) cells determined by differential staining. Follistatin treatment did not increase the rate of total cleavage and the proportion of late-cleaving embryos when compared to control. However, supplementation with 1 and 10, but not 100, ng mL-1 follistatin increased the proportion of early-cleaving embryos (26.3 and 35.3% vs. 9.5%) and development to the blastocyst stage (28.6 and 31.7% vs. 18.4%) relative to controls (P < 0.05). Treatment with 10 ng mL-1 follistatin increased total cell numbers (130.1 vs. 110.9) and proportion of trophectoderm cells (61.6% vs. 48.4%) and decreased the ICM/total cell ratio (38.4% vs. 51.5%) in Day 7 blastocysts relative to controls (P < 0.05). The results indicate that exogenous follistatin treatment during the early stages of in vitro bovine embryo development can enhance time to first cleavage, development to the blastocyst stage, and cell allocation in favor of increased trophectoderm cells, and can support a potential functional role for follistatin in early embryogenesis.
15 EFFECT OF BULL EPIDIDYMIS STORAGE CONDITIONS ON CRYOPRESERVED EPIDIDYMAL SPERM IN VITRO FERTILITY AND LIPID PEROXIDATION STATUS
Authors: Nichi M. , Rijsselaere T. , Van Soom A. , De Clercq J. B. P. , Goovaerts I. G. F. , Barnabe V. H. , Bols P. E. J.
Published: December 2006

Although cryopreservation of epididymal sperm has been studied extensively in several species, some factors that could negatively influence its quality are still unknown, such as the storage conditions of the epididymides prior to sperm collection. Studies indicate that the lower the storage temperature, the better the sperm quality after collection (Kaabi et al. 2003 Theriogenology 60, 1249–1259). An additional factor is lipid peroxidation in which sperm membrane resistance against reactive oxygen species (ROS) attacks is an important component. The objective of this experiment was to study whether the epididymis storage temperature following slaughter, as well as the intrinsic resistance against oxidative stress, affects the fertilizing capacity of cryopreserved epididymal bull sperm in vitro. Twelve epididymides (6 bulls) were collected after slaughter and divided into 2 groups, (stored at either 4 or 37�C for 2), after which semen was collected from the caudae epididymides and cryopreserved. Subsequently, one aliquot of the frozen–thawed semen samples was subjected to induced lipid peroxidation with ferrous sulfate and ascorbate (37�C; 2 h), after which tiobarbituric acid-reactive substances (TBARS), as an index of lipid peroxidation, were measured according to a method previously described (Beorlegui et al. 1997 Andrologia 29, 37–42). A second aliquot was used for in vitro fertilization in a routine IVF–IVC setup in duplicate (24-h maturation, SOF culture medium in 5% CO2, 5% O2, and 90% N2). In vitro embryo production results at Day 7 and TBARS levels were statistically analyzed using SAS (SAS Institute, Inc., Cary, NC, USA). No influence of storage temperature was observed at either TBARS level (4�C: 943.6 � 173.4; 37�C: 751.4 � 136.2 ng of TBARS/108 spermatozoa; P = 0.3) or on blastocyst rates (4�C: 23.0 � 2.8; 37�C: 18.7 � 3.6% of blastocysts; P = 0.2). However, the percentage of hatched blastocysts tend to be higher for epididymides stored at 4�C when compared to those stored at 37�C (6.4 � 1.8 and 2.3 � 0.9, respectively; P = 0.06). In addition, a negative correlation was found between TBARS concentrations and blastocyst rates (R = –0.57; P < 0.05). Compared to fresh samples collected from epididymides under the same conditions (unpublished data), levels of TBARS were two- to threefold higher for the cryopreserved sperm, indicating that lipid peroxidation appears to play a role in the decrease in quality of cryopreserved epididymal sperm. In conclusion, temperature during the epididymides short-term storage prior to sperm cryopreservation did not seem to influence the sperms' in vitro fertilizing capacity. On the other hand, an alternative to improve cryopreserved epididymal sperm in vitro fertility (or fertilizing capacity) could be the addition of antioxidants to semen extenders. Further studies are necessary to confirm this hypothesis.
150 THE DISTRIBUTION PATTERN OF PRIMORDIAL GERM CELLS IN EARLY CHICK EMBRYOS
Authors: Nakamura Y. , Yamamoto Y. , Usui F. , Ono T. , Takeda K. , Nirasawa K. , Kagami H. , Tagami T.
Published: December 2006

In all vertebrates, primordial germ cells (PGCs) appear during early stages of development in extragonadal sites, then they migrate to the gonad and give rise to ova or spermatozoa. Unlike in other species, however, in avian and reptile embryos, PGCs use the vascular system as a vehicle to transport them to the future gonadal region where they leave the blood vessels. The present study was carried out to know the details of this unique migration pathway and the proliferation of endogenous PGCs in chicken embryos. Whole of the chicken embryos during stages X [Roman numerals refer to the staging system of Eyal-Giladi and Kochav (1976 Dev. Biol. 49, 321–327) to 17 (Arabic numerals refer to the staging system of Hamburger and Hamilton (1951 J. Morphol. 88, 49-82))] or embryonic blood during stages 12 to 17 were immunohistochemical stained using specific antibody raised against chicken vasa homolog (CVH), which could be recognized as a marker for chicken PGCs. The distribution patterns and populations of PGCs in embryos were observed under a stereomicroscope. The numbers of PGCs were presented mean and standard deviation (mean � SD). Anti-CVH staining revealed the distribution and population of chicken PGCs in early chick embryos. PGCs existed mainly in the area pellucida and concentrated in the central zone at stage X. The mean number of PGCs per embryo at this stage was 130.4 � 31.9. With the formation of primitive streak, PGCs were carried anteriorly to the edge of the blastoderm. The PGCs scattered anteriorly began to concentrate to the anterior point of the head on the dorsal side of stage 10 embryos. The average number of PGCs per embryo at stage 10 was 439.3 � 93.6. The mean numbers of PGCs per embryo during stages X to 10 increased gradually as development progressed to stage 10. We found the entrance point of PGCs from anterior edge of the blastoderm to the vascular network during stages 10 to 11. In the blood, PGCs could be detected from all of the samples during stages 12 to 17. In contrast, no PGC was recognized in the future gonadal region before stage 14, and then they began to appear in the same region at stage 15. The mean numbers of PGCs that located in the future gonadal region during stages 15 to 17 increased intensively and were 97.3 � 57.3, 200.3 � 113.5, and 327.6 � 102.4, respectively. Interestingly, the numbers of PGCs within future gonadal region during stages 15 to 17 were consistently and significantly different (P < 0.05) between the left and right side of the region. The results suggest that chicken PGCs move from extraembryonic area to the vascular network during stages 10 to 11, circulate in the blood stream, and finally, they begin to leave the blood vessels actively and migrate to the future gonadal region at stage 15.
154 UNDERNUTRITION DURING THE PERICONCEPTIONAL PERIOD IN EWES AFFECTS THE BLOOD PRESSURE AND WEIGHT OF THEIR NEONATAL LAMBS
Authors: Smith N. A. , Lonergan P. , Duffy P. , Crosby T. F. , Quinn P. , McAuliffe F. , Evans A. C. O.
Published: December 2006

Evidence from epidemiological and experimental studies has shown that maternal undernutrition during pregnancy can alter fetal growth and development and is associated with increased risk of hypertension and cardiovascular disease in the offspring. However, there is a paucity of data examining the effect of undernutrition during the periconceptual period on offspring health. Therefore, we investigated the impact of ewe undernutrition during the periconceptional period on lamb birth weight and blood pressure. Crossbred ewes (n = 166) were individually penned and randomly allocated to 1 of 2 treatments and fed either 0.7 (restricted) or 1.1 (control) maintenance energy requirements from 28 days prior until 7 days post-mating. After Day 7, ewes in both treatments were managed similarly until parturition. Following parturition, lamb gender, birth weight, and blood pressure were recorded. Lamb weight and blood pressure were subsequently measured at 4 and 10 weeks of age. Restricted ewes lost 2.6 � 0.2 kg over the treatment period compared with control ewes which gained 1.7 � 0.58 kg (P < 0.01). There was no treatment effect on lamb birth weight in either singleton or twin gestations. However, at 4 and 10 weeks of age singleton lambs born to nutritionally restricted ewes were heavier than those born to control ewes (males, n = 17: 19.4 � 0.1 vs. 16.4 � 0.1 kg; females, n = 16: 34.9 � 1.0 vs. 30.9 � 1.6 kg, P < 0.05). Blood pressure, measured within the first 24 h of life, was higher (P < 0.05) in singleton lambs born to nutritionally restricted mothers compared to controls (males: systolic 126 � 11 vs. 94 � 5; diastolic 99 � 12 vs. 58 � 5; mean 112 � 11 vs. 77 � 5 mmHg; P < 0.05; females: systolic 126 � 11 vs. 94 � 5; diastolic 99 � 12 vs. 58 � 5; mean 112 � 11 vs. 77 � 5 mmHg; P < 0.05). This difference was no longer apparent at 4 or 10 weeks of age. Mean blood pressures of twin lambs at birth and 4 weeks of age were numerically greater in the restricted than in the control treatment but were significantly different only among male twin groups at 4 weeks of age (diastolic 77 � 4 vs. 94 � 4; mean 96 � 4 vs. 110 � 3 mmHg; P < 0.01). Maternal undernutrition during the periconceptional period was associated with increased offspring weight at 4 and 10 weeks of age in singletons, as well as alterations in offspring cardiovascular function in both singletons and twins. These findings suggest that maternal nutrition at the time of conception influences offspring health. Exact mechanisms remain to be elucidated but may involve genetic modification.
This research was funded by IRCSET and University College Dublin.
155 LAPAROSCOPIC EVALUATION OF OVARIAN REACTION TO HORMONE STIMULATION IN CYNOMOLGUS MONKEYS (MACACA FASCICULARIS)
Authors: Tsuchiya H. , Iwatani C. , Yamasaki J. , Okahara J. N. , Okahara N. , Torii R.
Published: December 2006

Oocyte collection is a key step to development of a system for modeling human regenerative medicine and assisted reproductive technology in cynomolgus monkeys (Macaca fascicularis), but collecting oocytes at a suitable developmental stage (metaphase II) is difficult. Metaphase II (MII) oocytes can be collected from cynomolgus ovaries stimulated by hormone injection. In this study, we developed a useful method for collecting a large number of MII oocytes by monitoring the morphology and size of ovaries with laparoscopic observation. Controlled ovarian stimulation and oocyte recovery in mature cynomolgus monkeys have been previously described by Torii et al. (2000 Biochemistry 39, 3197–3205). Beginning at menses, levels of estrogens were down-regulated by the subcutaneous injection of a GnRH antagonist (Leuplin, 0.9 mg animal-1; Takeda Chemical Industries, Ltd., Osaka, Japan). Two weeks later, human follicle stimulating hormone (FSH, Fertinorm, 25 IU kg-1; Serono, Canton Zug, Switzerland) was administered for 9 days. On the day after that of the last FSH administration, human chorionic gonadotropin (hCG, Puberogen, 400 IU kg-1; Sankyo Co., Ltd., Tokyo, Japan) was intramuscularly injected. Follicular aspiration was performed at 404-41 hours post-hCG injection. Oocyte collection was monitored using a 3-mm laparoscope attached to a video system. Oocytes were aspirated from the follicles using a 20-guage needle. Follicle development of the ovary was rated morphologically as A (small follicles), B (many small follicles), or C (many large follicles), and size relative to the uterus was rated as 1 (no response), 2 (smaller), 3 (equal), or 4 (larger) at oocyte collection. Regarding morphology, the highest ratio of MII/total oocytes was obtained from B–C ovaries (rating of 2 ovaries, one each left or right with a rating of B or C, n = 8, 26.9 � 22.4, 70.7%), followed by B–B (n = 21, 19.5 � 14.3, 55.9%), and C–C (n = 19, 10.6 � 5.8, 51.9%). No MII oocytes were collected from A–A (n = 1, 0%) ovaries. Ovaries appeared to be over-stimulated in the ovaries rated C–C but under-stimulated in B–B. Regarding ovary size, the highest ratio of MII/total oocytes was obtained from 4–4 (both ovaries with rating of 4, n = 10, 21.9 � 11.2, 68.8%), followed by 3–3 (n = 21, 19.1 � 14.3, 58.2%), and 2–2 (n = 9, 11.6 � 6.5, 53.3%). No MII oocytes were collected from 1–1 ovaries (n = 1, 0%). The number of MII oocytes collected was directly related to ovary size: more MII oocytes were collected from larger ovaries. These data demonstrate that the number of oocytes collected is directly related to ovary size. Our results suggest that the ratio of MII oocytes can be predicted by the morphology and the size of ovaries. In addition, we found that ovarian development can be controlled by adjusting FSH dosage. Therefore, laparoscopic observation of ovaries during FSH treatment and adjusting FSH dosage are necessary to collect MII oocytes efficiently.
156 DIFFERENTIAL REORGANIZATION OF HISTONE ACETYLATION THROUGH ATP-DEPENDENT REMODELING FACTOR DURING PRONUCLEAR FORMATION IN PORCINE ZYGOTIC CHROMATINS
Authors: Wee G. , Koo D.-B. , Kim J.-S. , Song B.-S. , Park J.-S. , Jin X.-L. , Lee Y.-Y. , Han Y.-M. , Lee K.-K.
Published: December 2006

ATP-dependent remodeling complexes and histone acetyltransferase/deacetylase complexes in chromatin modification can be characterized by the use of energy from ATP hydrolysis or covalent modification. But they have similar functions during the transcriptional process. After fertilization, histone acetylation in paternal and maternal chromatin is reprogrammed to obtain transcriptional activity during chromatin remodeling such as decondensation. However, these mechanisms in zygotic chromatin are poorly understood. In the present study, the reorganization process of histone H4 acetylation after fertilization was investigated through co-localization in the nucleus of ATP-dependent remodeling factors and histone acetyltransferases during parental chromatin decondensation. The molecules were monitored by immunofluorescence analysis with specific antibodies directed against AcH4K5, HAT1, P300, Tip60, Brg-1, and Mi-2. Fluorescence signals of Brg-1 and Mi-2 in porcine embryonic fibroblasts and HeLa cells co-localized with chromatin during interphase and M phase, although the Mi-2 signal existed around chromosomes at metaphase. However, Brg-1 and Mi-2 in porcine oocytes did not interact with chromosomes during meiosis, despite their existence in the oocyte cytoplasm. At 6 h after fertilization, signals of Brg-1 and Mi-2 were observed in most parental chromatin and remained until syngamy of the pronuclear stage. In histone acetylation and chromatin remodeling, acetylation of H4K5 was generated from sperm chromatin at 4 h after fertilization and preceded the appearance of Brg-1. Additionally, HAT1 showed stronger intensities compared with P300 and Tip60, correlating with acetylated-H4K5, and also appeared earlier than Brg-1. In contrast to that in sperm chromatin, Brg-1 in maternal chromatin anteceded HAT1. Consequently, paternal chromatin remodeling was completed after histone acetylation, but maternal chromatin remodeling was preceded by histone acetylation. Our findings indicate that paternal and maternal chromatin undergo differential remodeling and reprogramming during pronuclear formation, suggesting that gene expression in the chromatin of each parent may be regulated separately.
159 LOCALIZATION OF CLAUDIN FAMILY PROTEINS IN PIG EMBRYOS DURING PRE-IMPLANTATION DEVELOPMENT
Authors: Xu S. , Lee J. , Harayama H. , Miyake M.
Published: December 2006

Tight junctions (TJ) are critical for blastocoel formation in mammalian pre-implantation embryos. Claudin family proteins, which are TJ proteins, are generally important for the barrier function of TJ. However, the expression and localization of claudin proteins are not clarified in mammalian pre-implantation embryos. The present study was designed to examine changes in the localization of claudin isoforms, claudin-1, -2, and -4, in pig parthenogenetic diploids through pre-implantation development. Oocyte–cumulus–granulosa cell complexes were collected from the follicles 4–6 mm in diameter, and then maturation-cultured. Only oocytes with a prominent first polar body were subjected to electro-stimulation after maturation, and they were treated with cytochalasin B to produce parthenogenetic diploids. Presumptive diploids were then cultured for 168 h and observed every 24 h. Embryos of each developmental stage from 2-cell to blastocyst were subjected to immunofluorescence staining of the anti-TJ proteins. Zona-free embryos were fixed and treated with rabbit anti-claudin-1 and -2 polyclonal antibodies or mouse anti-claudin-4 monoclonal antibody, followed by treatment with Alexa fluor 488-labeled goat anti-rabbit IgG antibody or goat anti-mouse IgG antibody. Some embryos were treated with rabbit anti-occludin antibody or anti-ZO-1 antibody for the detection of the TJ-net work. All embryos were counterstained with Hoechst 33342 and observed under an epifluorescence microscope after whole-mounting. The specific fluorescence for all TJ proteins examined was observed in embryos at all pre-implantation stages. Occludin and ZO-1 were detected in the cytoplasm before the morula stage (96 h), and localized toward the boundary region among cells from the morula and early blastocyst stages, reflecting the distribution of the tight junction in embryos. Claudin-1 and -2 localized in the cytoplasm at early-cleaving stages. They were detected in the nucleus after compaction, and the distribution of these proteins in the nucleus was dominant at the blastocyst stage. The distribution of the 2 claudin proteins in the nucleus was quite different; claudin-1 distributed rather homogeneously, but claudin-2 formed several bright spots in the nucleus. Claudin-4 also showed a unique distribution pattern in embryos; it was detected in the cytoplasm with strong fluorescence at the periphery of the nucleus of the 2- and 4-cell embryos. Claudin-4 changed its localization toward the boundary region of cells around the early blastocyst stage (120 h), and then the distribution of claudin-4 was restricted to the boundary region later in the blastocyst stage (144 h). These results indicate that claudin-4, but not claudin-1 and -2, is responsible for the formation of TJ in the pre-implantation embryos, although the function of these claudin proteins in the nucleus is unknown.
161 EFFECT OF MELOXICAM TREATMENT ON PREGNANCY RATES IN HOLSTEIN HEIFERS
Authors: Guzeloglu A. , Erdem H.
Published: December 2006

We previously reported that administration of flunixin meglumine two times at a critical stage approaching pregnancy recognition associated with corpus luteum maintenance increased early embryo survival and pregnancy rate via an additive antiluteolytic effect with the conceptus (Guzeloglu et al. 2006 Reprod. Fertil. Dev. 18, 183). In this study, the objective was to determine if a single administration of meloxicam, a non-steroid anti-inflammatory drug (NSAID) with a longer half-life, could be used instead of flunixin meglumine to avoid repeated injections in heifers at a critical stage following insemination to increase pregnancy rate due to its inhibitory effect on PGF2� synthesis. Eighty-five 15-18-month-old Holstein heifers were synchronized; following insemination (Day 0), the heifers were randomly assigned to receive a subcutaneous meloxicam injection (0.5 mg kg-1; n = 37) in the afternoon of Day 15 or left untreated as controls (n = 48). Pregnancy rates were defined as the percentage of heifers diagnosed pregnant by ultrasound between Days 31 and 38 after AI. The effect of treatment on pregnancy rates was analyzed by chi-square test. Meloxicam treatment on Day 15 after insemination dramatically decreased the pregnancy rates in the heifers (25 of 48 (52%) in the control group vs. 9 of 37 (24.3%) in the meloxicam-treated group; P < 0.01). This result indicates that administration of meloxicam around a time associated with pregnancy recognition and corpus luteum maintenance was harmful to the pregnancy even though the drug is considered to be safe during pregnancy in cattle. It is concluded that direct or indirect effects of meloxicam on the uterine environment and/or embryo are not favorable at least during early pregnancy in cattle. How meloxicam exerted its detrimental effect on pregnancy warrants further investigation.