bluuebunny/crossref_metadata_2025 huggingface dataset:
171 EFFECT OF LONG-TERM TRANSPORTATION OF OVARIES ON THE DEVELOPMENT OF IN VITRO-PRODUCED BOVINE EMBRYOS
Authors: Nakatate M. , Tsuchiya K. , Adachi I. , Takahashi K. , Aisan A. , Sugulle A. , Dochi O. , Koyama H.
Published: December 2006

The transportation of bovine ovaries would allow the shipment of oocytes for research purposes after the slaughter of valuable cows. The objective of this study was to investigate the effect of long-term transportation of ovaries on the development of in vitro-produced bovine embryos. After collection of the ovaries from a slaughterhouse, they were placed inside a thermos flask and transported to the laboratory. The thermos flask was covered with a freezer pack in a foam polystyrene box. The transportation time was 17–18 h, and the temperature of the thermos flask changed from 20°C to 28°C (average 23.8°C) during the transportation. Cumulus–oocyte complexes (COCs) were collected by the aspiration of follicles with a diameter of 2–6 mm. The COCs were matured for 20 h in IVMD101 (RIFP: Research Institute for the Functional Peptides, Yamagata, Japan) containing DM199 supplemented with 5.56 mM glucose, 0.91 mM pyruvate, 5 mM taurine, 5 mM selenium, 5 mM HEPES, and 10 µg/mL gentamicin at 38.5°C under an atmosphere of 5% CO2 in air (Hoshi 2003 Theriogenology 59, 675–685). The matured COCs were inseminated with 5 × 106 sperm/mL in IVF100 (RIFP) medium comprising a modified BO medium supplemented with 1.25 mM sodium pyruvate, 0.5 mM cysteine, 5 mg/mL BSA, 7.5 µg/mL sodium heparin, 5 mM caffeine, and 10 µg/mL gentamicin. After 6 h of gamete co-culture, the presumed zygotes were cultured in IVD101 (RIFP) medium comprising DM199, 2.48 mM lactate, 0.27 mM pyruvate, and 2.22 mM of glucose for 9 days at 38.5°C under an atmosphere of 5% CO2, 5% O2, and 90% N2 in air. As controls, bovine ovaries were transported to the laboratory within 1–1.5 h. Embryo development was evaluated based on the cleavage rate, blastocyst rate, and total number of cells on Days 7–9 after in vitro fertilization. The experiment was replicated five times, and data were analyzed by chi-square test and ANOVA. Results are presented in Table 1. There were no differences in the cleavage rate between the treatments. The blastocyst rate and the number of cells in the blastocyst after long-term transportation of ovaries were significantly lower than those in the controls. These results suggest that the long-term transportation of bovine ovaries does not affect on the cleavage; however, the blastocyst rate and the quality of blastocysts may be affected. Therefore, additional experiments are required to determine suitable conditions for long-term transportation of bovine ovaries.

Table 1.
Effect of long-term transportation of ovaries on the development of bovine IVM/IVF embryos
176 EFFECT OF HYPERTHERMIA ON DEVELOPMENTAL COMPETENCE OF GERMINAL-STAGE OOCYTES: IN VIVO AND EX VIVO STUDIES IN MICE
Authors: Roth Z. , Aroyo A. , Yavin S. , Arav A.
Published: December 2006

Mammalian oocytes are susceptible to thermal stress at various stages of follicular development. The present study was performed to determine the effects of maternal hyperthermia on germinal vesicle (GV)-stage oocytes in mice. Evaluation parameters included (a) the oocyte's ability to become fertilized, (b) ex vivo embryonic development, (c) pregnancy outcome, and (d) quality of offspring. Female mice (CB6F1) were synchronized with pregnant mare serum gonadotropin (PMSG) followed by hCG 48 h later. One hour after hCG administration, control mice were kept under normo-thermal conditions (22�C, 45% RH), whereas the treated mice were exposed to thermal stress (40�C, 70% RH) for 1.5 to 2 h to induce a rise of 1.8�C in rectal temperature. After a 1-h recovery period, both treated and control mice were paired with stud males overnight. In Exp. 1, mated mice were sacrificed 20 h after hCG administration and embryos were recovered and cultured in vitro. The number of putative zygotes recovered was counted, and cleavage and blastocyst-formation rates were recorded on Days 1 and 5 post-fertilization, respectively. Examination of oocyte developmental stage during hyperthermia induction revealed that oocytes were at the GV stage. The percentage of putative zygotes that cleaved into the two-cell stage was higher (P < 0.05) in the control (91.2 � 1.3) than in the treated group (84.5 � 1.7). In addition, blastocyst-formation rate was higher in the control (P < 0.05; 84.3 � 1.8%) than in the treated group (57 � 2.6%). In Exp. 2, females were left with the stud males until litter delivery. The date of delivery and the number of pups were documented. Eight-week-old offspring were submitted to behavioral (locomotor activity and episodic memory) tests. The one-way ANOVA procedure of JAMPIN (SAS Institute, Inc., Cary, NC, USA, 2004) was used for statistical analysis. The percentage of females that gave birth during the first and second estrous cycles after hyperthermia induction was 59% and 71% for treated and control groups, respectively. The average number of pups in the first estrous cycle was lower (P < 0.05) for the treated group, and only became equal to that in the control group in the third estrous cycle. Behavioral tests did not reveal any differences between pups from the two experimental groups. In summary, GV-stage oocytes from stimulated ovaries are highly sensitive to thermal stress, since induction of maternal hyperthermia during this stage disrupts oocyte developmental competence both in vivo and ex vivo. However, oocytes that survive thermal stress exhibit normal developmental potential, since heat-stressed mice produced normal pups, at least as reflected by offspring behavioral tests. Thus, strategies designed to protect the ovarian pool of oocytes from thermal stress are likely to improve fertility in heat-stressed females.
18 DO TEMPORARY CALF REMOVAL AND/OR EQUINE CHORIONIC GONADOTROPIN ADMINISTRATION INCREASE PREGNANCY RATES IN LACTATING NELORE COWS TREATED WITH A PROGESTERONE-RELEASE INTRAVAGINAL DEVICE?
Authors: Pinheiro V. G. , Souza A. F. , Pegorer M. F. , Ereno R. L. , Barros C. M.
Published: December 2006

Reports indicate that either temporary calf removal or equine chorionic gonadotropin administration can increase the efficiency (pregnancy rate) of hormonal treatments with progestins during postpartum anestrus. This experiment evaluated effects of TCR and/or eCG administration in a protocol with progesterone that is frequently used for fixed-time artificial insemination (FTAI) in cows during postpartum anestrus. The protocols were tested at three farms in lactating Nelore cows (40 to 70 days post-partum, n = 361) with body condition scores from 2.5 to 3.0 (0- to 5-point scale). At a random stage of the estrous cycle (Day 0), animals received a basic PEPE (progesterone-estrogen-prostaglandin-estrogen) protocol with insertion of an intravaginal device with 1.0 g progesterone (DIB�, Syntex, Buenos Aires, Argentina) and an i.m. injection of 2.5 mg of estradiol benzoate (EB, Estrogin�, Farmavet, S�o Paulo, Brazil). Eight days later (Day 8) cows were treated i.m. with 150 mg D-cloprostenol (PGF2� Prolise�, ARSA S.R.L., Buenos Aires, Argentina), and the DIB was removed. Twenty-four hours after DIB removal, cows received 1.0 mg EB i.m. and 30 to 36 h later all animals were FTAIed without estrus detection. Cows were allocated randomly to four groups: PEPE, PEPE/TCR, PEPE/eCG, and PEPE/TCR/eCG. In Group PEPE/TCR, calves were removed temporarily for 54 h (from DIB removal until FTAI). In Group PEPE/eCG, animals received PEPE treatment plus one dose i.m. of 300 UI eCG (Novormon�, Syntex) following PGF2� administration (Day 8). In Group PEPE/TCR/eCG, animals were treated as in protocol PEPE/TCR plus eCG on D8. All animals were examined by ultrasonography (Aloka SSD 500, 7.5 MHz probe) 10 days before and at the beginning of hormonal treatment in order to detect anestrous cows (absence of corpus lutcum in both exams). Pregnancy diagnosis was performed by ultrasonography 30 days after FTAI. The data were analyzed by logistic regression. The following variables were considered in the model and did not affect pregnancy rates: farms, inseminators, and semen (sire). A total of 75% of the animals were in anestrus (absence of CL) and the pregnancy rates were similar (P > 0.05) among the four groups: PEPE (43/85 = 50.6%), PEPE/TCR (42/98 = 42.9%), PEPE/eCG (41/88 = 46%), and PEPE/TCR/eCG (39/90 = 43.3%). The results indicate that in Nelore cows, in postpartum anestrus and good body condition, TCR and/or eCG administration do not improve the efficiency (pregnancy rate) of the PEPE protocol.
This work was supported by FAPESP. V.G.P. and A.F.S. received fellowships from CNPq, Brazil.
184 EFFECT OF LIVE CONFOCAL FLUORESCENCE IMAGING ON EMBRYONIC DEVELOPMENT
Authors: Ross P. , Cibelli J.
Published: December 2006

The aim of this study was to examine the effects of fluorescent confocal imaging on embryo development. DNA damage, caspase activation, and reactive oxygen species (ROS) production were also measured. In vitro-generated bovine 8-cell embryos and in vivo-produced mouse 2-cell embryos were stained with Mitotracker Red and individually imaged using a spinning-disc confocal module (CARV) attached to a Nikon TE2000-U microscope. The sample was excited with a 120 W metal halide lamp (X-Cite 120) using a fluorescent filter set (Excitation: 575 nm; Emission: 640 nm). A variety of number and durations of exposures were tested. Continuous variables were analyzed by t-test, whereas categorical data were analyzed using the homogeneity chi square test. Bovine embryos showed no differences in blastocyst development (Day 7.5; Day 0 = IVF) or hatched blastocysts (Day 11) between groups of imaged and non-imaged embryos (Table 1). Despite numerous attempts, we were unable to find a level of exposure that disturbed embryo development, indicating that bovine embryos have a high degree of tolerance to the imaging system. After exposure, mouse embryos were cultured in vitro to the blastocyst stage and transferred into surrogate synchronized females. Embryos subjected to five exposures of 50 ms developed normally both in vitro and in vivo (Table 1). However, exposing the embryos 30 times significantly affected in vitro and in vivo development. Subsequently, we examined the effect of confocal imaging on DNA damage (Comet assay), ROS production (dichlorofluoresceindiacetate, DCF), and caspase 3 activation (PhiPhiLux®-G1D2). Bovine embryos were exposed to 60z–50 ms or 60z–500 ms, and mouse embryos to 5z–50 ms or 30z–50 ms after Mitotracker Red staining. We observed that caspase 3 was not activated when measured 1.5 h after imaging. Similarly, DNA damage was not different among embryos exposed at different levels of imaging. However, ROS production significantly increased in both bovine and mouse embryos after exposure to confocal imaging, and this increase correlated with fluorescence exposure level. We conclude that imaging bovine pre-implantation embryos with this system increased ROS production but did not disturb in vitro development (even at unnecessary high levels of exposure). In mouse embryos, in vitro development after imaging directly correlated with in vivo development. It remains to be determined whether the increase in ROS production and decreased developmental potential is due to the fluorescence exposure alone or the combined effect of fluorescence exposure and Mitotracker labeling.

Table 1.
Embryonic development after confocal fluorescence imaging
189 PREGNANCY RATES IN HEAT-STRESSED DAIRY CATTLE RECEIVING ONE OR TWO IN VITRO-PRODUCED EMBRYOS IN A TIMED EMBRYO TRANSFER PROGRAM
Authors: Franco M. , Block J. , Jousan F. D. , de Castro e Paula L. A. , Brad A. M. , Franco J. M. , Grisel F. , Monson R. L. , Rutledge J. J. , Hansen P. J.
Published: December 2006

The objective was to determine whether transfer of two embryos would increase pregnancy rates in heat-stressed dairy recipients receiving an in vitro-produced embryo transferred into the uterine horn ipsilateral to the corpus luteum (CL). Such a treatment would increase the likelihood that the cow receives at least one embryo competent for sustained development. In addition, transfer of two embryos into the ipsilateral uterine horn is likely to increase the amounts of interferon-tau and other embryonic-signaling molecules in the uterus. A total of 32 virgin crossbreed heifers and 26 lactating crossbreed cows were used as timed embryo transfer recipients after being subjected to an ovulation synchronization protocol as follows: GnRH (100 �g) and insertion of previously used progesterone-containing CIDR on Day -10, prostaglandin F2� and CIDR removal on Day -3, and GnRH (100 �g) on Day 0 (day of anticipated ovulation). All recipients had a palpable CL on Day 6 and were randomly selected to receive one (n = 31 recipients) or two (n = 27) embryos on Day 7. At Day 64, the pregnancy rate tended to be higher (P = 0.07) for cows than for heifers. While not significant, heifers that received two embryos tended to have lower pregnancy rates than those that received a single embryo (20% vs. 41%); there was no difference in pregnancy rate in cows (50% for two embryos vs. 57% for one embryo). Pregnancy losses between Day 64 and Day 127 occurred in one group only cows receiving two embryos. In that group, pregnancy rate was 50% at Day 64 but 17% at Day 127. Overall, there was no difference in pregnancy rates at day 127 between cows and heifers, but recipients that received two embryos (17% for cows and 20% for heifers) had lower pregnancy rates (P < 0.03) than recipients that received one embryo (57% for cows and 41% for heifers). Only one animal, a cow, had twin fetuses at day 127. In conclusion, unilateral transfer of two embryos failed to improve pregnancy rates of dairy cattle exposed to heat stress. The fact that fetal loss occurred sooner for heifers than for cows points out the importance of uterine capacity as a limiting factor for maintenance of fetal development of two conceptuses. The suitability of timed embryo transfer was evident from the high pregnancy rates achieved with crossbreed females that received a single embryo.
This work was supported by BARD Grant No. US-1551-14, USDA TSTAR Grant No. 2004-14135-14715, Grant No. 2001-12101-11318 from the USDA-IFAFS Program, and CAPES Grant No. 134202-1).
190 EFFECT OF THE PROPERTIES OF CERVICAL MUCUS ON PREGNANCY RATES IN HOLSTEIN HEIFERS AND COWS AFTER EMBRYO TRANSFER
Authors: Maekawa T. , Morita S. , Douchi O. , Koyama H.
Published: December 2006

Selection of animals as recipients of embryo transfer is an important procedure of embryo transfer on farms. Most animals are evaluated for their acceptability as recipients based on the quality of their corpus leteum (CL). However, since rectal palpation is a subjective evaluation method, a more objective method of assessing the suitability of the recipient is required. Cervical mucus may be able to be used to evaluate the condition of the uterus indirectly. The purpose of this study was to investigate the relationship between the properties of cervical mucus and pregnancy rates after embryo transfer in Holstein heifers and cows. Cervical mucus was collected using a swab off the ostium uteri externum and was stained with 5% Giemsa's solution for 20 min one day before embryo transfer. The stained cervical mucus were classified based on the type of staining pattern (Kitamura et al. 2003 Theriogenology 59, 307) into five groups: filiaceous (Type 1), taenia (Type 2), claustral (Type 3), nubecula (Type 4), or aqueous (Type 5). Proportions of the types of cervical mucus and pregnancy rates were analyzed by chi-square test. In Experiment 1, 113 heifers and 266 cows were examined for cervical mucus type. No significant difference was observed in the proportions of the types of cervical mucus between heifers and cows (heifers: 35.4%, 18.6%, 16.8%, 25.7%, and 3.5%; cows: 24.4%, 14.3%, 20.3%, 30.8%, and 10.2% for Types 1∼5, respectively). In Experiment 2, either a fresh or frozen-thawed embryo was implanted in vivo in 84 heifers and 163 cows 7 days after estrus. The heifers and cows were judged to have normal sized CLs (normal, 17 mm or more) and have no vaginal abnormalities such as cervical mucus contaminated with pus and urovagina as per vaginal examination. The proportions of acceptable Type 5 recipients was lower than that of Type 1 (P < 0.05). The pregnancy rates were 47.6% for heifers and 45.4% for cows (Table 1). The pregnancy rates of Types 1–3 (53.5%) were significantly higher than for Types 4 and 5 (29.9%) in the cows (P < 0.05). Although there was no statistically significant difference, the same tendency was observed in the heifers. Pregnancy was unsuccessful in Type 5 recipients, both heifers and cows. The total pregnancy rates of Types 1–3 were significantly higher than for Types 4 and 5 (53.5% vs. 29.9%, P < 0.001). These results suggest that cervical mucus type can serve as an objective selection criterion for embryo recipients. Further, embryo transfer should be avoided in Type 5 recipients.

Table 1.
Cervical mucus type and pregnancy rates (%) in dairy cattle
194 ESTABLISHMENT OF GOAT EMBRYONIC STEM CELL-LIKE LINES DERIVED FROM IN VIVO-PRODUCED BLASTOCYST-STAGE EMBRYOS
Authors: Behboodi E. , Begin I. , Rao K. , Neveu N. , Pierson J. , Baldassarre H. , Karatzas C.
Published: December 2006

Pluripotent embryonic stem cells (ESC) derived from the inner cell mass (ICM) of mammalian blastocysts provide AN unlimited number of cells that can be used in gene targeting and be of great value to agriculture and medicine. Embryonic stem cells with capacity for germ line transmission have been verified only in mouse despite many efforts to derive ESC from other mammalian species. The methods for the derivation, propagation and differentiation of ESC from domestic animals have not been fully established. The objective of this study was the generation and initial characterization of goat embryonic stem cells (GESC) derived from in vivo-produced blastocyst-stage embryos. Goat compact morulae and blastocysts were collected from superovulated adult Saanen crossbreed donors 7 days after insemination with a fertile male. Embryos were collected by uterine flushing using a 12Fr. Foley catheter by means of a laparoscopically assisted mid-ventral laparotomy under general anesthesia. Twenty eight in vivo-derived blastosyst-stage embryos were cultured on a goat fetal fibroblast feeder layer (inactivated by Mitomycin C) in a medium of DMEM containing 0.1 mm �-mercaptoethanol, 0.1 mm MEM nonessential amino acids, 200 mm l-glutamine, and 10% FCS. Following three days in culture 25 of 28 embryos hatched. Ten embryos attached to the feeder layer, nine degenerated, and nine embryos were floating in the medium and expanding in size. After 5-7 days in culture four of the tehn attached embryos appeared with a prominent an ICM outgrowth and 3 of the nine floating embryos formed structures resembling ICM disc surrounded by trophectoderm cells. The ICM and the embryonic discs were isolated mechanically and cultured on goat feeder cells in DMEM medium containing 10 ng/mL horse leukemia inhibitory factor (hLIF) and 10% FCS. The ICM and embryonic disc outgrew into colonies on Day 4 post-culture. Compact colonies of cells from these outgrowths were isolated mechanically and passed onto fresh goat feeder cells every 4-5 days with the addition of 10 ng/mL hLIF to the culture media. Established colonies at passage 6 were tested for immunoreactivity against alkaline phosphatase (AP) and Oct-1 using standard protocols. The result was the estabilshment of embryonic stem cell-like colonies (57%) from both ICM and embryonic discs cultured on goat fetal fibroblast feeder cells. Colonies forming from these outgrowths (50%) of either ICM or embryonic disc stained positive for both AP and Oct-4. Embryoid bodies formed from colonies of either ICM or embryonic disc in suspension (DMEM containing 10% FCS with no hLIF). Two cell lines (one from ICM and one from embryonic disc) have been maintained so far through passage 8 and have been cryopreserved. These GESC-like lines will be used in further characterization and ultimately in transgenic animal production studies.
2 IDENTIFICATION OF GENES INDUCED BY THE CONCEPTUS IN THE BOVINE ENDOMETRIUM DURING THE PRE-IMPLANTATION PERIOD
Authors: Klein C. , Bauersachs S. , Ulbrich S. , Meyer H. , Schmidt S. , Reichenbach H. , Vermehren M. , Blum H. , Sinowatz F. , Wolf E.
Published: December 2006

Early embryonic development, implantation, and maintenance of a pregnancy are critically dependent on an intact embryo-maternal communication. So far, only few signals involved in this dialogue have been identified. In ruminants, interferon tau (IFN�) plays a key role in the process of maternal recognition of pregnancy by exhibiting antiluteolytic activity. Even though many experimental findings indicate a pivotal role of IFN� in the context of embryo-maternal communication in ruminants, a number of other systems may be involved. To identify genes induced in the bovine endometrium by the signaling of the embryo, a combination of subtracted cDNA libraries and cDNA array hybridization was applied. Monozygotic twin pairs (n = 5) were used as the biological model. Pregnancy was created in one twin by transferring two in vitro-produced embryos on Day 7 of the estrous cycle; the other twin received a sham embryo transfer. Pregnant and nonpregnant twins were slaughtered at Day 18; endometrial tissue samples were recovered and processed for transcriptome analysis as described (Bauersachs et al. 2005 J. Mol. Endocrinol. 34, 889-908). Screening of 4608 clones of two subtracted libraries revealed 90 different up-regulated genes and mRNAs, of which almost 50% are known to be stimulated by type I interferons. Among these interferon-stimulated genes, the ISG15 system is assumed to be of particular interest, and several components were studied in more detail using in situ hybridization. The pattern of mRNA expression suggests that modification of endometrial proteins through ISG15ylation plays a fundamental role during the pre-implantation period. A classification of the identified genes based on Gene Ontologies revealed the prevalence of genes involved in regulation of gene expression, cell communication, cell growth, cell differentiation, cell proliferation, and cell adhesion, and also the prevalence of genes with immune-related functions. These results underline the intense response of the endometrium to the presence of a conceptus, culminating in the preparation of the maternal environment for embryonic implantation. Further, for eleven selected genes the expression in the endometrium was quantified by the use of real-time RT-PCR. Overall, the results of quantitative RT-PCR and array hybridization correlated very well. To our knowledge this study provides the first holistic gene expression analysis of the bovine endometrium during the pre-implantation period. The results underline the importance of IFN� as an embryo-derived pregnancy recognition signal and depict the molecular mechanisms at the mRNA level underlying the intense embryo-maternal dialog taking place at Day 18 of gestation.
205 DERIVATION OF CAT EMBRYONIC STEM-LIKE CELLS FROM IN VITRO-PRODUCED BLASTOCYSTS AND THEIR SUPPORT BY INTRASPECIFIC VS. INTERSPECIFIC FEEDER CELLS
Authors: Serrano M. A. , Gómez M. C. , Lopez M. , Dumas C. L. , Smith K. E. , Leibo S. P. , Dresser B. L. , Pope C. E.
Published: December 2006

Interspecific nuclear transfer has been successfully demonstrated in nondomestic cats (Gomez et al. 2004 Cloning Stem Cells 6, 247); however, the efficiency remains low and may be attributable to nuclear reprogramming errors. Embryonic stem cells (ESC) may complete nuclear reprogramming more efficiently than somatic cells and, therefore, are potentially useful for increasing cloning success (Jaenisch et al. 2002 Cloning Stem Cells 4, 389). The objectives of this study were to: (1) compare efficiency of immunosurgery vs. mechanical separation for isolating the inner cell mass (ICM) of in vitro-derived cat blastocysts; and (2) determine the influence of mouse (MEF: CF-1) and cat (CEF) embryonic fibroblast feeder layers on ICM attachment and growth of ES-like cells. After ICMs were isolated from in vitro-derived blastocysts (n = 142) by immunosurgery or mechanically, they were plated either on mitotically inactivated CEF (40 �L/mL Mitomycin-C; 5 h) or MEF (30 �L/mL Mitomycin-C; 2.5 h). Cells were cultured in DMEM-F12, 1 mM L-Glutamine, 0.1 mM 2-mercaptoethanol, 1.25% nonessential amino acids, 15% knock-out replacement serum, 5% fetal bovine serum, 40 ng/mL leukemia inhibitory factor, 5 ng/mL basic fibroblast growth factor, 100 IU penicillin, 100 �g/mL streptomycin, and 25 �L/mL amphotericin-B in a humidified atmosphere of 5% CO2 in air at 38�C. Our results indicated that ICM isolation and attachment were not affected by either the method of isolation (immunosurgery: 75.8 � 6.9% vs. mechanical: 89.5 � 6.4%) or the feeders (MEF: 74.6 � 6.7% vs. CEF: 90.7 � 6.6%). However, the incidence of ES-like cell colony formation was significantly affected by the feeder layer (CEF: 55.4 � 7.2% vs. MEF: 12.7 � 7.2%; P < 0.001). A total of 32 ES-like cell lines were derived on CEF (n = 26) and MEF (n = 6), of which 50% were alkaline phosphatase (AP)-positive. One ES-like cell line derived on MEF spontaneously differentiated into myocardiocytes after 14 days in culture. Three ES-like cell lines derived on CEF were immunostained for ESC-markers Oct-4, SSEA-1, and SSEA-4, and for AP. Positive results for all markers were observed in a few colonies of each line, with colonies from one cell line appearing on Day 23 and remaining in culture for 102 days (12 passages). Colonies from the other two cell lines appeared on Day 17 and remained in culture for 78 days (9 passages). Colonies derived on MEF appeared on average at 17.9 days and remained in culture an additional 15 to 61 days without further characterization. The present results describe the first isolation of cat ES-like cells. We have demonstrated an important species-specific relationship between feeder layers and the derivation of cat ESCs. Further studies are in progress to improve culture conditions for the derivation and expansion of stable cat ESC lines.
206 COMPARATIVE EVALUATION OF VARIOUS PROTOCOLS FOR NEURAL DIFFERENTIATION OF MOUSE EMBRYONIC STEM CELLS
Authors: Tas A. , Arat S. , Dalcik H.
Published: December 2006

Mouse embryonic stem (ES) cells derived from the inner cell mass of blastocysts can differentiate into neuronal cells by treatment with retinoic acid (RA). ES cells cultured as aggregates and as single cell suspensions were then exposed to RA which induced multiple phenotypes of neuronal cells. Differentiation was dependent on the concentration of RA and the time of exposure. In this study, we cultured ES cells as a suspension in which they formed embryoid bodies (EBs). The EBs were treated with varying concentrations of RA for differing times. We used increasing concentrations of RA (50 nM, 100 nM, 1 �M, and 3 �M) prepared from a stock of 10 mM RA in DMSO. Immunocytochemistry staining was carried out on 2, 5, 7, and 9 days of culture. We formed EBs for 4 days with standard ES cell medium (without LIF) plus an additional 4 days of treatment with 1 �M RA. ES cells were treated with 1 �M RA for 2 days in suspension culture. Two-day-old EBs plated on culture dishes were treated with 1 �M RA for 3 days. To test for the effect of RA concentration on embryonic differentiation, 2-day-old EBs were treated with 50 nM, 100 nM, 1 �M, and 3 �M RA for 3 days. The time-dependent effects of RA on the 4-/4+ RA group were investigated. Results showed that neuronal precursor cells appeared on the second day of culture; they were stained with nestin antibody. On the 5th day of culture, neurons were detected with NCAM antibody. On the 7th day of culture, glial cells were observed with GFAP, and on the 9th day of culture GFAP, expression increased. In EBs that were plated and then treated with RA, the same results were obtained. RA induced neuronal differentiation in a concentration-dependent manner. Low concentrations (50 nM and 100 nM) of RA induced neuronal differentiation besides mesenchymal differentiation; however, higher concentrations (1 �M and 3 �M) of RA did not induce mesenchymal differentiation. The most efficient neuronal differentiation was obtained at 3 mM RA concentration.
This study was performed in TUBITAK Research Institute for Genetic Engineering and biotechnology.
215 EPIDIDYMAL SPERM CRYOPRESERVATION OF ONE SOMALIA WILD ASS (EQUUS AFRICANUS SOMALIENSIS) USING SIX DIFFERENT EXTENDERS
Authors: Álvarez M. , Martínez-Pastor F. , García-Macías V. , Borragán S. , Celada M. , Bernardo J. , Gonzalez N. , Alves S. , Anel L.
Published: December 2006

The Somalia wild ass (Equus africanus somaliensis) is a critically endangered taxon (IUCN 2004 red list) which could benefit from biological resource banking. In this work, we studied the effect of different extenders applied to the cryopreservation of epididymal sperm obtained from one male of this subspecies. This animal (13 years old; housed in Cabarceno Park, Cantabria, Spain) was castrated because of very aggressive behavior with other mature males. Genitalia were dissected and weighed (testicles: right, 166 g, and left, 179 g; cauda epididymis: right, 9.3 g, and left, 11.8 g). Sperm were flushed from the cauda epididymis, yielding 15 mL of sample. Sperm concentration was 15 × 109 spermatozoa/mL, totaling 225 × 109 (allowing 4500 doses at 50 × 106 sperm/dose). Sperm motility (TM = % total motile; PM = % progressive; VAP = average path velocity) was assessed by CASA (Microptic, Barcelona, Spain). Viability (VIAB = % viable sperm) and acrosomal status (ACR = % viable spermatozoa with intact acrosomes) were assessed using propidium iodide (37 μmol/L) and PNA-FITC (1 ng/L) and flow cytometry. Chemicals were purchased from Sigma (Madrid, Spain). Part of the sample was divided into six aliquots and diluted 1:1 with different extenders: UL4: Tes-Tris-Fructose (TTF), 10% egg yolk (EG), and 4% glycerol (G); UL8: TTF, 20% EG, and 8% G; AND4: Andromed® (Minitüb, Tiefenbach, Germany) and 4% G; AND7: Andromed® and 7% G; GENT: Gent 1045; and INRA: INRA96 and 4% G. Andromed, Gent, and INRA are commercial extenders. Samples were cooled to 5°C (−0.2°C/min) and then diluted to 200 × 106 sperm/mL. Samples were packed (0.5-mL straws) and frozen using a biofreezer (from 5°C to −15°C at −15°C/min, and from −15°C to −100°C at −25°C/min). Samples were thawed at 65°C for 6 s, and assessed as for pre-freezing (Table 1). Post-thawing motility recovery using AND7 was excellent. The highest viability recovery was achieved by UL4, although that in AND7 was similar. The poor results of equine commercial extender Gent 1045 in this species are remarkable. Our results highlight the importance of species differences in the field of sperm cryopreservation. It is necessary to carry out continuous research for optimizing cryopreservation protocols in order to create germplasm banks for wild species.

Table 1.
Quality assessment results
229 OOCYTE RECOVERY, IN VITRO FERTILIZATION AND EMBRYO TRANSFER IN THE SERVAL (LEPTAILURUS SERVAL)
Authors: Pope C. E. , Gómez M. C. , Cole A. , Dumas C. , Dresser B. L.
Published: December 2006

Servals are medium size (9 to 18 kg) spotted cats found in sub-Saharan Africa that are protected by CITES under Appendix II regulations. There are at least six sub-species, one of which is listed as Endangered by the U.S. Endangered Species Act. In vitro-derived embryos have been produced in at least one-half of the 36 species of nondomestic cats, and kittens have been born after embryo transfer in six species. In the present study we evaluated (1) ovarian response of servals to repeated exogenous gonadotropin stimulation, and (2) in vitro and in vivo developmental ability of in vitro-derived embryos. One two-year-old and one five-year-old female were treated six and three times, respectively, over a 3.5-year period, with a total of 20 or 25 IU of porcine FSH (i.m.; Sioux Biochem, Sioux City, IA, USA) administered daily over four days during interestrus. On Day 5, 15 IU of porcine LH (i.m.; Sioux Biochem) was given, and laparoscopic oocyte retrieval was performed 24 h later. A total of 234 preovulatory oocytes were recovered: 182 (mean = 30.3) from the two-year-old and 52 (mean = 17.3) from the five-year-old female. A total of 91 and 91 oocytes were recovered at retrievals 1 through 3 and 4 through 6, respectively, from the two-year-old donor. Eighty oocytes from the two-year-old donor were inseminated with cooled (24 h, 4°C) semen. Frozen semen from the same male was used to inseminate 102 oocytes from the two-year-old female and 52 oocytes from the five-year-old female. Overall, 136 embryos (58% cleavage frequency) were produced: 119 (65% cleavage frequency) from the two-year-old and 17 (33% cleavage frequency) from the five-year-old female. Cleavage frequency of oocytes from the two-year-old female inseminated with cooled or frozen semen was similar, 68% (54/80) and 64% (65/102), respectively. Embryos were cultured for 5 or 6 days before controlled rate cryopreservation or uterine transfer (Gómez et al. 2003 Theriogenology 60, 239–251). On Day 5, 66 early to mid-stage morulae were cryopreserved at a slow controlled rate. Sixty Day 5 and 18 Day 6 embryos were auto-transferred to a recipient (8 to 26/transfer) in a total of six surgical procedures, of which five were with fresh embryos (n = 70) and one was with cryopreserved embryos (n = 8). The sixth embryo transfer procedure (26 fresh embryos) resulted in the unassisted birth of a live male kitten on Day 77 of gestation. We have shown that in vitro-derived embryos can be generated in the serval and that oocyte retrieval rates and cleavage frequencies are comparable to those reported for other species of mid-sized nondomestic cats. The nominal incidence of pregnancy and frequency of embryo survival may be improved by transferring early cleavage staged embryos into the oviduct, as demonstrated in the African wildcat (Felis silvestris lybica; Gómez et al. 2004 Cloning and Stem Cells 6, 247–258).
This work was partially funded by the Dan Heard Conservation Challenge Grant.
23 NEONATAL PHYSIOLOGICAL STATUS OF CLONED AND CONTROL CALVES
Authors: Batchelder C. A. , Whitcomb M. B. , Bertolini M. , Mason J. B. , Petkov S. G. , Hoffert K. A. , George L. W. , Anderson G. B.
Published: December 2006

Health problems and mortality rates of cloned calves are major limitations of cloning technology and represent substantial economic losses as well as justifiable animal health and welfare concerns. The objectives of this study were to compare neonatal viability and physiological status of cloned and control calves. Cloned (Holstein, n = 5; Hereford, n = 3) and control (embryo transfer: Holstein, n = 3; Hereford, n = 3) calves were carried in the same group of Hereford × Angus crossbred recipient dams and were delivered by Cesarean section at term (Days 273–280) following induced labor. Additional calves (Holstein, n = 3; Hereford, n = 2) resulting from AI and delivered vaginally by their natural dams (Days 269–279) following spontaneous initiation of parturition were included as normal controls to evaluate the effects of the induction procedure. Physical evaluations and measurements of blood biochemistry (19 parameters), gases and electrolytes (9 parameters), and complete blood counts (18 parameters) were performed within 10 min of birth and at 1, 6, 12, 18, 24, 36, and 48 h after birth. Cloned calves were observed with increased occurrence of flexural limb deformities (4/8 clones, 0/9 controls; P < 0.05) and developmental delays of physical adjustment parameters such as time to suckle and stand (5/8 clones requiring >3 h; P < 0.05). Cloned calves were more variable than, but not different from, controls for most blood parameters measured. Compared with controls, at birth clones exhibited reduced red blood cell counts (6.8 and 8.6 × 109 cells/mL, clones and controls, respectively; P < 0.01), plasma bicarbonate (23.1 vs. 26.2 mmol/L; P < 0.05), and plasma glucose (39.4 vs. 73.6 mg/dL; P < 0.05). Blood urea nitrogen concentrations in clones tended to be elevated beginning 24 h after birth and were significantly greater than those in controls by 48 h (13.4 vs. 7.4 mg/dL; P < 0.01). Echocardiographic measurements at 24 h of age varied between groups (Table 1) and were indicative of circulatory abnormalities likely originating in utero for clones. The results of this study identified the physiological differences between clones and controls at birth and may be useful in the development of clinical-care protocols to maximize the health and survival of cloned calves.

Table 1.
Echocardiographic characteristics of cloned and control calves
239 HORMONE PROFILE AND OVARIAN FOLLICLE DIAMETER IN EARLY POSTPARTUM LACTATING COWS
Authors: Park S. J. , Baek K. S. , Park S. B. , Ahn B. S. , Kim H. S.
Published: December 2006

The purpose of this study was to assess the ovarian follicle diameter and hormone status during early postpartum in cows. Eighteen Holstein cows, aged 3.5–5.5 years and of 400–600 kg BW with normal calving, were examined every 3rd day starting from Day 15 until Day 33 postpartum. Blood samples for hormonal (progesterone (P4) and estradiol-17β (E2)) analyses were withdrawn from the coccygeal vein every 3rd day until the end of the experiment. The ovarian follicular number and size were verified and measured using a sector vaginal transducer ultrasound scanner (6.5 MHz probe, SA 600; Medison Co., Seoul, Korea). The least squares means (±SEM) are presented for each day (GLM of SAS). In postpartum cows, E2 levels were 27.09 ± 5.22 pg, 24.50 ± 5.76 pg, 26.92 ± 4.62 pg, 25.53 ± 3.74 pg, 27.47 ± 5.72 pg, 26.48 ± 8.06 pg for Days 15, 18, 21, 24, 27, 30, and 33, respectively, 28.01 ± 7.52 pg; P4 levels were 1.35 ± 1.14 ng, 2.41 ± 1.70 ng, 2.85 ± 2.26 ng, 2.88 ± 1.90 ng, 2.91 ± 1.91 ng, 3.04 ± 2.31 ng, and 4.04 ± 3.70 ng for Days 15, 18, 21, 24, 27, 30, and 33, respectively. The ovarian follicle diameter was 12.36 ± 3.31 mm, 11.91 ± 2.99 mm, 13.09 ± 3.37 mm, 13.55 ± 2.76 mm, 14.55 ± 3.68 mm, 15.64 ± 5.53 mm, 17.45 ± 3.22 mm for Days 15, 18, 21, 24, 30, and 33, respectively. The present results show that mean plasma concentration of P4 and mean follicle diameter gradually increased from Day 15 to Day 33 postpartum. It is concluded that monitoring of P4 and E2 during early postpartum period would be useful to predict the ovarian starts for early rebreeding in dairy cows.
245 EXPRESSION AND FUNCTIONAL ROLE OF CELL DIVISION CYCLE 42 IN MOUSE OOCYTES MATURATION AND PRE-IMPLANTATION DEVELOPMENT
Authors: Cui X.-S. , Li X.-Y. , Kim N.-H.
Published: December 2006

Cell division cycle 42 (Cdc42), a member of the Rho family of small guanosine triphosphatase (GTPase) proteins, regulates multiple cell functions, including motility, proliferation, apoptosis, and cell morphology. In order to gain insight into the role of Cdc42 in embryo development, we first characterized mRNA and protein levels of Cdc42 in mouse oocytes and early embryogenesis. We then examined the possible role of the gene in oocyte maturation and pre-implantation development using RNA interference analysis. The relative abundance of Cdc42 transcripts were measured by real time RT-PCR. After normalization with histone H2a mRNA levels, the mRNA expression of Cdc42 was abundant in immature oocytes and reduced slightly in zygotes and 2- to 8-cell stage embryos. The expression levels were significantly increased during the morula and blastocyst stages. Indirect immunocytochemistry showed protein synthesis of Cdc42 in oocytes and embryos of all stages. Introducing small interference RNA (siRNA) of Cdc42 into germinal vesicle stage oocytes or zygotes specifically reduce both mRNA expression and protein synthesis of Cdc42 in metaphase II stage oocytes and early embryos developing in vitro. Meiotic maturation was significantly reduced following siRNA injection into germinal vesicle stage oocytes. It is evident that actin distribution in siRNA treated blastocysts is morphologically abnormal following injection of siRNA for Cdc42. Injection of siRNA into zygotes did not influence cleavage, but significantly decreased in vitro development to morulae and blastocysts. While housekeeping genes such as tissue plasminogen activator were not altered by siRNA, wiskott-aldrich syndrome protein family 1 (WASP1) mRNA was down-regulated in the morula. Interestingly, mRNA of WASP1, tubulin alpha 1 (Tuba1), and actin-related protein 2/3 complex subunit V (Arpc5) increased at the blastocyst stage following siRNA injection. These results suggest that Cdc42 plays an important role during oocyte maturation and early pre-implantation development, likely through linkage with several other genes.
This work was funded by a grant from National Research Laboratory Program in Korea.
249 EXPRESSION OF GROWTH-RELATED IMPRINTED GENES IN DECEASED NEWBORN AND SURVIVING CLONED PIGLETS
Authors: Jiang L. , Jobst P. , Ayares D. , Prather R. S. , Yang X. , Tian X. C.
Published: December 2006

Different from cloned mice and cattle, which are often born with large offspring syndrome (LOS), pigs produced through nuclear transfer have not been found to be overly large compared to their age-matched controls. In our study, significant differences (P < 0.05) were found in the body weight of both deceased newborn cloned piglets (n = 9) (1.2 � 0.8 vs. 2.1 � 0.2 kg) and cloned pigs at one month of age (n = 7) (6.1 � 1.3 vs. 8.0 � 0.8 kg), when compared to their controls (n = 5 for each age group). Because imprinted genes are important regulators of fetal growth and are subject to reprogramming during nuclear transfer, we aimed to determine the expression levels of both growth-enhancing and growth-inhibiting imprinted genes in these cloned pigs by real time quantitative reverse transcription polymerase chain reaction (RT-PCR). These genes include: Igf2 and Peg3 (growth-promoting), and Igf2r and Grb10 (growth-inhibiting). Tissues from six major organs were collected from clones and controls of both age groups. SYBR Green I was utilized as a reporter for the PCR reactions and 18S ribosomal RNA was used as the internal reference for quantifying the relative expression level of all genes. With the exception of Igf2, the other three imprinted genes were found to have statistically different levels of expression in certain organs of both groups of pigs when compared to their age-matched controls. However, no strong correlation was found between the levels of gene expression and the low-body-weight phenotype of the cloned pigs. Contrary to the notion that nuclear transfer can reduce the variation in the resulting animals due to their identical genetic make-up, larger variances of gene expression were found in clones of both groups, indicating the randomness of the nuclear reprogramming process. For all four imprinted genes studied, the coefficient of variation (CV) decreased greatly from newborn to one month old in both clones and controls, suggesting a more stringent regulation on imprinted gene expression after birth.
251 COMPARATIVE ANALYSIS OF GENE EXPRESSION PATTERNS IN PORCINE PRE-IMPLANTATION EMBRYOS DERIVED FROM DIFFERENT ORIGINS
Authors: Kim J.-G. , Jin H.-F. , Mohana B.-K. , Song H.-J. , Jeong Y.-J. , Balasubramanian S. , Choe S.-Y. , Rho G.-J.
Published: December 2006

The amount of information gathered on the kinetics and quantitative profile of gene expression in nuclear transferred (NT) pre-implantation embryos is still scarce and limited to a handful of genes in pig. In the present study, we compared the relative abundance (RA) of six development-related genes of pre-implantation embryos from different origins. Cumulus-oocyte complexes (COCs) were matured, fertilized and cultured by the method of Abeydeera et al. (2000 Theriogenology 54, 787-797). Parthenogenetic (PA) and NT embryos were produced as described by Kim et al. (2005 Mol. Rep. Dev. 70, 308-313). Sets of 10 embryos each at 4-cell, 8-16-cell, morula, and Day 7 blastocyst stages were used to extract total RNA for analyzing the expression pattern of Bax (pro-apoptotic), Bcl-xl (anti-apoptotic), Oct-4 (pluripotent transcription), Stat3 (cytoplasmic transcription), IFN-tau (implantation) and VEGF (vasculogenesis) genes (three replicates) with real-time quantitative PCR (LightCycler�; Roche Diagnostics, Mannheim, Germany) following RNA isolation (with Dynabeads� mRNA Direct" kit; Dynal, Oslo, Norway) and cDNA amplification (Oligo (dT)12-18 primer and Superscript" III cDNA synthesis kit; Invitrogen, Carlsbad, CA, USA). The expression of each gene was normalized to Histone H2A expression. Statistically significant (P < 0.05) differences in gene expression were analyzed by ANOVA. Oct-4, Stat3, Bax, and Bcl-xl were expressed at the 4-cell stage. However, IFN-tau and VEGF were expressed at the morula stage. The expression patterns of all genes throughout the embryonic development in all types of embryos were similar. However, the relative abundance (RA) of Bax in the 8-16 cell stage of NT and PA was significantly (P < 0.05) increased as compared to that in IVP counterparts. The RA of Oct-4 and Bcl-xl did not differ throughout all stages in NT and PA embryos. There were no significant differences in all types of embryos with respect to the RA of Oct-4 and Bcl-xl with exception of the blastocysts in NT (significantly decreased, P < 0.01). The RA of Stat3 was significantly (P < 0.05) increased in the 8-16 cell stage (NT and PA) and in blastocysts (NT). Similarly, the RA of IFN-tau was significantly (P < 0.05) increased in NT blastocysts. The RA of VEGF was not significantly different in all stages and types of embryos except NT blastocysts (decreased expression, P < 0.01). These results suggest that expression patterns of Bax, Bcl-xl, Oct-4, Stat3, IFN-tau, and VEGF can be used as markers to assess the developmental competence of porcine nuclear transfer embryos.
This work was supported by Grant No. 1000520040020000 from Biogreen 21, Republic of Korea.