bluuebunny/crossref_metadata_2025 huggingface dataset:
207 ESTABILSHMENT OF CYNOMOLGUS MONKEY EMBRYONIC STEM CELL LINES AND CONFIRMATION OF THE POSSIBILITY FOR GERMINAL COMPETENCY
Authors: Teramura T. , Kawata N. , Fujinami N. , Takenoshita M. , Sagawa N. , Matsumoto K. , Saeki K. , Hosoi Y. , Iritani A.
Published: December 2006

Embryonic stem cells (ESCs) of nonhuman primate are important tools for human gametogenesis research. Generally, ESCs, embryos, and fetuses of nonhuman primates are similar to these of human. Recently, germ cell formation of mouse ESCs in vitro has been reported. In this study, we established new cynomolgus monkey ES (cyES) lines and determined germinal competency by assessing expression of mRNA markers. CyES lines were established using blastocysts produced by intracytoplasmic sperm injection (ICSI). For inducing super-ovulation, females were treated with 25 IU/kg pregnant mare serum gonadotropin (PMSG) once a day for 9 days, followed by 400 IU/kg hCG. Oocytes were collected 40 h after injection of hCG. After sperm injection, embryos were cultured in mCMRL medium to the blastocyst stage. For ES line establishment, inner cell masses (ICMs) were isolated by immunosurgery. ESC colonies emerged at about 10 days after ICM plating; three cyES cell lines were successfully obtained (3/11; 27.3%). We characterized these lines by immunocytochemistry for Oct-3/4, SSEA-3, and SSEA-4, which are diagnostic markers for primate ESCs, and by assay for alkaline phosphatase (ALP) activity. All cell lines expressed Oct-3/4, SSEA-4 and ALP activity. The previously reported SSEA-3 weak expression in cyES cells was not observed. These lines differentiated spontaneously when they were replaced in non-adherent culture (embryoid body: EB) or injected into SCID mice subcutaneously. To assess germ cell competency in vitro, we analyzed for the presence of vasa mRNA which shows a restricted expression pattern to germ cell formation, and DMC1 and SYCP1 which show specific existence on synaptonema complex in meiosis. Detection of these germ cell markers was performed by RT-PCR with total cDNA from ESCs and EBs. Nanog mRNA was detected only in ESCs. Oct-4 was detected in gonadal tissue of both sexes, ESCs, and EBs. Vasa was expressed in testis, but not in ESCs or somatic cells. Interestingly, we recognized weak expression of Vasa in Day 12-16 EBs. DMC1 and SYCP1 as meiosis markers were not detected. Because Oct-4 and Vasa mRNA are transcribed simultaneously, similar to that in the early part of gametogenesis such as the latter period of primordial germ cell (PGC) migration, PGC formation in cynomolgus EBs could occurr as in some cases of mouse or human EBs previously reported. Although detailed properties such as the functions of these Vasa-positive cells have not been confirmed, these results demonstrate that cyES cells obtained in the current study might contribute to putative germ cells in vitro by differentiating to EBs.
This study was supported by a Grant-in-Aid for the 21st Century COE Program of the Japan Mext and by a grant for the Wakayama Prefecture Collaboration of Regional Entities for the Advancement of Technology Excellence of the JST.
209 RISKS OF TRANSMITTING MYCOBACTERIUM AVIUM SSP. PARATUBERCULOSIS BY TRANSFER OF IN VIVO-DERIVED AND IN VITRO-FERTILIZED BOVINE EMBRYOS
Authors: Bielanski A. , Algire J. , Randall G. , Surujballi O.
Published: December 2006

Paratuberculosis (Johne's disease) is a chronic infectious disease of cattle and other domestic and wild ruminants caused by Mycobacterium avium ssp. paratuberculosis (Map) which is widespread throughout the world. The disease is characterized by persistent diarrhea, weight loss, and eventually death. In addition to interest in the economic losses, there has been an increasing interest in eradicating the disease due to the potential involvement of the causative microorganism in Crohn's disease, a debilitating chronic enteritis in man. Experiments were conducted to determine the possibility of transmission of Map by embryo transfer (ET) and the association of Map with in vivo-derived and in vitro-fertilized (IVF) embryos. These experiments involved (1) collection of embryos from naturally infected donors (n = 5) and transfer of those embryos to uninfected recipients (n = 12) and testing of others for Map by culture on Herrold's medium with mycobactin or by PCR; and (2) in vitro exposure of in vivo-derived and IVF embryos to Map and their subsequent transfer to uninfected recipients. Experiment 1 revealed the presence of Map in the uterine horns of all five subclinically infected donors, but it was not detected in association with sequentially washed in vivo-derived embryos by culture or by PCR. In Experiment 2, a high proportion of both in vivo-derived and IVF embryos exposed to Map in vitro tested positive for Map even after sequential washings as recommended by IETS. Transfer of in vivo-derived and IVF embryos that had been exposed to Map, and then washed, into 18 recipients resulted in 13 pregnancies and 8 calves born without evidence of disease transmission to either the recipients or the offspring over the following 5 year period. In conclusion, it is unlikely that Map will be transmitted by ET when the embryos have been processed according to the washing protocols recommended by the IETS.
21 EFFECT OF SEVERAL PARAMETERS ON PARTHENOGENETIC BOVINE EMBRYO DEVELOPMENT IN VITRO
Authors: Arat S. , Bagis H. , Tas A. , Akkoc T.
Published: December 2006

The activation of oocytes is one of the most important steps for a successful cloning and has great importance on embryo development in vitro. The objective of this study was to examine the different parameters affecting parthenogenetic embryo development in vitro. In the first experiment, two activation protocols were compared to examine the effect of electrical pulse on activation. Bovine oocytes isolated from slaughterhouse ovaries were matured in TCM-199 supplemented with fetal bovine serum (FBS), sodium pyruvate, penicillin/streptomycin, rat insulin-like growth factor (rIGF-1), bovine follicle-stimulating hormone (bFSH), and bovine luteinizing hormone (bLH). A group of oocytes was exposed to a DC pulse of 133 V/500 �m for 25 �s, and then activated by calcium ionophore (5 �M) for 10 min, cytochalasin D (CD) (2.5 �g/mL) + cycloheximide (CHX, 10 �g/mL) for 1 h, and CHX alone for 5 h (Group 1). Another group of oocytes was activated only by chemicals without electrical pulse. Activated oocytes were cultured for 72 h in G1-3 and then 4-6 days in G2-3 medium. In the second experiment, oocytes activated by electrical pulse and chemicals were cultured in Barc medium for 7-9 days or 72 h in G1-3 and then 4-6 days in G2-3 medium. In the third experiment, oocytes activated by electrical pulse and chemicals were cultured for 48 h or 72 h in G1-3 and then 5-7 days or 4-6 days in G2-3 medium. The differences among groups were analyzed by one-way ANOVA after arcsin square transformation. In the first experiment, cleavage rate (75.6%), development rate (37.3%), and blastocyst cell number (78.4 � 3.2) of oocytes activated by electrical pulse was higher than for the group without electrical pulse (28.7%, 8.0%, 59.5 � 4.3, respectively; P < 0.05). This result showed that activation was started more effectively by electrical pulse than by chemicals. In the second experiment, there was no significant difference on cleavage rate between the two groups (66.6%, 65.0%, respectively), and the blastocyst development rate of parthenogenetic embryos cultured in G1-3/G2-3 (36.6%) was higher than in the Barc medium group (16.6%; P < 0.05). This result showed that G1-3/G2-3 medium was more effective for parthenogenetic embryo development than Barc medium. In the third experiment, although significant differences could not be found between the two groups in the development rate of parthenogenetic embryos cultured for a total of 7-9 days (30.8%, 39.2%, respectively), the development rate of embryos cultured for 72 h in G1-3 was higher (26.4%) than for the 48-h group (15%; P < 0.05) on Day 7. This result showed that embryos developed more slowly when cultured for a shorter time in G1-3 medium before transfer to G2-3 medium.
This study was supported by a grant from TUBITAK, Turkey (VHAG-1022).
210 EFFECTS OF INTERFERON-τ ON REPLICATION OF BOVINE VIRAL DIARRHEA VIRUS AND BOVINE HERPESVIRUS-1
Authors: Galik P. , Waldrop J. , Marley S.
Published: December 2006

Bovine viral diarrhea virus (BVDV) and bovine herpesvirus-1 (BHV-1) are the most likely viruses to be associated with abattoir-origin materials used in in vitro embryo production. Further, both viruses are known to associate with zona pellucida-intact embryos after exposure and washing, and limited evidence indicates that developing, transferable embryos are able to inhibit viral replication in adjacent cells. Interferon-τ is known to have anti-BVDV and anti-BHV-1 activities, but it is not known whether interferon-τ which is secreted by developing embryos has the same effects. The objectives of this study were to evaluate the cytotoxicity and anti-viral effect of interferon-τ against a non-cytotoxic high affinity strain of BVDV (SD-1) and against a strain of BHV-1 (Colorado) in cell culture. Madin Darby bovine kidney (MDBK) cells were seeded in 96-well plates and then inoculated with serial dilutions (1:10) beginning with an initial concentration of 0.2 �g of interferon-τ. Cells and interferon were incubated at 37.5�C in 5% CO2 and air for 24 h prior to addition of virus. Five concentrations of BVDV were added to the wells to give 500, 50, 25, 10 or 5 cell culture infective doses (50% endpoint) per well. Three concentrations of BHV-1-50, 10, and 5 plaque-forming units were evaluated in separate cell cultures. Virus isolation (for BVDV) or plaque assays (for BHV-1) were utilized to determine if the addition of interferon-τ decreased the amount of infective virus. The interferon-τ produced no observable cytotoxicity in MDBK cells in any of the assays. At its three highest concentrations, the interferon-τ significantly decreased the amount of BVDV but it had no significant effect on the amount of BHV-1 in cell cultures. Thus, it is possible that interferon-τ produced by developing embryos might limit or prevent transmission of BVDV to recipients if this virus were to be inadvertently associated with the embryos that are transferred. However, a similar effect is not expected for BHV-1.
211 LACTOFERRIN INHIBITS BOVINE HERPESVIRUS-1 IN CELL CULTURE AND ALLOWS NORMAL DEVELOPMENT OF IN VITRO-PRODUCED EMBRYOS
Authors: Givens M. , Marley M. , Galik P. , Riddell K. , Stringfellow D.
Published: December 2006

Lactoferrin is an iron-binding glycoprotein found in milk, saliva, tears, and other exocrine secretions. It is known to have in vitro antiviral effects against human, feline, and canine herpesviruses. In addition, lactoferrin is known to be safe in cell culture. Bovine herpesvirus-1 (BHV-1) is a likely contaminant of in vitro embryo production. Further, trypsin treatment is not completely effective in removing the virus from these embryos. We hypothesized that a nontoxic concentration of lactoferrin might prevent replication of BHV-1 within in vitro embryo production systems. Thus, the specific objectives of this research were to determine if lactoferrin from bovine milk would inhibit BHV-1 in cell culture and to determine if in vitro-produced embryos could develop normally when cultured in lactoferrin. Two-fold dilutions of lactoferrin (from 10 to 0.625 mg/mL) were added to Madin Darby bovine kidney cells, followed in 15 min by the addition 104 PFU/mL of BHV-1 (Colorado strain). Samples of cell lysate were taken at Day 2 and virus was quantified by plaque assay. The percent of virus inhibited by the antiviral agent at each concentration was determined by comparison to equivalent samples from temporal control cultures in which no compound was added before or after inoculation (Percentage of virus inhibited = [Quantity of virus in the control sample - Quantity of virus in the compound sample]/Quantity of virus in the control sample � 100). Next, the effect of lactoferrin was determined on in vitro-produced embryos. Cumulus oocyte complexes were received from an abattoir, matured in transit, placed in fertilization drops for 6 h, and then placed in culture drops containing lactoferrin (10, 5, and 2.5 mg/mL). At Day 3.5, embryos > 4 cell stage were placed into fresh culture drops containing lactoferrin. On Day 7.5, blastocyst development was noted and the developed embryos were stained to count viable cells. Blastocyst development rate and nucleated cell count of the treated embryos were compared to those of the controls using Chi square test, and ANOVA and Tukey-Kramer HSD, respectively. Lactoferrin (10 mg/mL) inhibited 2 to 5 logs of virus. At concentrations of 5 and 2.5 mg/mL, 1 to 3 logs of virus were inhibited, and concentrations of 1.25 and 0.625 mg/mL inhibited 0 to 2 logs of virus. Lactoferrin did not affect the nucleated cell count of the treated embryos. In addition, unlike 10 and 5 mg/mL, 2.5 mg/mL of lactoferrin did not affect blastocyst development. These preliminary results indicate that lactoferrin from bovine milk can significantly inhibit BHV-1 in cell culture. Furthermore, supplementation of in vitro culture with 2.5 mg/mL of lactoferrin does not affect blastocyst development or cell count of in vitro-produced embryos.
219 MORPHOMETRIC CHARACTERIZATION OF EPIDIDYMAL AND EJACULATED SPERMATOZOA FROM BROWN BEAR (URSUS ARCTOS)
Authors: Garcia-Macias V. , Martinez-Pastor F. , Alvarez M. , Paz P. , Borragan S. , Celada M. , Anel E. , Anel L.
Published: December 2006

Sperm morphology is an useful characteristic for estimating potential fertility. Currently, we are obtaining baseline information on various aspects of reproduction in the brown bear (Ursus arctos) with the intention of using the knowledge to establish a germplasm bank for the species. In the present report, we describe the results obtained using assisted sperm morphology analysis (ASMA, Sperm Class Analyzer®; Microptic S.L, Barcelona, Spain) to analyze the morphological differences in epidydimal (caput, corpus, and cauda) and ejaculated brown bear spermatozoa. A post-mortem epididymal sperm sample was obtained from an adult brown bear after accidental death. The epididymides were excised, washed, and dissected into the three major segments; caput, corpus and cauda. Then multiple incisions were made in the tissue to allow migration of spermatozoa into the surrounding medium. Semen was collected by electroejaculation from five adult brown bears living in a semi-free ranging environment in the Cabarceno Park (Cantabria, Spain). Anesthesia was induced using tiletamine + zolazepan (Zoletil 100®; Virbac, Carras, France; 7 mg/kg), and ketamine (Imalgene 1000®; Rhone Merieux, Lyon, France; 2 mg/kg). The electroejaculation unit (PT Electronics®; Boring, Oregon) was connected to a 3-lateral electrode transrectal probe (26 mm in diameter, 320 mm in length). Ejaculation occurred at 6–10 V/250–300 mA. For head morphometry assessment, sperm samples were fixed in glutaraldehyde and slides were smeared and air-dried for 2 h. The samples were then stained with Diff-Quik® staining (37°C; 10 min in the red component and 15 min in the blue component). The area, perimeter, length and width, and ellipticity (length/width) of heads were measured from at least 100 spermatozoa/slide. As shown in Table 1, values obtained for each measure were similar in both epididymal and ejaculated spermatozoa. These results provide normal morphometry values for brown bear spermatozoa, a potentially useful characteristic for predicting fertility.

Table 1.
Head morphometry for epididymis and ejaculated bear sperm (mean ± SD)

This work was supported in part by CANTUR S.A. and CICYT (CGL 2004–0278/BOS).
22 EFFECT OF OOCYTE AGE ON THE DEVELOPMENT OF MOUSE PARTHENOGENETIC EMBRYOS AND EFFECT OF OXYGEN CONCENTRATION ON EMBRYO DEVELOPMENT IN VITRO
Authors: Bagis H. , Arat S. , Odaman H. , Tas A.
Published: December 2006

The objective of this study was to investigate the effects of two parameters on mouse embryo development in vitro. These parameters were the effect of oocyte age on activation and the effect of O2 concentration in culture. In the first experiment, oocytes were recovered from superovutated mice at 15 h (group 1) or 20 h (group 2) after human chorionic gonadotropin (HCG) injection. All oocytes were activated for 6 h with 10 mM Sr2+ in Ca2+ free medium in the presence of 5 �g/mL of cytochalasin B. After activation, embryos were cultured in KSOM.aa medium for 4.5-5.5 days. Zygotes from naturally bred mice were used as control. Differences in blastocyst formation rate and blastocyst cell number among treatments were analyzed by one-way ANOVA after arcsin square transformation. In the first experiment, blastocyst formation rate in the first group was higher than in the second group (62.6% vs. 47.1%; P < 0.05). In addition, blastocyst cell number was also higher in the first group than in the second one (69.4 � 3.2 vs. 52.4 � 2.2; P < 0.05). However, both values were higher in control group (80%, 76.2 � 1.2; P < 0.05) than in the experimental groups. These results showed that young oocytes were activated more effectively than aged oocytes. In the second experiment, mouse zygotes were cultured in a humidified atmosphere of 5% CO2 in air (group 3) or 5% CO2, 5% O2, and 90% N2 (group 4). Blastocyst formation rate and blastocyst cell number of zygotes cultured in low O2 concentration (group 4) for 4.5 days were higher than for group 3 (76.3% vs. 56.4 and 69.0 � 3.4 vs. 52.8 � 2.3; P < 0.05). There was a significant difference in blastocyt formation rate of embryos for 5.5 days between the two groups (25.8% for group 4 vs. 14.4% for group 3; P < 0.05). This suggests that the embryos developed more slowly in high O2 concentration. These results showed that low O2 concentration provided a more suitable environment for mouse embryo development in vitro. The same experiment was repeated with parthenogenetic embryos recently in our laboratory.
This study was supported by a grant from TUBITAK, Turkey (VHAG-1022).
222 IN VITRO FERTILIZATION OF OOCYTES OBTAINED THROUGH TRANSVAGINAL OOCYTE RETRIEVAL FROM CYCLIC MURRAH BUFFALOES (BUBALUS BUBALIS)
Authors: Manik R. S. , Chauhan M. S. , Gupta V. , Singla S. K. , Palta P.
Published: December 2006

Very limited information is available in the literature on in vitro maturation (IVM), in vitro fertilization (IVF) and in vitro culture (IVC) of oocytes collected through the transvaginal oocyte retrieval (TVOR) technique in buffaloes. Therefore, the present study was undertaken to examine the post IVF cleavage rates and embryonic development up to the blastocyst stage in the Murrah breed of buffalo subjected to TVOR. The five cyclic Murrah buffaloes were synchronized for estrus by a single prostaglandin injection. The animals were subjected to TVOR once weekly for seven weeks. TVOR was performed using an ultrasound machine with a transvaginal convex transducer (5 MHz), a needle guide, a single-lumen 19-gauge 60-cm-long needle, and a vacuum pressure of 50 mmHg. The number and size of follicles in each ovary was determined before puncture. The follicles were characterized on the basis of their diameter as small (3-5 mm), medium (6-9 mm), and large (e10 mm). The oocytes recovered were classified as grade A, cumulus-oocytes complexes with e5 layers of cumulus cells; grade B, those with two to four layers; grade C, partially denuded oocytes; and grade D, completely denuded oocytes. IVM, IVF and IVC were carried as reported by Chauhan et al. (1999 J. Dairy Science 82, 918-926). Briefly, the oocytes were cultured for 24 h in a CO2 incubator (5% CO2 in air) at 38.5�C for in vitro maturation. Frozen-thawed semen was used in Bracket and Olyphant medium for capacitation and fertilization. The in vitro-fertilized and cleaved embryos were cultured further for 9 days in modified synthetic oviductal fluid. The small follicles constituted a major proportion (60%) of the total observed follicles, although a substantial proportion of medium (19%) and large (21%) follicles were also present. A total of 76 oocytes were recovered by aspiration of 110 follicles, with an overall recovery rate of 70% (range 67-74%). Of these, 45 (59%) were of grades A and B, and 31 (41%) were of grades C and D. The mean number of total follicles and the oocytes recovered per session did not differ significantly among individual donors. Out of the 37 oocytes subjected to IVM and IVF, 19 (51%) cleaved at Day 2 post-insemination. A total of four embryos (11%) developed into morulae/blastocysts. This study demonstrates the use of TVOR as a mean of obtaining oocytes, their fertilization, and further embryo development in the Murrah breed of buffalo.
223 DESCRIPTION OF GENITALIA AND SPERM RECOVERED POSTMORTEM FROM A PYGMY SPERM WHALE, KOGIA BREVICEPS
Authors: Martinez-Pastor F. , Garcia-Macias V. , Garcia J. , Alvarez M. , Anel E. , Herraez P. , de Paz P. , Anel L.
Published: December 2006

A pygmy sperm whale (Kogia breviceps; adult male; 350 kg) was stranded and died on a beach near Cabo Bustos (Asturias, North of Spain) on March 12th, 2005. Finding specimens of this species is a rare event on Spanish shores, although this whale is not considered endangered. Postmortem examination was performed 24 h later. Genitalia (testicles and epididymides) were extracted. The postmortem report indicated that vas deferens and seminal glands seemed to contain an important amount of semen, which was not recovered. Refrigerated genitalia were send to our laboratory, arriving around 40 h postmortem. The refrigerated testicles were in poor physical condition upon arrival, indicating advanced tissue detoriation. The epididymides (very long) were not closely attached to the testicles, but were connected by a loose conjunctive membrane. We divided the epididymides into four regions that approximated the (1) caput, (2) mid-region, (3) corpus, and (4) cauda. Physical characteristics of the genitalia are described in Table 1. The left testicle was larger, and possibly more active, than the right one. A sperm sample was obtained from the cauda region after incising the tissue. Osmolality and pH of the sample were 428 mOsm/kg and 6.62, respectively (maybe due to tissue breakdown) and the sperm concentration was 1194 × 106/mL. Spermatozoa were immotile, even after diluting in buffered medium; it is possible that postmortem damage occurred quickly. However, using flow cytometry we determined that 57% of cauda spermatozoa had intact plasma membranes and acrosomes (determined by staining with 37 mmol/mL propidium iodide and 1 μg/mL PNA-FITC; Sigma, Madrid, Spain). Examination by phase contrast microscopy (×600) showed many spermatozoa with abnormal heads and bent midpieces and flagella, even in the cauda (13% and 21%, respectively). Sperm head morphometry was studied using DiffQuick staining and an automated analysis system (SCA2000; Microptic, Barcelona, Spain). Mean sperm head size was 3.71 ± 0.19 × 2.61 ± 0.12 μm in width and length, respectively. Computer analysis (AnalySiS-GmbH, Cologne, Germany) of phase contrast images revealed that the mean size of the sperm midpiece and flagellum were 3.44 ± 0.19 and 40.95 ± 2.02 μm, respectively. The information obtained after postmortem recovery of the testes and epididymis should be useful to future conservation efforts of the pygmy sperm whale and similar species. The rapid deterioration of the testicular tissue by 40 h postmortem was not expected since good quality sperm samples have been obtained at similar postmortem intervals in other species. Therefore, we recommend that postmortem sperm recovery should be accomplished as rapidly as possible in this species.

Table 1.
Genitalia measurements
230 IN VITRO MATURATION OF OOCYTES FROM ENDANGERED DORCAS GAZELLE (GAZELLA DORCAS NEGLECTA)
Authors: Roldan E. R. S. , Berlinguer F. , Succu S. , Gonzalez R. , del Olmo A. , Espeso G. , Gomendio M. , Ledda S.
Published: December 2006

In vitro maturation of oocytes recovered from dead animals provides an opportunity for rescuing genetic material for biodiversity conservation. The dorcas gazelle (Gazella dorcas) is regarded by the World Conservation Union (IUCN) as ‘vulnerable’ but the subspecies G. dorcas neglecta is thought to be endangered due to excessive hunting. A captive breeding program for dorcas gazelles has been developed at the Estacion Experimental de Zonas Aridas (CSIC) in the South of Spain where efforts have so far concentrated on natural breeding and on the development of sperm cryopreservation protocols. The aim of the present study was to explore the possibility of recovering and maturing in vitro healthy oocytes from animals that die suddenly for the establishment of a program to rescue female gametes. Ovaries of a dorcas female that died unexpectedly were collected about 7 h after death of the animal. Cumulus–oocyte complexes (COCs) were recovered by slicing the ovaries. Collection and washing of COCs were performed in warmed TCM-199-HEPES with antibiotics and polyvinyl alcohol. Degenerated oocytes or those with expanded cumulus cells were removed. A total of 15 COCs were cultured in TCM-199 with 10% heat-treated fetal calf serum, 10 μg/mL ovine FSH/LH, 1 µg/mL estradiol, and 0.1 mg/mL glutamine at 38.5°C under 5% CO2/air with high humidity. After 24 h of culture, matured oocytes, as revealed by the presence of a polar body, were activated with 7% ethanol for 10 min and further incubation for 3 h. Meiotic progression and activation were evaluated by staining with Hoechst 33342 and propidium iodide (1 μg/mL each) and visualization under a fluorescence microscope. Results at the end of incubations showed that 4/15 oocytes were degenerated, 4/15 were arrested at the MI stage, and 7/15 (46.7%) progressed to the MII stage. One oocyte was found to be at the 2-cell stage but it could not be established whether this was the result of the activation method used. These results demonstrate that it is possible to recover viable oocytes several hours after death and rescue them for subsequent in vitro maturation and fertilization. More studies are needed to characterize suitable conditions for oocyte maturation, fertilization, and culture in the dorcas gazelle. This would, in turn, help in the effort to rescue biomaterials from wildlife for generating offspring.
This work was supported by the Spanish Ministry of Education and Science (REN 2003–01587) and Acciones Integradas (HI20030336).
232 EFFECT OF DIETARY FAT SUPPLEMENTATION ON OOCYTE AND EMBRYO QUALITY AND ON EARLY EMBRYONIC DEVELOPMENT OF SHEEP
Authors: Cancino-Arroyo G. , Ake-López R. , Herrera J. , Centurion F. , Ordoñez-León A.
Published: December 2006

The objective was to evaluate the effect of fat supplementation on oocyte quality and in vitro embryonic development (48 h). A total of 18 ewes, with a body condition score of 2.5 to 3 points out of 5, having had three to four lamb births, and at three to four months post-lamb birth, were distributed between an experimental oil group (OG; n = 9) that received corn oil (4% of the MS/diet) and a control group (CG; n = 9) that didn't receive oil. The two groups were maintained in confinement for 21 days (the duration of the experiment) and fed first with a concentrate diet followed by forage. The animals had access to minerals and water ad libitum. The diets were similar in energy (10.3 ± 0.05 MJ/s/d) and protein (141.75 ± 5.7 gPC/s/d) for both groups. The estrous cycle was synchronized (14 days) with intravaginal sponges (40 mg of fluorogestone acetate), inserted 7 days after the beginning of the diets. The end of the diet coincided with the retirement of the sponges. One day before sponge retirement, 75 mg prostaglandin F2α per sheep was administered, followed by ovarian stimulation with 1000 IU of pregnant mare serum gonadotropin (PMSG). Follicular diameter was determined by ventral laparotomy with the aid of a micrometer. Follicles were classified as small (2 to 2.9 mm), medium (3 to 4.9 mm), and large (>5 mm); oocytes were collected in TCM-199 medium. Oocytes were classified as excellent, good, fair, or low quality and transferred to Petri dishes in drops (50 mL) of TCM-199. Oocytes were matured and fertilized in vitro and cultured for 48 h. Oocyte quality as well as maturation, fertilization, and cleavage rates were compared by ANOVA. Ewes from the OG group presented a statistically higher proportion of oocytes with excellent quality (42%; P < 0.05) than GT ewes (26%). The proportion of good quality and fair quality oocytes was similar among groups (P > 0.05). A higher proportion of oocytes of low quality was found in the control group than in the OG group (40% vs. 18%); however, there was no significant difference (P > 0.05). Higher rates of maturation, fertilization, and early development were found in the OG compared with the CG (81.8, 60.6, and 36.4 vs. 68.6, 42.9, and 17.1, respectively); however, the differences were not significant (P > 0.05). In conclusion, the addition of 4% corn oil in the diet improved the quality of the oocytes; however, it had no significant effect on early embryonic development.
233 TEMPOROSPATIAL EXPRESSION PROFILES OF GROWTH AND DIFFERENTIATION FACTOR-9 AND BONE MORPHOGENETIC PROTEIN-15 GENES IN THE BOVINE OVARY
Authors: Hosoe M. , Kaneyama K. , Ushizawa K. , Akagi S. , Noguchi J. , Takahashi T.
Published: December 2006

Bone morphogenetic protein (BMP) family proteins regulate the folliculogenesis and ovulation rate in mammals. Of the members of BMP family, growth and differentiation factor-9 (GDF-9) and BMP-15 are oocyte-derived proteins that play critical roles in follicular development. In the present study, we characterized the temporospatial expression patterns of GDF-9 and BMP-15 in bovine ovaries. Bovine ovaries were obtained from local abattoir. Cumulus–oocyte complexes (COCs) and mural granulosa cells were collected by aspiration of follicles 2–5 mm in diameter. Follicular aspiration was done the day following slaughter. The COC and mural glanulosa cells were separated and cultured for 20 h according to the protocol for oocyte maturation. Total RNA was isolated from COCs, cumulus cells, denuded oocytes, and mural granulosa cells and used for PCR. For in situ hybridization, collected ovaries were immediately fixed with 4% formaldehyde-PBS and embedded in a paraffin block. In situ hybridization was carried out with digoxigenin-labeled probes. In COCs and cumulus cells, the transcripts encoding GDF-9 and BMP-15 were detected immediately after follicular aspiration and still remained at the end of maturation culture. However, in mural granulosa cells, only GDF-9 expression was detected. Germ cell/oocyte markers, ZAR1 and VASA, were not detected in cumulus and mural granulosa cells. Quantitative real-time RT-PCR revealed extensive expressions of both GDF-9 and BMP-15 in oocytes. In situ hybridization study showed that GDF-9 and BMP-15 were co-localized to oocytes in primary, secondary, and antral follicles. The ovular co-expression of GDF-9 and BMP-15 appeared to commence at fetal life. Expression of GDF-9 and BMP-15 was not clear in cumulus cells possibly because of low expression level. In conclusion, our results show that GDF-9 and BMP-15 are co-localized to oocytes from an early stage of folliculogenesis. Since the transcripts encoding GDF-9 and BMP-15 are detected in cumulus cells, it is suggested that the oocyte is not the sole source of them. Additionally, the cumulus and mural granulosa cells are suggested to be functionally differentiated relative to BMP-15 expression.
235 FOLLICULAR WAVE EMERGENCE AND DEVELOPMENT FOLLOWING ESTRADIOL PLUS PROGESTERONE AT A GROWING OR A STATIC PHASE OF THE DOMINANT FOLLICLE IN CIDR-TREATED, LACTATING DAIRY COWS
Authors: Kim U. H. , Suh G. H. , Kim I. H.
Published: December 2006

Treatment with estradiol benzoate (EB), compared to GnRH, in a controlled internal drug release (CIDR)-based timed AI protocol induced relatively asynchronous follicular wave emergence, resulting in ovulation of smaller ovulating follicles (Kim et al. 2005 Theriogenology 63, 260-268). In this study, we compared new follicular wave emergence and development following treatment with EB (2 mg) plus progesterone (50 mg) at a growing (Day 3 after ovulation) or a static (Day 8) phase of the dominant follicle during the first follicular wave in CIDR-treated, lactating dairy cows. Previously synchronized, lactating dairy cows received CIDRs (InterAg, Hamilton, New Zealand; Day 0) 3 days after ovulation and were assigned to two treatment groups and a control group. Cows in the T1 group (n = 11) received 2 mg EB (SY Esrone; Samyang, Seoul, Korea) and 50 mg P4 (SY Ovaron; Samyang) i.m., and those in the control group (n = 10) received no treatment at that time. Other previously synchronized cows received a CIDR (Day 0) with injections of 2 mg EB plus 50 mg P4 i.m. 8 days after ovulation (T2 group, n = 10). Thereafter, all cows received PGF2� (Lutalyse; Pharmacia & Upjohn, Puurs, Belgium) at the time of CIDR removal on Day 7, and GnRH (Conceral; Dongbang Co., Seoul, Korea) on Day 9. Ovaries of each cow were examined by transrectal ultrasonography (Sonoace 600 with 7.5 MHz linear-array transducer; Medison Co., Ltd, Seoul, Korea) every 24 h from Days 0 to 9, on Day 11, and on Day 14 of treatment to observe changes in ovarian structures (follicles and corpora lutea (CL)). Data were analyzed by Fisher's exact test, Kruskal-Wallis test, and ANOVA using an SAS program (SAS Institute, Inc., Cary, NC, USA). Follicular wave emergence occurred within 7 days in 3/10 of the control group, 9/11 of the T1 group, and 9/10 of the T2 group (P < 0.05). The interval to wave emergence did not differ (P > 0.05) among groups (3.7 � 1.2, 4.8 � 0.5, 5.9 � 0.3 days, respectively). While the diameters of dominant follicles on Day 0 were greater (P < 0.01) in the T2 group (14.3 � 1.2 mm) than in the control (9.4 � 0.6 mm) and T1 (10.5 � 0.7 mm) group, diameters of preovulatory follicles on Day 9 were significantly smaller (P < 0.01) in the T1 (13.6 � 0.7 mm) and T2 (12.3 � 0.5 mm) groups than in the control group (16.4 � 0.8 mm). Synchronized ovulation by 40 h after the GnRH injection (Day 11) occurred in 10/10 of the control group, 10/11 of the T1 group, and 9/10 of the T2 group (P > 0.05). In conclusion, administration of 2 mg EB plus 50 mg P4 at both a growing and a static phase of the dominant follicle during the first follicular wave results in synchronous follicular wave emergence and development and also synchronized ovulation in CIDR-treated, lactating dairy cows.
246 EXPRESSION PATTERN OF NANOG AND Par3 GENES IN IN VITRO-DERIVED BOVINE EMBRYOS
Authors: Gandolfi F. , Cillo F. , Antonini S. , Colleoni S. , Lagutina I. , Lazzari G. , Galli C. , Brevini T.
Published: December 2006

Homeobox genes have been demonstrated to be important in patterning and lineage specification during early embryogenesis. Nanog belongs to the family of DNA-binding transcription factors and has been shown to maintain pluripotency of embryonic stem cells, both in murine and human. Par3 plays an essential role in determining cell fate of the early mouse embryo, leading to the generation of the inner cell mass and the trophectoderm. No information is available on these genes in the bovine; therefore, the aim of the present study was to identify and characterize Nanog and Par3 expression in bovine embryos. Oocytes recovered from slaughterhouse ovaries were matured for 22 h, fertilized in vitro and then cultured in mSOFaa medium. RNA was extracted from pools of five oocytes and embryos at different stages of development (2-, 4-, 8-, 16-cell, morula and blastocyst). It was then reverse transcribed, and PCR runs were carried out with primers specifically designed for Nanog and Par3, based on the sequence data bank available. The amplified products were separated on a 2% TAE agarose gel, purified, sequenced and aligned using Clustal W. Comparison of the bovine Nanog cDNA sequence (EMBL AM039957) with databases revealed a 84% degree of homology with the human, 97% with the mouse, and 82% with the goat genes. IVF bovine embryos express Nanog only upon genome activation, becoming detectable from the 8-cell stage onward indicating that Nanog is zygotically expressed in the bovine similar to what happens in mouse, pig and goat. Bovine Par3 cDNA sequence (EMBL AM039956) shows a high degree of homology with human (83%), mouse (81%), and rat (79%). Also Par3 is expressed only upon the maternal to embryonic transition (MET) at the 8-cell stage. As opposed to the expression patterns of other early embryo genes, like Oct-4 and Zar-1, Nanog and Par3 expression patterns in bovine embryos closely resemble those described in the mouse. Since both are absent in the ooplasm and before MET, they represent useful markers for genome activation.
This work was supported by FIRB RBNE01HPMX, FIRST 2004 and ESF-EuroStells.
247 MESSENGER RNA EXPRESSION PATTERNS OF DNA AND HISTONE METHYLTRANSFERASES IN PREIMPLANTATION DEVELOPMENT OF IN VIVO- AND IN VITRO-PRODUCED BOVINE EMBRYOS
Authors: Höffmann K. , Niemann H. , Hadeler K.-G. , Herrmann D. , Wrenzycki C.
Published: December 2006

The effects of in vitro production (IVP) and/or somatic nuclear transfer on mRNA expression patterns have mostly been determined in morulae and blastocysts, i.e. after embryonic genome activation. Comparative data regarding mRNA expression patterns throughout the oviductal phase of pre-implantation development are scarce. Here we studied mRNA expression for genes related to DNA methylation and modification of histones which account for the major epigenetic reprogramming during development. Pertubated epigenetic reprogramming of the genome is a likely cause of developmental abnormalities and epigenetic diseases associated with assisted reproduction technologies. The objective of the present study was to compare mRNA expression of DNA methyltransferases Dnmt1, -3a, and -3b and histone methyltransferases SUV39-h1 and G9a between in vivo-derived bovine embryos and their IVP counterparts using a semiquantitative RT-PCR assay (Wrenzycki et al. 2002 Biol. Reprod. 66, 127-134) employing two embryos for each assay. In vivo-derived embryos were collected from 28 superovulated heifers by endoscopic flushing of oviducts (zygotes to 8-cell stages) (Besenfelder et al. 2001 Theriogenology 55, 837-845) or by uterine flushing (16-cell stages to blastocysts). Endoscopic flushing at different time points after AI (Days 1, 1.5, 2, 3, 4, and 4.5) yielded 31 zygotes; 15 two-cell, 5 three-cell, 13 four-cell, 1 five-cell, 2 six-cell, and 11 eight-cell embryos; 4 degenerated embryos; and 18 unfertilized ova. The recovery rate (corpora lutea counted per recovered embryos) was 58% and 62% for the endoscopic and uterine flushing, respectively. Differences in the relative abundance of each gene transcript between groups were tested using ANOVA with the main effects being origin (in vivo/in vitro) and developmental stage (zygote to blastocyst) and their interactions followed by multiple pairwise comparisons using a Tukey test (P < 0.05). Origin of embryos affected the relative abundance of transcripts for Dnmt1, Dnmt3a, and SUV39-h1, and developmental stage affected the relative abundance of transcripts for Dnmt1, -3a, -3b, SUV39-h1, and G9a. No interactive effects were observed for origin and developmental stage in the relative abundance of all transcripts. The relative abundance of Dnmt1 transcripts differed significantly between in vivo- and in vitro-produced morulae and blastocysts. For Dnmt3a, mRNA differences were determined between in vivo- and in vitro-produced 10-16-cell stages and morulae. Suv39-h1 transcripts differed significantly between in vivo- and in vitro-derived zygotes, 2-cell embryos, 8-cell embryos, 10-16-cell embryos, and blastocysts. The results suggest that IVP alters mRNA expression of genes related to epigenetic modifications very early in development, even before the embryonic genome has been activated.
248 ALTERED GENE EXPRESSION PROFILES IN PIG scNT-DERIVED UMBILICAL CORD
Authors: Hwang K. C. , Cho S. K. , Kim J. H.
Published: December 2006

Despite numerous studies performed on somatic cell cloned (scNT) animals, the factors behind the anomalies such as overgrowth, placental malformations, and heart failure remain unknown. These observations provide a cautionary note about the safety of current cloning procedures. Ovaries were collected from pubertal gilts at a local slaughterhouse, and oocytes with evenly granulated cytoplasm surrounded by at least three uniform layers of compact cumulus cells were selected and cultured in NCSU-23 medium. A single donor cell was injected into the perivitelline space of each egg and then electrically fused. Approximately 200 scNT embryos were transferred into the oviduct of each recipient. In the study, we generated 58 somatic cell cloned (scNT) piglets. Of these, six scNT piglets displayed severe abnormalities in several organs due to severe disruption of two umbilical cord arteries. Histologically, there was evidence of severe calcification in the kidney and placenta, peliosis in the sinusolidal space of the liver, abnormal stroma cell proliferation in lung, and tubular degeneration as a result of ischemic change due to lung congestion. To determine the genes responsible for disruption of umbilical cord arteries, a pig 13 K oligo cDNA microarray, developed in-house at GenoCheck Co., Ltd. (Ansan, Korea), was used for gene expression profiling studies. In scNT piglets that have severe disruption in the arteries of umbilical cord, 23 genes were up-regulated, but 127 genes were down-regulated, especially angiogenesis-related genes (VEGFa, PLANH1, CYR61, and SMAD4), cytoskeleton-related genes (14-3-3E, CCT3, DCTN1, KRT7, and MARK4), and hypoxia-related genes (HIG2 and DDIT4). These scNT piglets were chronically exposed to maternal and fetal blood circulation problems and, as a result, they died within the first week of life. The histological findings in this study have not been reported previously in scNT animals and demonstrate a possible correlation between early death of scNT clones and abnormality of two umbilical cord arteries.
250 DISTRIBUTION OF HISTONE macroH2A1 IN BOVINE PRE-IMPLANTATION EMBRYOS DERIVED FROM PARTHENOGENETIC ACTIVATION AND IN VITRO FERTILIZATION
Authors: Kim C. , Ma Y. , Chang C.-C. , Rasmussen T. , Yang X. , Tian C.
Published: December 2006

It is known that heterochromatin is characterized by the presence of the histone variant of macroH2A1. MacroH2A1 is a core variant histone with a hybrid structure consisting of a domain that resembles a full-length histone H2A1 followed by a large nonhistone domain. We have previously studied the dynamic changes of macroH2A1 accumulation during the pre-implantation developmental period in the mouse. In the present study, we investigated the distribution of microH2A1 in bovine metaphase II oocytes and pre-implantation embryos at 2-, 4-, 8-, 16-cell, and morula stages as well as blastocysts harvested at Days 8, 9, 10, 11, 12, and 13 following activation and in vitro fertilization (IVF). To generate parthenotes, denuded and in vitro-matured oocytes were activated using a combined treatment of calcium ionophore A23187, cycloheximide (CHX), and 6-dimethylaminopurine (6-DMAP). Five oocytes and pre-implantation embryos at each stage of development were used to follow the development expression pattern of microH2A1 by immunocytochemistry. The cross-reactivity of the primary antibody against mouse microH2A1 was verified by Western blot analysis with bovine fibroblasts. Another staining control included immunostaining with antibody against histone molecules. The stained embryos were observed by laser-scanning confocal microscopy and epiflourescence microscopy. No microH2A1 stain was observed in bovine oocytes or pre-implantation embryos up to the expanded blastocyst stages. In the IVF group, the macroH2A1 was first found in elongated blastocysts (Day 11) after hatching. We observed different expression patterns of macroH2A1 in activated vs. IVF bovine embryos. In the parthenote group, we failed to find robust expression even when embryos were cultured for 13 days. Moreover, the pattern of macroH2A1 expression in bovine embryos was different fromn that in the mouse, in which the onset of macroH2A1 expression occurred by the 16-cell morula stage. These results suggest species differences in the establishment of epigenetic signals.
This work was supported by grants from USDA to X. Y. and X. C. T.
256 LINES OF PIGS SELECTED FOR COMPONENT TRAITS OF LITTER SIZE EXHIBIT DIFFERENTIAL GENE REGULATION AT THE ONSET OF EMBRYO (TROPHECTODERM) ELONGATION
Authors: Miles J. R. , Blomberg L. A. , Freking B. A. , Zuelke K. A.
Published: December 2006

Significant embryonic (<18%) loss occurs in the pig as the pre-implantation embryo undergoes a dramatic morphological change from ovoid (8-10 mm) at gestational Day 11 to a long, thin filament (<150 mm) by Day 12. Lines of pigs selected for increased uterine capacity have improved embryonic survival, whereas pigs selected for increased ovulation rate have decreased embryo survival. Alterations in the expression of genes that play key roles in embryo elongation and maternal recognition of pregnancy may reflect differences in embryo survival observed in these lines of pigs. The objective of the current study was to evaluate the expression level of transcripts involved in developmentally important processes, such as steroidogenesis, cellular differentiation, cell-cell interaction, and immune modulation, at the onset of porcine embryo elongation (gestational Day 11) in control animals and lines selected for increased ovulation rate or uterine capacity. Total RNA was isolated from Day 11 ovoid embryos, and gene expression was measured via real-time PCR. Data were analyzed for analysis of variance using GLM procedures. Expression of steroidogenic acute regulatory protein mRNA was decreased (P = 0.02) in embryos from the ovulation rate and uterine capacity lines compared with controls. Similarly, expression of cytochrome P450scc and aromatase mRNA were decreased (P < 0.003) in embryos from the ovulation rate and uterine capacity lines, compared with the control line. Interestingly, 17�-hydroxylase mRNA expression was decreased (P = 0.0004) only in embryos from the ovulation rate line compared with the control and uterine capacity lines. In contrast, expression of cytokeratin-8 and -18 mRNA were increased (P < 0.02) in embryos from the uterine capacity and ovulation rate lines, compared with controls. Expression of galectin 1 mRNA was increased (P = 0.02) in embryos from the uterine capacity line, compared with the ovulation rate line. Expression of interleukin-1 receptor type 1 mRNA was increased (P = 0.03) in embryos from the uterine capacity line, compared with both the control and the uterine capacity lines. Constitutive expression of 17�-hydroxysteroid dehydrogenase, stratifin, and interleukin-1� mRNA were observed in embryos from all lines. These results demonstrate altered gene expression in embryos from pigs selected for increased uterine capacity and ovulation rate compared with controls, and may contribute to differences observed in embryo survival between these lines of pigs.