bluuebunny/crossref_metadata_2025 huggingface dataset:
250SPECIFIC GENE KNOCK DOWN OF OCT-4 IN MOUSE PREIMPLANTATION EMBRYOS USING SHORT INTERFERENCE RNA
Authors: Shin M.R. , Cui X.S. , Lee K.A. , Jun J.H. , Kim N.-H.
Published: February 2004

RNA interference is used to specifically and effectively inhibit the expression of cognate genes. In the present study we investigated the inhibitory effect of gene expression in mouse embryos developing in vitro by injecting short interference RNA (siRNA). Fertilized mouse zygotes were obtained from mated females 20–24h after hCG injection. Chemically synthesized 21-nt siRNA was commercially obtained and injected into mouse zygotes. The zygotes were then cultured in KSOM medium supplemented with 4% BSA at 37°C. Semi-quantitative RT-PCR was used to examine Octamer-binding transcription factor (Oct-4) gene expression in a single mouse embryo developing in vitro following siRNA-injection. In order to determine the expression and distribution of Oct-4 in mouse embryos, the mouse embryos were fixed in 4% paraformaldehyde for 20 min and permeabilized with 0.2% triton x-100 for 10min. Embryos were then incubated with rabbit Oct-4 polyclonal antibody for 1h and with FITC-labeled goat anti-rabbit antibody. Propidium iodide was used for DNA staining. siRNA injection did not retard the development of mouse zygotes. The number of blastocyst cells and the ICM/TE ratio did not differ in the siRNA injected blastocysts and the non-injected control group. Semi-quantitative RT-PCR revealed that Oct-4 expression was decreased at the 4-cell embryo stage and was significantly high at the morula and blastocyst stages. Injection of siRNA into oocytes inhibited RNA expression of Oct-4 and Nanog, but not of E-cadherin and Heat shock protein 70.1. Immunocytochemical staining showed inhibition of Oct-4 synthesis of the morulae and blastocysts following injection of siRNA. After culture of the embryos in the ES cell-derived conditioned medium, the embryos were stained for alkaline phosphatase (AP), a marker specific to pluripotent cells. AP was not detected in the inner cell mass of blastocysts following siRNA injection. These results suggest that siRNA injection into a mouse zygote specifically inactivates Oct-4 in mouse embryos developing in vitro.
251SEARCH FOR GENES INVOLVED IN DEVELOPMENTAL COMPETENCE IN
MOUSE OOCYTES USING SUPPRESSION SUBTRACTIVE HYBRIDIZATION
Authors: Suzuki O. , Hata T. , Koura M. , Noguchi Y. , Takano K. , Yamamoto Y. , Matsuda J.
Published: February 2004

During the first month after birth, synchronous follicular growth occurs in the ovary of immature mice (first wave). Previously, we showed that mouse oocytes during the first wave were more competent developmentally in older females (Suzuki O et al., 2002 Theriogenology 57, 628 abst), although the numbers of mature oocytes did not differ with female age (17, 18, and 24 days old). In this study, we examined the genetic factors that affect the developmental competence of mouse oocytes during the first wave using suppression subtractive hybridization (SSH). Oocytes collected from 17- and 24-day-old B6D2F1 females (D17 and D24, respectively) without hormonal treatment were matured in Waymouth medium supplemented with pyruvate (0.23mM), antibiotics, bovine fetuin (1mgmL−1), and polyvinylpyrrolidone (3mgmL−1). After 17-h culture at 37°C in an atmosphere of 5% CO2, 5% O2, and 90% N2, total RNA was isolated from oocytes whose germinal vesicles had broken down (mature oocytes), separately, in three independent culture groups per age (each group contained oocytes from four animals) using Cell-to-cDNA Cell Lysis Buffer (Ambion, Austin, TX, USA). Some of the total RNA from each independent group was pooled by age (total of RNA from approximately 100 oocytes per age) and used for SSH. A SMART cDNA Synthesis Kit (Clontech, Palo Alto, CA, USA) was used to reverse-transcribe total RNA to cDNA. SSH was performed with a PCR-Select cDNA Subtraction Kit (Clontech). The subtracted PCR products were cloned into pGEM-T vector (Promega, Madison, WI, USA). Clones from the subtracted library (D24−D17) were sequenced and their identities were examined using the NCBI BLAST search. The differential expression of candidate genes preferentially expressed in mature D24 oocytes suggested by SSH was confirmed with cDNA transcribed separately in the three independent culture groups per age using real-time quantitative PCR with an ABI Prism 7900HT with TaqMan technology (Applied Biosystems, Foster City, CA, USA). Of 513 clones sequenced, the top six preferentially-expressed candidate genes in more developmentally-competent D24 oocytes were spindlin (20 clones), bmi-1 (4 clones), cyclin B1 (4 clones), E330034G19Rik (4 clones), Jagged1 (4 clones), and Ndfip2 (4 clones). The expression of spindlin in mature D24 oocytes (relative threshold cycle: −3.8±0.7, mean±SD) was confirmed to be approximately 11-fold higher than in D17 oocytes (−0.3±1.5) when GAPDH was used as an internal control (P<0.05, t-test). Quantitative analyses of mRNA expression of the remaining genes are now under way. Our results suggest that spindlin is one of the key factors leading to the acquisition of developmental competence in mouse oocytes during folliculogenesis. Supported by JSPS KAKENHI (No.145716000) and MHLW.
254IDENTIFICATION OF OOCYTE SPECIFIC GENES USING SSH AND
MICROARRAY ANALYSIS
Authors: Vallée M. , Gravel C. , Palin M.-F. , Sirard M.-A.
Published: February 2004

The main objective of this project was to isolate and identify important genes specifically expressed in oocytes. These genes are characterized and may uncover the molecular mechanisms related to the unique functions found in the oocyte. Total RNA (1μg) from denuded germinal vesicle-stage (GV) oocytes and somatic tissues were used for cDNA production. The mRNAs were reverse-transcribed and the cDNAs were amplified using the Smart cDNA amplification kit (Clontech, BD Biosciences, San Jose, CA, USA). SSH was performed with the PCR-Select cDNA Subtraction Kit (Clontech). Briefly, pools consisted of bovine oocytes for the tester and bovine somatic tissues for the driver. The same procedure was repeated with mouse and xenopus tissues. All of the 3500 subtracted PCR products generated by SSH were cloned, PCR amplified and sequenced. The resulting sequences were compared against the GenBank database using online computer BLASTn program. For microarray analysis, purified PCR products were spotted on GAPS II glass slides (Corning, Corning, NY, USA) using a VersArray Chip WriterPro robot (BioRad, Hercules, CA, USA). Forward- and reverse-subtracted PCR products were used as probes labeled with Cy-3 and Cy-5 dyes (Amersham, Piscataway, NJ, USA) using the Amino Allyle cDNA Labeling Kit (Ambion, Austin, TX, USA). Slides were scanned and analyzed using the ChipReader and ArrayPro Analyser software (Media Cybernetics, Carlsbad, CA, USA). Detection of the oocyte-specific zona pellucida (ZP), growth differentiation factor-9 (GDF-9), bone morphogenetic protein 15 (BMP15), H1 histone family, member O, oocyte-specific (H1oo), and cyclin B1 transcript in the oocyte subtracted library increased confidence in the validity of the subtraction procedure. All of these transcripts account for 12 % of the clones for the mouse subtracted oocyte library. Microarray analysis performed with the mouse arrays revealed that 33% (382) of the clones were differentially expressed (ratio>10) in the oocyte for the mouse library. Of the 139 clones (12%) that seemed to be present only in the oocyte, 65 were found to be expressed in both the bovine and the xenopus subtracted oocyte library. The most interesting clones to date are #1518, a gene associated with pluripotency;; #1776, a cDNA associated with stem cells in mouse, and #1906, a protein that interacts with chromatin. Since they are conserved across species, the chances that their function is important are quite high. A validation step with RT-PCR analysis will need to be performed on genes identified as oocyte-specific, and the functionality of these genes will be determined with the RNAi technique. WEB page http://www.begc.crbr.ulaval.ca/ (Supported by NSERC).
255EXPRESSION OF TRANSCRIPTION FACTORS PRIOR TO THE
MATERNAL-TO-ZYGOTIC TRANSITION IN BOVINE EMBRYOS
Authors: Vigneault C. , McGraw S. , Bujold G. , Sirard M.-A.
Published: February 2004

During the first stages of bovine embryonic development, until the 8- to 16-cell stage, the zygote is maintained by the mRNA and proteins stored in the oocyte. New embryonic transcription is reported to begin only at the 8- to 16-cell stage even if some minor transcription is detected from the 2-cell stage. In order for this to occur, several factors are required to remodel the chromatin and activate the transcription machinery. Some regulating transcription factors are possibly present in the oocyte in their mRNA form, and their translation could enhance the maternal-to-zygotic transition (MZT). In our study, we observed the expression patterns of five transcription factors (ATF2, HMGN2, HMGB2, HUEL and MSY2) in bovine in vitro-produced embryos. Embryos were produced in vitro using selected cumulus-oocyte complexes from 3-5-mm follicles of slaughterhouse ovaries. Pooled GV or MII oocytes, and 2-, 4-, 8-cell and blastocyst-stage embryos (n=40/stage) were washed in PBS and frozen at −80°C. Each pool was spiked with 1 pg of GFP RNA containing a poly(A) tail. The RNA was extracted using the Absolutely RNA Microprep Kit (Stratagene, La Jolla, CA, USA), co-precipitated with linear acrylamide (Ambion, Austin, TX, USA) and reverse-transcribed with Omniscript (Quiagen). The quantitative amplification of the transcription factors was performed in triplicate using the equivalent of 1 oocyte or embryo per reaction on a Lightcycler (Roche, Indianapolis, IN, USA). Data were normalized with the GFP levels found in each pool and a Least-Significant-Difference method was used for statistical analysis. Immunocytochemistry studies were performed on oocytes and embryos fixed and permeabilized in a solution of paraformaldehyde and Triton X-100, and results were observed on a confocal microscope. Our results show that the transcripts of the transcription factors studied are found at higher levels in pre-MZT embryos and at lower levels in subsequent stages. For HMGN2 and MSY2, there is a decrease in mRNA during oocyte maturation. For both genes, the residual mRNA remains constant up to the 4-cell stage before another loss in transcript levels in the 8-cell stage. In the case of ATF2, HMGB2 and HUEL, the maternal transcript levels are maintained until the 4-cell stage, suggesting that the mRNA is protected from degradation until its possible translation at the MZT. These results, combined to immunolocalization of the proteins, suggest a possible implication of some of these factors in the bovine MZT.
257DIFFERENTIAL GENE EXPRESSION IN GERMINAL VESICLE AND
METAPHASE II MOUSE OOCYTES.
Authors: Yoon S.J. , Jun E.H. , Kim Y.H. , Lee H.Y. , Kim N.-H. , Lee K.A.
Published: February 2004

The present study was conducted to identify oocyte-specific genes according to developmental stage. We used an accurate Annealing Control Primer (ACP;; SeeGene, Seoul, Korea)-based GeneFishing technology. This new and innovative method has developed 120 ACPs so far to locate differentially expressed genes. This method is more specific and sensitive than differential display-PCR that is labor-intensive and results in a high degree of false positives. To identify genes differentially expressed in germinal vesicle (GV)- and metaphase II (MII)-stage oocytes, fully grown GV-intact and MII oocytes were obtained. The addition of 0.2mM IBMX in the collection medium inhibited GV breakdown. The mRNA samples were prepared from the same number of GV and MII oocytes using a Dynabeads mRNA DIRECT Kit. GeneFishing system utilizes a two-stage PCR to fish out authentic differentially expressed genes by regulating binding of each portion of ACP at a different stage of PCR. The differentially expressed bands were extracted and cloned into a TOPO TA cloning vector (Invitrogen, Carlsbad, CA, USA), sequenced, and analyzed by BLAST search. To confirm the differential expression of selected transcripts, RT-PCR and immunofluorescence staining were conducted. Using 12 different ACPs, we isolated 27 differentially expressed bands from GV and MII oocytes. BLAST results indicated that most of these bands had strong homology with known genes. We confirmed that pleckstrin homology, protein kinase D2 (PKD), and COP9 (constitutive photomorphogenic) homologs were GV-specific, while minichromosome maintenance deficient 2 mitotin (MCM2), malate dehydrogenase, soluble Mor2, growth arrest specific 6 (Gas-6), Bcl-2 homolog (Diva), and suppressor of cytokine signaling 4 were expressed highly in MII. Immunofluoresence staining results confirmed higher expression of Gas-6 protein in MII oocytes compared to GV oocytes. Immunohistochemistry results with ovarian tissues demonstrated that MCM2 protein was highly expressed in the nucleus of developing oocytes and granulosa cells, and Diva was highly expressed in the oocytes of primordial follicles. Immunofluoresence staining for these proteins is under investigation. The functions of many of these genes have been determined in systems other than gamete systems. Those data provide insight about their function in oocyte maturation. For example, PKD, COP9, and pleckstrin homology are genes in the axis of a newly identified PKD signaling system. Therefore, further investigation of the functions of each gene identified in this study will provide a basis for understanding the mechanism of oocyte maturation.
258ULTRASTRUCTURAL STUDY OF STALLION SPERM FOLLOWING THAWING,
CAPACITATING AND IVF PROCEDURES: EFFECT OF THE PRESENCE OF CUMULUS-ENCLOSED
OOCYTES
Authors: Abbate F. , Germanà P.G. , Colleoni S. , Gandolfi F.
Published: February 2004

The use of IVF in horses has a limited efficiency, reflecting low oocyte developmental competence and inadequate sperm capacitation procedures. In a preliminary study, using carboxyfluorescein diacetate/propidium iodide staining, we determined that the freezing-thawing procedure left only 56.6±3.4 % of the sperm cells with an intact membrane. The following incubation in TALP-IVF induced membrane damage at high rates with only 9.58±1.8 % of them intact after 18h. However, the presence of at least four cumulus-enclosed oocytes (CEO) in the medium significantly increased the number of membrane-intact spermatozoa at the end of incubation (53.87±1.99%). This indicated that the sperm thawing and capacitating procedures can damage the cell membrane but the presence of four or more CEO in TALP-IVF could prevent further damages. The aim of the study was to investigate in detail the membrane damages and to analyze the differences induced by the presence of CEO. Spermatozoa were thawed in water at 37°C, and centrifuged for 30 minutes at 600g in a 45–90% Percoll gradient made with modified Tyrode’s medium. The sperm pellet was washed once in the same medium and diluted to a final concentration of 1×106 spermatozoa/ml TALP supplemented with 0.6% (w/v) BSA fatty acid free and 12μgmL−1 heparin (TALP-IVF). Sperm cells were incubated with 0 or 4 in vitro-matured CEO. Sperm cells were examined after thawing, 0, 2 and 18h from the beginning of incubation in TALP-IVF. Each experiment was replicated at least 3 times. Both scanning and transmission electron microscopy were performed on sperm samples fixed in 2.5% glutaraldehyde in 0.1M phosphate buffer, pH 7.2, using standard procedures. Specimens for scanning electron microscopy were examined under a field emission gun JEOL JSM 6301 microscope. For transmission electron microscopy the samples were examined with a JEOL JEM 100 SX. A minimum of 25 cells were analyzed for each group. Immediately after thawing, damaged spermatozoa showed, on the surface of their heads, small vesicles correlated to a progressive process of vacuolisation and degeneration of membrane integrity. The same lesions were visible at all the successive time points taken into account. Moreover, a loss of the acrosome integrity with acrosomal swelling and a decrease of content homogeneity were observed particularly in the spermatozoa cultured for 18 h without CEO. When CEO were present in the IVF medium lesions were visible in a lower percentage of spermatozoa but the type of lesions did not differ from those observed in their absence. These observations confirmed our previous data and gave more details on the lesions that occur during the IVF procedures in the horse. Supported by MURST COFIN grant n. 2001078849.
265EFFECT OF SPECIFIC GRAVITY OF CULTURE MEDIUM ON IN VITRO EMBRYO
DEVELOPMENT IN BUFFALO
Authors: Arathy D.S. , Ashis S. , Sharma G.T. , Majumdar A.C. , Chauhan M.S.
Published: February 2004

In buffalo the success rate of transferable quality embryo production through in vitro procedure is very low as compared to cattle. Sub optimal culture conditions and physical conditions such as specific gravity of the culture medium may lead to a reduced rate of transferable buffalo embryo production from the oocytes matured and fertilized in vitro (Palta & Chauhan,1998 Reprod. Fertil. Dev. 10, 379–391). This experiment was therefore conducted to find out the role of specific gravity of the IVC medium on the development rate of the buffalo embryos in vitro. Follicles of slaughter house ovaries were aspirated and the collected oocytes with cumulus-oocytes complexes (COCs) were cultured in TCM-199 medium supplemented with 10% fetal calf serum, 10% buffalo follicular fluid and 0.5μgmL−1 FSH in 5% CO2 incubator at 38.5°C. The matured oocytes were then inseminated with frozen-thawed buffalo semen suspended in BO medium. After 42h of post-inseminations the cleavage rates were evaluated. The 2–4 cell-cleaved eggs (Day 2 of post-insemination) were randomly divided and cultured for eight days in vitro in 1) modified synthetic fluid (mSOF)+0.8 %BSA (control), 2) mSOF+0.8 % BSA+gelatin (1mgmL−1) 3) mSOF+0.8% BSA+1mgmL−1 gelatin+10ngmL−1 epidermal growth factor (EGF). Supplementation of gelatin increased the specific gravity of the mSOF medium from 0.9658±0.009 to 1.0331±0.013 without any change in pH (7.4). The development of embryos to the 8–16 cell-stage on day 4 of in vitro culture were significantly higher (P<0.05) in mSOF+0.8% BSA+1mgmL−1 gelatin (81.8%; 27/33) than that in mSOF + 0.8% BSA (75.7%; 28/37) and mSOF+0.8% BSA+10ng/mL EGF (68.7%; 22/32). When these embryos were further cultured for another four days (Day 8), the development of transferable quality embryos (morula/blastocyst) was 42.4% (14/33), 48.7% (18/37) and 46.9% (15/32), respectively. Supplementation of gelatin increased the cleavage of eggs up to the 8–16 cell-stage embryo, but did not significantly enhance the rate of development to the morula/blastocyst stage in comparison to control and EGF-supplemented group. However, the percentage of transferable quality embryos was slightly lower in the gelatin-added group but not statistically significant than other groups. The study concluded that increase in specific gravity of the in vitro culture medium enhanced initial cleavage rate but did not have any role in transferable embryo production in buffalo.
267BODY WEIGHT GAIN VARIATION, HORMONAL AND METABOLIC
STATUS AND IN VITRO EMBRYO PRODUCTION IN SUPEROVULATED DAIRY HEIFERS
Authors: Freret S. , Ficheux C. , Jeanguyot N. , Joly C. , Ponter A. , Ponsart C. , Grimard B. , Humblot P.
Published: February 2004

This study aimed to investigate the hormonal and metabolic status of heifers subjected to short-term variation of energy intake and associated growth rate;; oocytes were collected by ovum pick-up (OPU) for in vitro embryo production. Experimental scheme, diets and OPU protocol have been described previously (Freret et al., 2003 Theriogenology, 59, 445 abst). Briefly, oocytes from 16 Prim′Holstein heifers (14±1 months old, 340±25kg) were collected by OPU every two weeks after superovulation treatment (total dose of 250μg FSH (Stimufol®, Merial, France) divided into 5 i.m. injections 12 hours apart, at decreasing doses). They received individually for 6 weeks (Period 1=OPU 1 to 4) a diet aimed at a 1000g day−1 body weight gain (BWG). Heifers were then allocated to 2 groups (overfeeding or dietary restriction), for 8 weeks after OPU 4 (Period 2=OPU 5 to 8). COCs were collected 12h after the last FSH injection for IVF and IVC. Blood sample analyses were performed once a week to determine glucose, insulin, IGF1, non esterified fatty acids (NEFA), β-hydroxybutyrate (βOH) and urea concentrations, and at the time of follicular puncture for estradiol assay. Effects of period, group of growth rate and their interaction where analyzed using the mixed procedure of SAS (female effect as random) and least-squares means were subsequently compared with Scheffe’s test. Three groups of growth rate were determined according to results observed during period 2 (Table 1). A period effect was observed for glucose, insuline and estradiol (P<0.05). But Scheffe’s test showed a significant variation between periods only in the ‘600gday−1’ group, with more estradiol and less glucose in period 2 which was associated with more blastocysts and grade 1 embryos (Freret et al., 2003 Theriogenology, 59, 445 abst), and in the ‘1400g day−1’ group with more insulin in period 2 (associated with more follicles <8mm 2 days before FSH treatment). In period 2, βOH concentration was significantly higher in the ‘1400gday−1’ group when compared to the others (Table 1). For urea, NEFA and IGF1 concentrations, no difference between groups or periods was observed. These results illustrate the role of glucose and insulin as mediators of nutritional effects on reproduction in growing animals. These results suggest that nutritional requirements aimed at optimizing follicular growth and embryonic development may be different.

Table 1
268PREGNANCIES FROM FROZEN IVF CATTLE EMBRYOS USING
SEX-SORTED AND UNSORTED SPERM
Authors: Fry R.C. , Earl C.R. , Hollinshead F.K. , Wild D. , Lindemans W.
Published: February 2004

Previously we demonstrated that sex-sorted sperm could produce IVF embryos from juvenile and adult cattle at rates similar to those for unsorted sperm (Fry et al., 2003 Theriogenology 52, 198). In this study we investigated the pregnancy rates of recipient cattle following the transfer of frozen/thawed IVF embryos generated from young heifers using sex-sorted and unsorted sperm. COCs collected from FSH-stimulated Senepol or Beefex heifers by TVR were matured, fertilized with either sex-sorted or unsorted Senepol sperm and cultured for 6 days under our standard laboratory conditions (Fry et al., 2003 Theriogenology 59, 446, Earl et al., 1997 Theriogenology 47, 255). Embryos reaching the blastocyst or expanded blastocyst stage of development were frozen by the CL-V method of vitrification. Briefly, embryos were equilibrated for 5–10min in HEPES-199 media containing 20% FCS (HM), placed in HM containing 10% EG, 10% DMSO for approximately 2 minutes and then in HM containing 20% EG, 20% DMSO for between 20–60sec (Vatja et al., 1997 Cryoletters 18, 191). Vitrification was achieved by collecting between 5–10 IVF embryos in a 3-μL droplet and securing this droplet to a coded CL-V holder. The droplet was vitrified using the CL-V kit (Lindemans et al., 2004 Theriogenology in press) and then sealed in a precooled ‘‘straw’’ for storage in liquid nitrogen. To thaw, the ‘‘straw’’ with specimen was removed from storage;; the specimen droplet was withdrawn from the ‘‘straw’’ and placed directly into HM containg 0.2M sucrose (SM). After approximately 5–10min each embryo was assessed, loaded into a tomcat catheter in SM and transferred surgically into a recipeint cow within 10–15min of thaw. Of 129 Brahman and Brahman cross cows receiving 2 injections of 125μg cloprostenol 11 days apart, 60 exhibited oestrus 2–4 days after the second injection and 53 were deemed suitable for embryo transfer. Pregnancy was determined by ultrasound on Day 40. No difference in pregnancy rate was found between treatment groups (P>0.05; Table 1). The low submission rate (60/129) and pregnancy rate for the in vivo control group indicate that the fertility of the recipient cows may have been compromised by the drought conditions predominating in Central Queensland. Notwithstanding, the CL-V method for the vitrification of IVF embryos produced by either sex-sorted or unsorted sperm gave similar and very promising pregnancy results of around 40%. This provides new opportunities for the rapid banking of large numbers of sexed IVF embryos generated from elite cattle by TVR for user friendly embryo transfer programs.

Table 1
Pregnancies from IVF embryos derived from sex-sorted and unsorted sperm and frozen by the CL-V method of vitrification, or from in vivo embryos frozen in glycerol
276INFLUENCE OF SPERM TREATMENTS ON BLASTOCYST DEVELOPMENT IN VITRO AND
CELL NUMBER IN CATTLE
Authors: Jeon B.G. , Moon J.S. , Kim K.C. , Rho G.J.
Published: February 2004

Experiments were designed to examine the effects of developmental rate and cell numbers in embryos produced by in vitro fertilization (IVF) using sperm from 2 bulls (sperm A and B purchased from commercial sale) isolated by three methods. Cumulus-oocyte-complexes collected from ovaries harvested from a local slaughter house were matured in 50μL droplets of serum-free M199 medium supplemented with 1μgmL−1 estradiol-17β, 10μgmL−1 LH and FSH under silicone oil at 39°C in a humidified atmosphere of 5% CO2 in air. After 24h of culture, oocytes were fertilized with the sperm treated by three different methods of isolation;; percoll gradient, swim-up and glass wool filtration;; a final concentration of 2×106 cells mL−1 was used. At 16h after fertilization, presumptive zygotes were co-cultured in serum-free M199 with BOEC for up to 192h post-insemination. At 48h and 120h post-insemination, the cultures were fed with 25μL of serum-free M199. The embryos were compared for their rates of cleavage at 48h post-insemination, development to the blastocyst stage, and hatching, and also the cell number at 192h post-insemination. Differences between treatments were analyzed using one-way ANOVA after arc-sine transformation of the proportional data of cleavage, development into blastocyst stage and hatching. Comparisons of means among treatments were performed using Tukey-Kramer multiple comparisons test. The results are summarized below. The rates of cleavage in embryos produced by IVF using sperm from 2 bulls isolated by percoll, swim-up and glass wool were not significantly different. The blastocyst development and hatching rates between sperm treatment were not significant within bull sperm A and within sperm B. However, although the hatching rate in percoll treatment of bull sperm A was higher than in bull sperm B, the difference was not statistically significant. The mean cell numbers in percoll treatment of bull sperm A (176.5±7.1) were significantly higher (P<0.001) than the others. In bull sperm B the cell numbers in percoll treatment were higher than the other two treatments, but the differences were not statistically significant. In conclusion, these results support the concept that sperm preparation using percoll has beneficial effects on blastocyst cell number.

Table 1
Developmental rates of in vitro embryos using sperm from 2 bulls isolated by three methods with 4 replicates
277ENERGY REQUIREMENT DURING DEVELOPMENT TO THE BLASTOCYST STAGE OF
PORCINE EMBRYOS PRODUCED IN VITRO
Authors: Kikuchi K. , Ozawa M. , Fuchimoto D.-I. , Noguchi J. , Kaneko H. , Nagai T.
Published: February 2004

A successful in vitro production (IVP) of porcine blastocysts, which enables piglet production after transfer to recipients, was reported (Kikuchi et al., 2002 Biol. Reprod. 66, 1033–1041). Generally, in the IVP system, both glucose and glutamine as energy sources were included in vitro culture (IVC) medium from Day 2 (Day 0=the day of in vitro fertilization) until Day 6. However, the exact requirement of these substances for the development to the blastocyst stage of IVP embryos has not yet been clarified. The objective of the present study was to evaluate whether these two substances are necessary for embryonic development to the blastocyst stage in culture during the period. Porcine cumulus-oocyte complexes were matured for 46h and fertilized in vitro as reported by Kikuchi et al. (see above). After removal of cumulus cells and spermatozoa, the oocytes were cultured subsequently in NCSU-37 supplemented with pyruvate and lactate (IVC-PyrLac) for 2 days. Then they were cultured until Day 6 in other IVC medium prepared as follows (1–6); Basic IVC medium (BM) was a modified NCSU-37 consisting of 108.7mM NaCl, 4.8mM KCl, 1.7mM CaCl2, 1.2mMKH2PO4, 1.2mM MgSO4, 25.1mM NaHCO3 and 4mgmL−1 fatty acid-free BSA. Then one or more of the following energy sources were supplemented to BM;; (1) 12mM sorbitol (SigmaUltra), 5.55mM glucose (Wako special grade) and 1.0mM glutamine (Sigma) (NCSU-37/Gln+), (2) 19.2mM sorbitol and 1.0mM glutamine (IVC-Sorbitol/Gln+); (3) 19.2mM mannitol (SigmaUltra) and 1.0mM glutamine (IVC-Mannitol/Gln+), (4) 12mM sorbitol and 5.55mM glucose (NCSU-37/Gln−); 5) 19.2mM sorbitol (IVC-Sorbitol/Gln−); and 6) 19.2mM mannitol (IVC-Mannitol/Gln−). The osmolarity of these media was adjusted to 283–285 osmolg−1. All embryos were fixed as whole mounts, stained and evaluated. The rate of blastocysts in NCSU-37/Gln+ (26.8%) was significantly higher (P<0.05; by analysis of variance and Duncan’s multiple range test) than those in IVC-Sorbitol/Gln+, IVC-Mannitol/Gln+ and NCSU-37/Gln− (19.0%, 17.0% and 15.5%, respectively). A remarkable decrease in the rates in IVC-Sorbitol/Gln− and IVC-Mannitol/Gln− (P<0.05; 1.4% and 2.0%, respectively) was observed. The cell numbers of NCSU-37/Gln+, IVC-Sorbitol/Gln+, IVC-Mannitol/Gln+ and NCSU-37/Gln− (55.5, 52.0, 49.6 and 58.7, respectively) had a tendency to be higher than those of IVC-Sorbitol/Gln− and IVC-Mannitol/Gln− (38.0 and 35.2, respectively). These results confirm that the supplementation of maturation medium with at least one energy source (glucose or glutamine) promotes embryonic development in vitro to the blastocyst stage, that the combination of both sources improves the chance of the embryonic survival, and that porcine embryos do not utilize sorbitol or mannitol as an energy source. The importance of glucose and glutamine is suggested for the development to the blastocyst stage of porcine IVP embryos.
279VASCULAR MORPHOMETRY OF BOVINE PLACENTOMES IN LATE GESTATION FROM
EMBRYOS PRODUCED IN VIVO OR IN VITRO
Authors: Miles J.R. , Farin C.E. , Rodriguez K.F. , Alexander J.E. , Farin P.W.
Published: February 2004

The role of the vascular supply in the development of placentas from embryos produced in vitro is poorly understood. The objective of this study was to determine the effects of in vitro embryo production on morphometry of blood vessels within fetal (cotyledonary) and maternal (caruncular) components of the placentome during late gestation. In vivo-produced embryos were recovered from superovulated Holstein cows on Day 7 after estrus. For in vitro embryo production, oocytes were aspirated from the ovaries of Holstein cows, matured in vitro, and then fertilized. Presumptive zygotes with their cumulus cells were transferred into M-199 with 10% estrus cow serum and cultured for 168h post-insemination. Semen from the same Holstein sire was used for the production of in vivo and in vitro embryos. Single blastocysts from each production system were transferred into the uteri of heifers. On Day 222 of gestation, fetuses and placentas were recovered in utero (in vivo, n=12; in vitro, n=12). Placentomes were collected, fixed and sectioned. Fetal and maternal blood vessels were identified within placentome sections using immunocytochemistry for vascular endothelial growth factor (VEGF) protein. A total of 4.8×105μm2 of tissue were examined from each placentome. Stereological methods were used to determine the volume densities of fetal and maternal blood vessels. Data were analyzed by GLM procedures. Fetuses were heavier (P=0.03) in the in vitro group (20.7±1.0kg, LS mean±SEM) compared to the in vivo group (17.3±1.0kg). Placentas were also heavier (P=0.06) for the in vitro group (2.5±0.2kg) compared to the in vivo group (2.0±0.2kg). Placental efficiency, calculated as fetal weight/placental weight, was similar between the two treatment groups (9.0±0.5 and 8.9±0.5 for in vivo and in vitro, respectively). Fetal vascular volume density in placentomes was not different between the two treatment groups (5.4±0.3% and 5.4±0.3% for in vivo and in vitro, respectively). In contrast, maternal vascular volume density was greater (P=0.02) for placentomes in the in vitro group (5.9±0.3%) compared to in vivo controls (4.9±0.3%). In summary, compared to placentomes from embryos produced in vivo, placentomes from embryos produced in vitro had similar volume density of fetal vessels, but had significantly increased volume density of maternal vessels. Supported by the State of North Carolina.
287IN VITRO PRODUCTION OF HOLSTEIN EMBRYOS USING SEX SORTED SEMEN
Authors: Wilson R.D. , Weigel K.A. , Fricke P.M. , Leibfried-Rutledge M.L. , Matthews D.L. , Rutledge J.J. , Schutzkus V.R.
Published: February 2004

Our objective was to explore the synergy between sexed semen and in vitro embryo production and assess benefits of these technologies on commercial farms. Ovaries were collected from high genetic merit Holstein cull cows via colpotomy or at the time of slaughter. Oocytes were aspirated from the ovaries, fertilized 20–24h later, and matured to the morula or blastocyst stage. Embryos were transferred into recipient Holstein cows and heifers on the same farms. Seven Wisconsin herds participated, and 365 embryos were produced from 104 donor cows. Only 272 of these embryos were transferred due to limited availability of recipients. Sexed semen from three Holstein sires was used. On average, 3.5±0.37 transferable embryos were produced per donor, including 1.4±0.18 grade 1 embryos and 1.5±0.20 grade 2 embryos. Individual farms averaged from 1.6 to 5.8 transferable embryos per donor. Laboratory data also revealed interesting results. On average 43.7±4.0 oocytes were collected per donor, and the number of usable oocytes (33.9±3.4), and percent embryos cleaved (52.1±1.9), were significant predicators of the number of blastocysts developed. We divided the usable oocytes and embryos cleaved per donor into quartiles. The fourth quartile for embryos cleaved was significantly greater (P<0.05) than the lower three quartiles, and the usable oocyte quartiles all significantly differed from each other. Semen freeze date was also a significant predicator of the number of blastocysts developed, suggesting significant variation in the quality of sorted semen per ejaculate. To preliminarily test the effect of sorting on the percentage of embryos developing to blastocyst stage, oocytes were recovered from ovaries collected at a slaughterhouse and fertilized using non-sorted semen or sex-sorted semen from the same sires. Oocytes (n=3312) fertilized using non-sorted semen tended (P=0.06) to produce more embryos developing to blastocyst stage than oocytes (n=1577) fertilized using sex-sorted semen (20.1±2.9% v. 12.2±2.3%, respectively). Preliminary pregnancy results show strong farm and sire effects. Overall conception rate was 36% for heifer recipients and 18milking cow recipients. These results suggest that low cost in vitro embryo production may have promise as an early system for utilizing sexed semen in dairy cattle breeding programs.
289EFFECT OF FERTILIZATION TIME OF PIG OOCYTES MATURED IN-VITRO BY BOAR
SPERM STORED AT 4°C
Authors: Yi Y.J. , Kim M.Y. , Chang Y.J. , Jin D.I. , Park C.S.
Published: February 2004

The use of boar sperm stored at 4°C may be a useful tool for in vitro production of pig embryos. Therefore, this study was undertaken to investigate the effect of fertilization time of pig oocytes matured in-vitro by boar sperm. The sperm-rich fraction (30–60mL) was slowly cooled to room temperature (20–23°C) by 2h after collection. Semen was transferred into 15-mL tubes, centrifuged at room temperature for 10min at 800g, and the supernatant solution was poured off. The concentrated sperm was resuspended with 5mL of the LEN (11.0g lactose hydrate, 20mL egg yolk, 0.05g N-acetyl-D-glucosamine and 100mL distilled water) diluent to provide 1.0×109 spermmL−1 at room temperature. The resuspended semen was cooled in a refrigerator to 4°C. The medium used for oocyte maturation was TCM-199 supplemented with 26.19mM sodium bicarbonate, 0.9mM sodium pyruvate, 10μgmL−1 insulin, 2μgmL−1 vitamin B12, 25mM HEPES, 10μgmL−1 bovine apotransferrin, 150μM cysteamine, 10IUmL−1 PMSG, 10IUmL−1 hCG, 10ngmL−1 EGF, 0.4% BSA, 75μgmL−1 sodium penicillin G, 50μgmL−1 streptomycin sulfate and 10% pFF. After about 22h of maturation, oocytes were cultured without cysteamine and hormones for 22h at 38.5°C, 5% CO2 in air. Oocytes were inseminated with boar sperm stored at 4°C for 2 days after collection. Oocytes were coincubated for 1, 3, 6 and 9h in 500μL TBM fertilization media with 1×106mL−1 sperm concentration. Thereafter, oocytes were transferred into 500μL NCSU-23 culture medium containing 0.4% BSA for further culture of 6, 48 and 144h, fixed and stained for the evaluation of fertilization parameters and developmental ability. Data were analysed by ANOVA and Duncan’s multiple range test using the SAS program. The rates of sperm penetration and male pronuclear formation were higher in the fertilization times of 6 and 9h than in those of 1 and 3h. The percentage of polyspermic oocytes was highest in fertilization time of 9h compared with other incubation times. The rates of cleaved oocytes were higher in the fertilization times of 6 and 9h (85.0 and 84.6%) compared with those of 1 and 3h (61.1 and 76.8%). The percentage of blastocyst formation from the cleaved oocytes was highest in the fertilization time of 6h (33.6%) than in that of 1, 3 and 9h (11.4, 23.0 and 29.6%). Mean cell numbers per blastocyst were 32.9±3.3, 27.6±2.7, 26.3±2.2 and 24.4±1.8 in the fertilization times of 6, 9, 3 and 1h, respectively. In conclusion, we found out that boar sperm stored at 4°C could be used for in vitro fertilization of pig oocytes matured in vitro. Also, we recommend the coincubation time of 6h in 500μL TBM fertilization medium with 1×106mL−1 sperm concentration for in vitro fertilization of pig oocytes matured in vitro.
293QUANTITATIVE TESTICULAR HISTOLOGY IN THE COMMON WOMBAT (VOMBATUS URSINUS)
Authors: MacCallum C.A. , Johnston S.D.
Published: February 2004

The seminiferous epithelium cycle of the male Common Wombat ( Vombatus ursinus) is documented here for the first time. Testicular material was obtained from 10 common wombats from the Southern Highlands of NSW in June (n=5) and November (n=5), fixed in Bouins solution and prepared for standard histological processing. Eight stages of the seminiferous cycle where identified based upon relative cellular associations and development of the spermatid during spermiogenesis. Stage 1 was further subdivided into 1A and 1B based on changes in shape of the spermatid nucleus. The relative frequency of each stage was also calculated using observations from 500 seminiferous tubule cross-sections as was the proportion of the various testicular tissue types. The relative proportions of the various stages of the seminiferous epithelial cycle in the common wombat testis were: Stage 1A, 4.0±0.5; Stage 1B, 4.2±0.4; Stage 2, 21.3±1.9; Stage 3, 15.4±1.2; Stage 4, 16.8±1.2; Stage 5, 11.1±1.5; Stage 6, 13.7±1.5; Stage 7, 7.3±0.6; Stage 8, 6.1±0.6. Relative proportions of the various tissue types observed in testis included: seminiferous tubules (41.5%±4.1); seminiferous tubule lumen (33.3±3.4%); leydig cells (14.6±1.1%); connective tissue (10.4±0.9%) and blood vessels (0.2±0.03%).

Table 1
Cell types and cellular associations of the eight stages of the wombat seminiferous epithelial cycle
301EFFECT OF THE MII-AGE AND ACTIVATION PROTOCOL ON THE PARTHENOGENETIC
DEVELOPMENT OF PORCINE OOCYTES
Authors: Lagutina I. , Lazzari G. , Galli C.
Published: February 2004

The completion of porcine oocyte nuclear maturation (MII) in vitro, characterized by the time of polar body extrusion, starts at about 32h of maturation and lasts more than 12h. This leads to the simultaneous presence in the population of matured oocytes with differing abilities to be activated. We investigated age-dependent changes in pig oocyte maturation, activation and development in SOFaa in response to electric impulse (EL) in the presence of cytochalasin B (CB) and EL in combination with cycloheximide and cytochalasin B (EL+CHX+CB). Oocytes were matured in TCM 199 with 10% FCS, cysteine, LH, FSH (Pergovet, Serono, Geneva, Switzerland) for 36h and then decumulated. Matured oocytes were activated at 40 and 44h by double pulse of 30μs DC 1, 5kVcm−1 and cultured in 5μgmL−1 CB for 4h or by EL followed by incubation in 10μgmL−1 CHX+5μgmL−1 CB for 4h. According to the MII-age before activation oocytes were divided into 2 age classes: 3–7 and 7–11h after polar body extrusion. Embryos were cultured in SOFaa in 5% CO2, 5% O2 at 38.5°C. The rates of cleavage, blastocyst formation and cell number of BL on Day 7 (BLD7) were recorded. Our results showed that the average rate of maturation at 44h was 72% (n=1377). About 50% and 87% of oocytes, that eventually matured, extruded the polar body at 37 and 40h, respectively. The average cell number of BLD7 developed in SOFaa was 80±36 (n=52) and was not affected by activation protocol. Seventy-nine and 27% of BL had more than 50 and 100 cells per BL, respectively. Porcine oocytes activated by EL acquired their developmental competence gradually, achieving the highest rates of cleavage and blastocyst formation 7h after polar body extrusion. By contrast, oocytes activated by EL+CHX+CB showed their maximal developmental competence earlier (3–7h group). In conclusion, we demonstrate that electric impulse in combination with CHX+CB treatment permits earlier efficient activation of porcine oocytes (3–7h after polar body extrusion).
306SPONTANEOUS AND PARTHENOGENETIC ACTIVATION OF RAT OOCYTES
Authors: Yoo J.G. , Smith L.C.
Published: February 2004

Oocyte activation is one of the critical steps for the success in a mammalian cloning program. In rats, information regarding parthenogenetic activation of oocytes is limited and further studies are required to elucidate the phenomenon of spontaneous activation and to develop effective protocols of induced activation. The objectives of the study were (1) to characterize the kinetics of spontaneous activation and (2) to examine different activation regimens for rat oocytes. Oocytes were collected from 3–4-wk-old PMSG-primed Sprague Dawley rats at 14h after hCG injection. HEPES-mR1ECM was used for oocyte collection and mR1ECM for in vitro culture (Miyoshi et al., 1995 J. Reprod. Fert. 103, 27–32). Experiment 1: Cumulus-denuded oocytes were fixed and stained with bisbenzimide (Hoechst 33342) at 10, 40, 70, 100 and 130min after recovery. Most oocytes (92.9%) were still arrested in the metaphase II stage at 10min after recovery, but the proportion of activated oocytes increased in a time-dependent manner, i.e. at 40min after recovery 50% were at anaphase. At 70min, 32.4%, 40.5%, 8.1%; at 100min, 4.8%, 57.1%, 23.8%; and at 130min, 0%, 3.3%, 86.7% had progressed to anaphase II, telophase II and extruded a 2nd PB, respectively. Experiment 2: Oocytes were exposed to the following activation treatments: Two sets of electrical stimuli (ES) 1h apart composed of 3 DC pulses, 1s apart of 1.2kVcm−1 field strength and 60μs duration in 0.25M mannitol solution (Roh et al., 2003 Reprod. Fert. and Dev., 15, 135–140). After ES treatment, oocytes were exposed for 3h to either cycloheximide (CHX, 10μg mL−1, Group 1), 6-dimethylaminopurine (DMAP, 2mM, Group 2), or CHX (10μgmL−1) and DMAP (2mM) (Group 3). Group 4 was exposed to Strontium (10mM) for 1h and then incubated with CHX (10μgmL−1) and DMAP (2mM) for 3h. Oocytes were washed thoroughly after treatment, transferred to 50-μL droplets of mR1ECM and cultured at 37°C in a humidified atmosphere of 5% O2, 5% CO2 and 90% N2. Pronuclear formation and cleavage rates were significantly higher in Groups 2 and 3 than in Groups 1 and 4 (92.6%, 88.9% and 84.6%, 80.8% v. 49.1%, 45.6% and 62.8%, 55.8%, respectively). First, these results show that rat oocytes undergo spontaneous activation very rapidly after recovery from the oviducts. Second, activation protocols with two sets of triple DC electrical stimulation followed by exposure to DMAP or CHX/DMAP are effective means of activation. In conclusion, the above information will be useful in establishing effective protocols for cloning rats. (Financial support by NSERC and Canada Research Chairs.)
310ENRICHING A DEFINED MATURATION MEDIUM IMPROVES SUBSEQUENT EMBRYONIC
DEVELOPMENT OF BOVINE OOCYTES CULTURED IN SMALL AND LARGE GROUPS
Authors: Donnay I. , Verhaeghe B. , Neirinckx G.
Published: February 2004

When performing Ovum Pick Up (OPU) in unstimulated animals, the number of oocytes collected per donor is often reduced. Culturing oocytes and embryos in small groups impairs embryonic development by comparison with embryos cultured in large groups: less blastocysts are obtained and their appearance is delayed. The aim of the study was to evaluate the effect of an enriched define maturation medium on further embryonic development of oocytes and embryos cultured in large and small groups during IVM, IVF and IVC. Bovine cumulus-oocyte complexes (COC) were collected from abattoir ovaries, selected on morphological criteria, and then allocated to two maturation media: TCM 199+10ngmL−1 mEGF or the same medium enriched with 5μgmL−1 insulin, 5μgmL−1 transferrin, 5ngmL−1 selenium, 19ngmL−1 IGF-1, 2.2ngmL−1 FGF, 90μgmL−1 L-cystein, 28μM myo-inositol, 100μM β-mercaptoethanol, 75μgmL−1 ascorbic acid, 720μgmL−1 glycine, 0.1mgmL−1 glutamine, 5UImL−1 hCG and 10UImL−1 eCG. For both media, COC were cultured either in groups of 18 to 20 (large) or in groups of 4 to 5 (small) in 500μL of medium in 4-well plates. After 24h maturation at 39°C and in 5% CO2 in air, the COC were fertilized and then cultured in modified SOF medium with 5% FCS at 39°C and in 5% CO2, 5% O2 and 90% N2. No selection was performed after the maturation step and the oocytes and embryos were kept in small or large groups throughout the experiment. Blastocyst development was evaluated at Day 7 and 8 post insemination. Results are shown in Table 1. As expected, a significant decrease was observed in blastocyst rates when oocytes and embryos were cultured in small groups. Enriching the maturation medium led to an important increase in blastocyst rates regardless of the number of oocytes cultured together, but the increase was greater when culture was performed in small groups from the maturation step (56% increase at Day 8 v. 31% increase for embryos cultured in large groups). The enriched medium also accelerated the appearance of the blastocysts in embryos cultured in small groups (74% of the blastocysts appeared on Day 7 instead of 50% in the control medium) which could indicate an improvement in blastocyst quality. The rate of hatching was not significantly increased. In conclusion, enriching the maturation allowed an increase in the developmental competence of abattoir oocytes matured in small and large groups. Although further experiments are needed, this could be of particular interest to improve embryonic development from OPU oocytes.

Table 1
Effect of an enriched defined maturation medium on blastocyst development from oocytes cultured in small or large groups