bluuebunny/crossref_metadata_2025 huggingface dataset:
265 DICER GENE EXPRESSION IN PRE-IMPLANTATION MOUSE EMBRYOS: IMPLICATION OF TRANSCRIPTION REGULATION AT THE BLASTOCYST STAGE
Authors: Cui X. S. , Shen X. H. , Li X. Y. , Kim J. M. , Kim N. H.
Published: December 2006

Dicer is an RNAse III enzyme that is related to the generation of the microRNAs involved in the gene silencing pathway. In order to obtain insight into the role of Dicer in early embryo development, we first evaluated its gene expression levels in mouse oocytes and embryos during in vitro development. The relative abundance of Dicer1 transcripts was established by real-time RT-PCR using the 2-ddCt method. H2a was applied as an internal standard to normalize the real-time RT-PCR reaction efficiency and quantify Dicer1 mRNA. Relatively high expression levels of mRNA in germinal vesicle-stage oocytes steadily decreased up to the 2-cell stage embryo, and then expression remained during morulae and blastocyst formation. Protein synthesis of Dicer was also observed in the mouse oocytes and early embryos. Specific silencing of mRNA expression and protein synthesis by RNA interference (siRNA) did not inhibit developmental events up to the blastocyst (BL) stage. However, Dicer1 siRNA reduced (P <0.05) total nuclei numbers in the BL-stage embryos (Dicer1: 77.2�4.2 vs. control: 62.7�3.1). Real-time RT-PCR also confirmed that, following Dicer1 siRNA microinjection into zygotes, transcription levels of several non-target genes, Cdc42, Cdh1, Dbc2, ILK, Tuba1, Plat, and Tie1, were not changed in blastocyst-stage embryos. However, selected transcription factors, Pou5f1 (P <0.01), Nanog (P <0.005), and Sox2 (P <0.01), in blastocysts were significantly down-regulated. Additionally, POU5F1 protein synthesis was also reduced. Using Applied Biosystem microarray technology, we compared gene expression profiles in control and Dicer1 siRNA microinjected blastocysts. This technique confirmed that 397 or 737 of 16354 genes were up- or down-regulated, respectively, following siRNA microinjection (P <0.05), including 52 transcription factors. The results suggest that expression of Dicer regulates gene expression at the blastocyst-stage embryo for cell fate, possibly by the transcription control.
269 EXPRESSION OF TANDEM-PORE DOMAIN K+ CHANNELS IN BOVINE OOCYTES AND PRE-IMPLANTATION EMBRYOS
Authors: Hur C. G. , Kang D. , Park J. Y. , Hong S. G. , Han J.
Published: December 2006

Tandem-pore domain K+ (K2P) channels that contribute to setting the resting membrane potential of excitable and nonexcitable cells are expressed in many kinds of cells and tissues. Recent studies have shown that TASK [TWIK (Tandem of P domains in Weak Inward rectifying K+ channels)-related acid-sensitive K+ channels] and TREK (TWIK-Related K+ channels), members of K2P channel family that are involved in a variety of cellular functions, are expressed in human myometrium, placenta, and cytotrophoblast cells. However, their expression in bovine oocytes and embryos has not yet been reported. In this study, we examined whether TASK and TREK channels are expressed in bovine immature (germinal vesicle-stage) and mature (metaphase II-stage) oocytes and in pre-implantation (2-cell- and 16-cell-stage) embryos using RT-PCR and immunocytochemistry. RT-PCR data showed that TASK-1, TASK-3, TREK-1, TREK-2, and TRAAK channels were expressed in bovine immature and mature oocytes. Interestingly, the expression levels of TREK channels were 2-fold higher than those of TASK channels as judged by semiquantitative RT-PCR and real-time PCR with cDNA synthesized from 50 individual immature and mature oocytes (P < 0.05, n = 4). Intensity of genes was normalized with respect to that of GAPDH. Consistent with RT-PCR data, immunocytochemical data showed that TASK-1, TASK-3, TREK-1, TREK-2, and TRAAK channels were expressed in bovine immature and mature oocytes. The fluorescence intensity of TREK channels was higher than that of TASK channels (P < 0.05, n = 5). TASK and TREK channels were also expressed in pre-implantation embryos. Of TREK channels, the TREK-2 channel was strongly expressed in immature and mature oocytes and in pre-implantation embryos (P < 0.05, n = 5). For statistics, Student's t-test was used, with P < 0.05 as the criterion for significance. Our results show that TASK-1, TASK-3, TREK-1, TREK-2, and TRAAK channels were expressed in bovine immature and mature oocytes and pre-implantation embryos. These results suggest that TASK and TREK channels could be involved in various physiological processes in mammalian oocytes and embryos.
270 IN VITRO MATURATION ALTERS THE EXPRESSION OF SPECIFIC PUTATIVELY IMPRINTED GENES IN BOVINE MII OOCYTES
Authors: Katz-Jaffe M. G. , McCallie B. R. , Preis K. , Seidel G. E. , Gardner D. K.
Published: December 2006

Genomic imprinting is an epigenetic form of gene regulation resulting in only one parental allele being expressed. Imprinted genes have diverse functions including the regulation of growth and development in mammals. Errors in genomic imprinting have been associated with human disease (e.g. Beckwith-Wiedemann Syndrome) and large offspring syndrome of in vitro-produced ruminant fetuses. The aim of this study was to investigate the effect of in vitro maturation (IVM) on the expression of specific putatively imprinted genes in the bovine oocyte. Cumulus-enclosed immature oocytes were collected from cattle after 6 injections of 50 mg FSH with transvaginal aspiration (TVA) performed 48 h post-final FSH injection. The oocytes were cultured in groups of 10 for 23 h in a defined maturation medium (G-Mat) with 5 mg of HSA and 100 ng mL-1 of epidermal growth factor (EGF; group A) or with 20% serum and 100 ng mL-1 of EGF (group B) at 38.5�C in 6% CO2 in air. In vivo-matured oocytes (group C) were also collected via TVA after the administration of 6 FSH injections (50 mg), prostaglandin (PG) with last FSH injection, and GnRH 37 h post-PG. Total RNA was extracted from individual MII oocytes in all 3 groups, and expression profiles of putatively imprinted genes (Igf2r, Peg3, and Snrpn) were assayed by real-time PCR (Roche Applied Biosciences, Indianapolis, IN, USA), relative to the housekeeping gene GAPDH. Statistical analysis of expression profiles was performed using REST software. Expression of the Igf2r and Peg3 putatively imprinted genes was significantly up-regulated in individual in vitro-matured MII oocytes (groups A and B, n = 5 replicates per group) when compared with control, individual in vivo-matured MII oocytes (group C, n = 5 replicates; P < 0.05). Gene expression did not differ between in vitro- and in vivo-matured MII oocytes for the putatively imprinted gene, Snrpn. In conclusion, following in vitro maturation of bovine oocytes, the putatively imprinted genes Igf2r and Peg3 were aberrantly expressed in individual oocytes relative to in vivo controls. Both of these putatively imprinted genes have been implicated in the regulation of growth and apoptotic pathways during mammalian development. Analysis of such putatively imprinted genes will facilitate the development of more suitable oocyte maturation conditions.
This research was supported by the Serono Research Institute.
271 IDENTIFICATION OF DIFFERENTIALLY EXPRESSED GENES IN A 35-DAY-OLD CLONED PIG FETUS USING THE ANNEALING CONTROL PRIMER SYSTEM
Authors: Ko Y. G. , Chung H. J. , Lee N. Y. , Chung H. J. , Park H. J. , Kim M. J. , Choi M. S. , Kim J. S. , Yang B. C. , Yang B. S. , Seong H. H. , Im G. S.
Published: December 2006

Somatic cell nuclear transfer in pig has limitations due to the high incidence of fetal failure after embryo transfer to recipients. Reasons for the inefficient cloning are assumed to be due to abnormal and poorly developed placenta. Thus, this study was designed to determine possible genetic causes of neonatal deaths and other related abnormalities. Genes expressed specifically or prominently on Day 35 were identified in cloned pig placenta utilizing PCR technology regulated by annealing control primers (ACPs). The RNA was isolated using Trizol reagent. By utilizing 120 ACPs, 53 expressed sequence tags (ESTs) of genes that are differentially expressed in cloned pig placenta compared with normal placenta were cloned and sequenced. The cloned genes or ESTs exhibited significant sequence similarities to known genes or ESTs of other species. Ten of the total known genes, i.e., pregnancy associated glycoprotein, H19, 60S ribosomal protein L12, 20-beta hydroxysteroid dehydrogenase, beta galactosidase precursor, aldehyde reductase, glypican 3 precursor, heme oxygenase 2, granulin precursor, and placenta-expressed transcript protein, were selected and their specific expression levels were confirmed by real-time RT-PCR in the normal and cloned pig placentas in triplicate using beta-actin for determining relative expression. The 60S ribosomal protein L12 and heme oxygenase 2 were highly expressed in the cloned pig placenta, whereas pregnancy associated glycoprotein, H19, 20-beta hydroxysteroid dehydrogenase, beta galactosidase precursor, aldehyde reductase, glypican 3 precursor, granulin precursor, and placenta-expressed transcript protein were low or void. Our data suggest that the ACP system effectively identified tissue-specific genes in cloned pig placenta. Furthermore, identified genes would assist in developing insight into the genetic basis of fetal failure and help in resolving low pregnancy rate in the production of cloned pigs.
280 EXPRESSION OF HSP70.1 GENE IN IN VITRO-PRODUCED BOVINE EMBRYOS CULTURED IN CR2 MEDIUM SUPPLEMENTED WITH KNOCKOUT™SR
Authors: Serapião R. V. , de Almeida Camargo L. S. , de Almeida Ramos A. , de Moura Folhadella I. , Polisseni J. , Lopes E. , Moreira Viana J. H. , de Moraes Ferreira A. , Guimarães M. F. M. , Ferreira de Sá W. , Fonseca F. A.
Published: December 2006

The exposure of embryos to serum during in vitro culture can affect morphology, metabolism, tolerance to cryopreservation, and expression of specific transcripts. On the other hand, serum-free medium seems to avoid some of those serum effects. KnockoutTMSR (GIBCO Laboratories, Grand Island, NY, USA) is a serum replacer optimized to support embryonic stem cells in culture and can also be used to replace serum during culture of in vitro-fertilized bovine embryos. The expression of genes associated with stress response, such as heat shock proteins (HSP), can be affected by in vitro culture conditions, being easily induced by a variety of stress agents, including culture medium components. This study aimed to determine whether KnockoutSR or serum in culture medium alters the relative abundance of HSP70.1 transcripts in in vitro-fertilized bovine embryos. Cumulus–oocyte complexes obtained from slaughterhouse ovaries were matured and feritlized in vitro. Presumptive zygotes were randomly cultured with their own cumulus cells in CR2aa medium supplemented with 10% fetal calf serum (GIBCO-BRL, Paisley, UK; FCS group), 10% KnockoutSR (GIBCO-BRL; KSR group), or 3 mg mL-1 of polyvinyl alcohol (PVA group). All steps were performed at 38.5�C, under 5% CO2 in air and 95% humidity. Blastocysts on Day 8 post-fertilization were rapidly frozen in liquid nitrogen and subsequently thawed for RNA extraction (3 replicates for each group). Total RNA extraction was performed using an Rneasy� Micro kit (Qiagen, Valencia, CA, USA), and the first strand was synthesized using SuperscriptTM III First Strand Synthesis kit (Invitrogen, Chicago, IL, USA). Relative quantification was performed in duplicate using real-time PCR (ABI Prism� 7000 Applied Biosystems, Foster City, CA, USA); reactions consisted of a mixture of iTaqTM SYBR� Green Supermix with ROX (Bio-Rad, Waltham, MA, USA) with cDNA equivalent to 0.8 embryos and gene-specific primers. Expression of the glyceraldehyde 3-phosphate dehydrogenase gene was used as endogenous reference. Calculations of relative quantification were performed by comparative Ct method, using the value found in the PVA group as calibrator. Expression levels for the FCS and KSR groups were 1.2 � 0.06- and 1.4 � 0.08-fold differences relative to the PVA group without differences (P > 0.05). These data show that bovine embryos cultured in medium supplemented with KSR have the same HSP70-1 expression pattern as those in medium with added FCS, suggesting that embryos in both groups are under the same stress conditions.
This work was supported by FAPEMIG, MG, Brazil, and CNPq, DF, Brazil. Thanks to Agrogenetica, Vi�osa, Brazil, for the real-time PCR machine.
294 IN VITRO DEVELOPMENT OF RAT OOCYTES FOLLOWING MICROINJECTION OF A CUMULUS CELL INTO THE OOPLASM AND CHEMICAL ACTIVATION
Authors: Fujii W. , Funahashi H.
Published: December 2006

If diploid zygotes constituted with a somatic and a maternal genome could successfully develop to term, a new reproductive method would be developed to produce animals. However, there appears to be little information on this subject. In the present study, in vitro early development of the constituted zygotes was examined. A cumulus cell was microinjected into a rat non-enucleated oocyte, the reconstructed oocyte was chemically activated, and the pronuclear formation and in vitro development of the embryo was observed. Prepubertal Wistar female rats (21–27 days old) were induced to superovulate with an IP injection of 15 IU of eCG, followed by 15 IU of hCG 48 h later. Cumulus cells were removed from oocytes by pipetting with 0.1% hyaluronidase. Experiment 1: The DNA content of cumulus cells for microinjection was evaluated by flow cytometry. Experiment 2: The optimal concentration of SrCl2 for activation of rat oocytes was examined. Experiment 3: Cumulus cells were injected into mature oocytes in BSA-free HEPES-buffered mKRB containing 0.1% polyvinyl alcohol (PVA) and cytochalasin B (5 �g mL-1), and were then chemically activated by treatment in Ca2+-free mKRB containing 5 mM SrCl2 for 20 min at 0 to 0.5 (A), 1 to 1.5 (B), or 3 to 3.5 h (C) after injection. Activated embryos were cultured in droplets of mKRB in an atmosphere of 5% CO2 in air at 37�C for 9 to 12 h. After being observed for pronuclear formation, the embryos were transferred into mR1ECM-PVA, and the cleavage and blastocyst formation rates were examined 24 and 120 h later, respectively. Results from 3 to 7 replicates were analyzed by ANOVA and Duncan's multiple range test. A total of 90.0 and 9.5% of cumulus cells derived from ovulated oocyte–cumulus complexes contained 2C and 4C DNA contents, respectively. Survival rates did not differ among oocytes stimulated with 0 to 5 mM SrCl2 (96.7–100%) but did differ between those stimulated with 1.25 and 10 mM SrCl2 (100 and 72.9%, respectively). Activation rates of oocytes increased at higher SrCl2 concentrations and were higher at 5 and 10 mM (92.6 and 98.5%, respectively) than at other concentrations. When cumulus-injected oocytes were activated after various periods after the injection, the incidences of pronuclear formation and cleavage did not differ among the periods (A: 95.0 and 81.3%; B: 85.6 and 85.0%; and C: 82.7 and 84.6%, respectively). Although a majority of the embryos developed to the 2- to 4-cell stages (78.7%; 152/208), the blastocyst formation rate was very low (0.8%; 2/208). In conclusion, rat non-enucleated oocytes injected with a cumulus cell can form pronuclei and cleave following chemical activation, but blastocyst formation of the embryos is very limited.
295 INFLUENCE OF FERTILIZATION MEDIUM ON THE INCIDENCE OF POLYSPERMY IN THE PIG
Authors: Galli C. , Crotti G. , Turini P. , Lagutina I. , Lazzari G.
Published: December 2006

The in vitro production of embryos is well established in most domestic species including cattle, pigs, sheep, and goats. However, a major problem of IVF in the pig is the high incidence of polyspermy. In our laboratory, we investigated the effect of 2 different media, TALP and SOFaa, on the rate of fertilization and polyspermy of pig oocytes. Preliminary experiments indicated that TALP provided the highest fertilization but also the highest polyspermy rates, as reported in the literature (Coy et al. 2002 Reproduction 124, 279–288). By contrast, much lower polyspermy rates but also much lower fertilization rates were obtained in SOFaa. Therefore, we made a direct comparison between the 2 media and a third medium prepared by mixing TALP and SOFaa equally (1 : 1 TALP–SOF) and using 2 different boars for IVF. Porcine oocytes were recovered from slaughtered donors and matured in vitro for 40 to 44 h in DMEM-F12 supplemented with 10% FCS, 0.05 IU of LH and FSH (Menogon; Ferring, Milan, Italy), 0.3 mM cystine, 0.5 mM cysteamine, 50 ng mL−1 of long-EGF, 100 ng mL−1 of long-IGF1, and 5 ng mL−1 of bFGF (Sigma-Aldrich, Milan, Italy) in 5% CO2 at 38.5°C. Boar frozen–thawed semen was separated on a Percoll gradient (45–90%) and diluted in TALP or in SOFaa with PHE (penicillamine, hypotaurine, epinefrine) and heparin (1 µg mL−1) to concentrations ranging from 0.01 to 0.15 million sperm/mL. The concentration was optimized for each boar and medium: For boar A, the concentration was 0.015 million sperm/mL for medium TALP and TALP–SOF and 0.15 million sperm/mL for medium SOF; for boar B, the concentration was 0.1 million sperm/mL for medium TALP and TALP–SOF and 0.15 million sperm/mL for medium SOF. The oocytes were co-incubated with the sperm suspension for 18 h and then were denuded of the surrounding cumulus and fixed in acetic acid–ethanol (1 : 3) for 48 h. Finally, they were stained with lacmoid and observed under phase-contrast microscopy. The data are shown in Table 1 and were compared by a chi-squared test. Our results indicated that TALP was the most efficient medium for pig IVF but over 50% of the oocytes were polyspermic. By contrast, very low polyspermy, but also very low fertilization, was observed in SOF medium for both boars A and B. Interestingly, the empirical approach of mixing the 2 media 50% each provided a dramatic reduction of the polyspermy rate while maintaining the fertilization rate at over 60% in both boars. At present, experiments are ongoing to clarify the role of specific components of the 2 media on the fertilization and polyspermy rates of pig oocytes.

Table 1.Effect of different media on fertilization and polyspermy rates with 2 different boars

This work was supported by grants from EUROSTELLS-ESF (ERAS-CT-2003-980409).
300 BLASTOCYST PRODUCTION BY IN VITRO MATURATION AND DEVELOPMENT OF PORCINE OOCYTES IN DEFINED MEDIA FOLLOWING INTRACYTOPLASMIC SPERM INJECTION
Authors: Kobayashi Y. , Fukui M.
Published: December 2006

Most culture media for in vitro production (IVP) of porcine embryos contain serum or bovine serum albumin (BSA); especially in the case of in vitro maturation (IVM) porcine follicular fluid is added. The present study was carried out to establish porcine-defined IVP. In Experiment 1, we investigated the efficacy of additional 0.6 mM cystine, 100 �M cysteamine (Cys), or cystine + Cys to a defined TCM-199 maturation medium with regard to the developmental competence of in vitro-matured porcine oocytes following intracytoplasmic sperm injection (ICSI). The control medium was a modified TCM-199 containing 0.05% (w/v) polyvinyl alcohol (PVA). In Experiment 2, the effect of epidermal growth factor (EGF) addition to a modified porcine zygote medium (mPZM) for in vitro culture (IVC) medium was investigated on embryonic development following ICSI. As positive and negative controls, 0.3% BSA (mPZM-3) or 0.3% PVA (mPZM-4), respectively, was added to the basic medium. Five or 10 ng mL-1 of EGF was supplemented in the negative control medium (mPZM-4). All percentage data on cleavage and blastocyst development were subjected to arcsine transformation before statistical analysis using the STATVIEW program (Abacus Concepts, Berkeley, CA). The mean cell numbers per blastocyst (assessed by Giemsa staining method) were analyzed by one-way ANOVA, and the differences among the groups were also analyzed using Tukey-Kramer multiple comparison tests. In Experiment 1, there were no significant differences in the rates of cleavage (31.4 to 64.3%) and blastocyst formation (6.5 to 22.9%) among the treatment and control groups. The mean cell numbers per blastocyst ranged from 30 to 48 among the groups, without significant differences. In Experiment 2, the rates of cleavage (43.0 to 58.0%) and blastocyst formation (14.0 to 23.6%), and the mean cell numbers per blastocyst (30 to 37) were not significantly different among the groups. However, the addition of 5 ng mL-1 of EGF to the mPZM-4 resulted in a significantly (P d 0.05) higher blastocyst rate (48.6%) to cleaved embryos than the other two defined groups (mPZM-4 and mPZM-4 with 10 ng mL-1 EGF: 23.4% and 23.1%, respectively), but not significantly different with mPZN-3 (40.1%). The present results indicate that TCM-199 with added cysteamine (100 �M) can be used as a defined IVM medium for porcine oocytes, and further addition of cystine into the IVM medium is not necessary. The addition of 5 ng mL-1 of EGF to a defined IVC medium enhanced subsequent development after ICSI. These results show that porcine blastocysts can be produced by defined media throughout the steps of IVP (IVM, ICSI, and IVC).
This study was partly supported by a grant-in-aid for Scientific Research (17580242) from the Japan Society for the Promotion of Science.
301 EFFECT OF CO-CULTURE WITH DIFFERENT STAGE EMBRYOS ON DEVELOPMENT OF ALGINATE-ENCAPSULATED SMALL NUMBER BOVINE EMBRYOS
Authors: Kobayashi S. , Sakatani M. , Inaba Y. , Kobayashi S. , Imai K. , Takahashi M.
Published: December 2006

Previous studies show that embryos cultured in large numbers have better developmental competence than those in small numbers in mice, sheep, and cattle. We have reported that co-culture of bovine embryos encapsulated in calcium-alginate gel (microcapsule) improves the development of embryos cultured in small numbers (Kobayashi et al. 2006 Reprod. Fertil. Devel. 18, 248). This method is beneficial for culture of small numbers of embryos such as OPU-derived embryos by recognizing the individual donor cows with abattoir-derived unidentified IVF embryos. In the previous study, we used the same stage embryos for co-culture of encapsulated embryos. However, in the case of unavailability of the same stage embryos, encapsulated embryos may be co-cultured with different stage embryos. Effect of different stage embryos on co-culture of encapsulated embryos is not clear. In the present study, we investigated the effect of co-culture of different stage embryos on development of encapsulated small number embryos. In vitro-matured and fertilized zygotes from abattoir derived ovaries were used for the experiment. Small numbers of zygotes were encapsulated by alginate-gel microcapsule to distinguish from co-cultured embryos. Encapsulation was carried out by putting the 1% sodium alginate solution containing zygotes slowly into 0.1% calcium chloride solution (microcapsule). The embryos used for co-culture were produced by IVF 1-3 days before preparation of encapsulated zygotes (Day 1, Day 2, and Day 3). Five encapsulated zygotes were cultured with 15 embryos for co-culture in one droplet (100 �L) made by CR1aa + 5% CS, at 38.5�C, CO2 in air. Encapsulated zygotes co-cultured with the same stage of zygotes were assigned as a control (Day 0). The rates of cleavage on Day 2 and development to blastocyst stage on Day 9 were recorded. Data were analyzed by Student's t-test. No significant difference was observed in the rate of cleavage in all experimental groups compared with control (Day 1: 72.5% (n = 80) vs. control: 75.7% (n = 70); Day 2: 76.3% (n = 80) vs. control: 82.5% (n = 80); and Day 3: 78.7% (n = 75) vs. control: 70.8% (n = 65). There was not a significant difference in the rate of development to the blastocyst stage in all experimental groups compared with control (Day 1: 42.5% vs. control: 44.3%; Day 2: 43.8% vs. control: 38.8%; Day 3: 44.0% vs. control: 35.4%). These results indicate that co-culture of different stages of embryos can normally support the development of small numbers of encapsulated embryos. These methods are useful to improve the development of small numbers of embryos derived from OPU-IVF embryos without synchronization of the developmental stage of co-cultured embryos.
309 DIFFERENCES IN PRONUCLEUS FORMATION RATES BETWEEN BULLS IN RESPONSE TO GAMMA IRRADIATION OF FROZEN - THAWED SEMEN
Authors: Petersen M. M. , Boe-Hansen G. B. , Birck A. , Avery B. , Jensen M. , Bøgh I. B.
Published: December 2006

Gamma irradiation induces DNA damage to mature bovine spermatozoa but does not affect motility, membrane integrity, or function (Fatehi et al. 2006 J. Androl. 27, 176–188), making it a useful model to evaluate the effect of fertilization with DNA-damaged spermatozoa. The objective of the present study was to analyze the fertilizing capacity of frozen–thawed gamma-irradiated sperm from 2 bulls: a bull with a high sperm quality and fertilization rate (bull A), and a bull with a low sperm quality and fertilization rate (bull B). To ensure that DNA damage was induced, frozen–thawed semen was exposed to a high dose of irradiation (90 Gy). Cumulus–oocyte complexes were obtained from abattoir ovaries and were in vitro-matured (IVM) using standard procedures (23 h in DMEM with 5% serum and eCG/hCG). For each treatment group, 3 to 5 straws of semen from the same ejaculate were used. After thawing, semen from the 2 bulls was either irradiated or held at room temperature before 1:1 dilution in sperm-TALP. During irradiation, semen was kept in the straws. The irradiated and nonirradiated sperm were used for IVF (23 h in IVF-TALP) within 1 h of thawing. IVM and IVF were carried out at 38.5�C in 5% CO2 in air. After IVF, presumptive zygotes were whole mount-fixed and 24 h later were stained with 1% aceto-orcein followed by determination of fertilization status. Fertilization was considered normal if 2 pronuclei (PN) were present. A total of 4 replicates were performed and 286 zygotes analyzed. No difference was detected between replicates, and the results were pooled. Fisher's exact test was used to determine effect of treatment. More zygotes had 2 PN after fertilization with semen from bull A (47/67 = 70%), compared with that from bull B (16/71 = 23%; P < 0.0001) using nonirradiated sperm. Irradiation of sperm significantly increased the fraction of zygotes with 2 PN from bull A (61/70 = 87%; P = 0.03), but decreased the 2 PN fractions in zygotes fertilized with sperm from bull B (2/75 = 3%; P = 0.0002). Ideally, to avoid straw variation, semen should have been pooled and divided into groups before irradiation. The variation between straws might explain the higher fertilization rates in bull A using irradiated sperm compared with nonirradiated sperm. In conclusion, there appear to be differences in fertilizing ability between bulls after irradiation of frozen–thawed sperm. This could be due to suboptimal DNA packaging, which made sperm from bull B more susceptible to radiation-induced damage. The potential irradiation-induced increase in DNA fragmentation in sperm from bull B compared with that from bull A might delay or prevent the formation of the 2 PN. Further studies are needed to investigate differences in fertilization and early embryonic development using sperm with intact or damaged DNA.
310 DETERMINATION OF THE PORCINE OVIDUCTAL GLYCOSIDASES DURING THE ESTROUS CYCLE
Authors: Romar R. , Carrasco C. , Marcos J. , Avilés M. , Coy P.
Published: December 2006

Carbohydrates play a key role in different reproductive events, such as the sperm–oviductal cell interaction and sperm–oocyte recognition. For example, β-d-galactose and α-d-mannose residues contained in the zona pellucida have been identified as sperm receptors in porcine oocytes (Song et al. 1999 J. Mamm. Ova Res. 16). The glycosidases, enzymes that remove carbohydrates, could play an important role in the reproductive tract, modulating decisive physiological events mediated by carbohydrates. However, the enzymatic activity level of these enzymes or their fluctuations throughout the estrous cycle in the porcine oviductal fluid (POF) has not been studied. The objective of this work was to compare the enzymatic activity level of 7 glycosidases in the POF at different stages of the estrous cycle. Oviducts were collected from the abattoir and classified according to the macroscopic aspect of the genital tract (Grippo et al. 1995 J. Reprod. Fertil. 105, 57–64) as early follicular please (presence of growing follicles), late follicular phase (presence of several grown follicles), early luteal phase (ovaries showing corpora hemorrhagica or recent corpora lutea), and late luteal phase (old corpora lutea or corpora albicans). After classification, oviducts were dissected and oviductal fluid samples were collected by aspiration with an automatic pipette while applying manual pressure from the isthmus toward the ampulla. Samples (6 per group) were centrifuged (7000g, 10 min) and the supernatant was stored at −20°C until assay. Total activity levels were measured fluorimetrically at 450 nm with the corresponding substrate conjugated to 4-methylumbelliferyl for each enzyme (Abascal et al. 1998 Biochem. J. 333, 201–207) using a FLUOstar Galaxy fluorometer (BMG Lab Technologies, Offenburg, Germany). Enzymatic assays were done in duplicate for 4 h at 37°C, and the reactions were stopped by adding glycine–calcium carbonate buffer. Fluorescence was corrected for tissue and substrate blanks. Fluorescence results of each enzyme and oviduct phase were analyzed using a one-way ANOVA with estrous cycle phase being the main factor. Results (mean counts of fluorescence, see Table 1) showed that changes of enzymatic activity during the estrous cycle and activity of some enzymes were modified during or after ovulation, suggesting a role of some glycosidases in the fertilization process. Preliminary assays for neuraminidase were negative in all samples. Future studies are necessary to identify the biological role played by the glycosidases present in the POF.

Table 1.Changes in porcine oviductal glycosidases during the estrous cycle

Supported by Fundacion Seneca (03018/PI/05) and Ministerio de Educacío y Ciencia (Project code 3495).
312 EXPOSURE OF SPERMATOZOA TO SOLUBILIZED EXTRACTS OF THE OVIDUCTAL EPITHELIUM APICAL PLASMA MEMBRANE ENHANCES FERTILIZATION IN PORCINE IN VITRO FERTILIZATION
Authors: Satake N. , Alhaider A. K. , Holt W. V. , Watson P. F.
Published: December 2006

In vitro production (IVP) of porcine embryos is currently suboptimal compared with IVP in species such as mice and cattle. In vitro fertilization (IVF) usually involves the co-culture of oocytes and spermatozoa in a medium droplet. Oocyte quality is the focus of many studies. In vivo, the quality of spermatozoa is as important as the oocyte, and females have many mechanisms to select the highest quality spermatozoa for their oocytes. Oviductal proteins have been shown to affect sperm motility of subpopulations within an ejaculate. The present study was carried out to investigate normal and polyspermic fertilization rates of spermatozoa exposed to oviductal epithelial apical plasma membrane (APM) proteins, a mixture of peripheral proteins extracted by 1 M NaCl from isolated oviductal apical plasma membranes, prior to co-culture with oocytes in IVF. Porcine oocytes were aspirated from ovaries and grade I quality oocytes (cumulus–oocyte complexes with a spherical shape, visible nucleus, even-density cytoplasm, and multiple layers of cumulus cells) were selected and matured for 48 h in TCM-199 supplemented with LH (0.5 �g mL-1), FSH (0.5 �g mL-1), and EGF (10 ng mL-1). Ejaculates were washed through a Percoll gradient to obtain a concentrated pellet. Spermatozoa were diluted in capacitation–fertilization medium in the presence or absence of APM proteins (100 �g mL-1), incubated for 10 min, and then co-cultured with oocytes for 6 h in modified Tween medium B with milk powder medium (Abeydeera and Day 1997 Theriogenology 48, 537–544) supplemented with BSA (0.4%) and sodium bicarbonate (5 mM). Presumptive zygotes were cultured in NCSU23 medium for a further 48 h. The oocytes/zygotes were then fixed and stained with propidium iodide for evaluation by confocal microscopy for fertilization and cleavage (n = 1235 oocytes). Fertilization rates were compared between treatments in a chi-squared test using the Mantel-Haenszel approach. The overall fertilization rate was significantly higher (78 vs. 86%) when spermatozoa were incubated in the presence of APM proteins (P < 0.05), and in the group of fertilized oocytes, polyspermic fertilization (47 vs. 21%) was significantly reduced when spermatozoa were exposed to APM proteins (P < 0.01). However, cleavage rates were not different. These results suggest that exposure of spermatozoa to APM proteins prior to IVF increases the fertilization rate and decreases the incidence of polyspermic penetration.
328 FLUORESCENT PROBE ASSESSMENT OF POST-THAW QUALITY OF RAM SPERM TREATED WITH SEMINAL PLASMA
Authors: Rovegno M. , Feitosa W. B. , Brandão A. C. , Nascimento A. B. , Peres M. A. , Visintin J. A. , Assumpção M. E. O. A.
Published: December 2006

A recently suggested alternative to improve post-thaw ovine semen quality is the addition of seminal plasma (SP). This is thought to be capable of improving sperm resistance to thermal shock, reverting cryo-capacitation and helping sperm survival in the female tract. The aim of this study was to evaluate the effect of thawed ram semen incubation with seminal plasma for 15 or 30 min as assessed by the fluorescent probes, Hoechst 33342 (40 µg mL−1), propidium iodide (0.5 mg mL−1), JC-1 (76.5 µM in DMSO), and FITC-PSA (100 µg mL−1 in Dulbecco's PBS (DPBS)), for cellular viability, plasmatic damage, mitochondrial activity, and acrosomal damage, respectively. Five ejaculates were collected from 4 different animals via artificial vagina and were pooled to eliminate individual differences. After thawing, semen was divided into 2 groups, one diluted with seminal plasma (1 : 1, 30% in DPBS) and the other in DPBS (1 : 1). After 15 and 30 min, fluorescent probes were added to 25 µL of each group and 100 cells were counted under a epifluorescence microscope (Olympus IX81, motorized inverted research microscope). Spermatozoa were classified under 8 categories: alive, damaged acrosome, and high mitochondrial activity (ADH); dead, damaged acrosome, and high mitochondrial activity (DDH); alive, damaged acrosome, and poor mitochondrial activity (ADP); dead, damaged acrosome, and poor mitochondrial activity (DDP); alive, intact acrosome, and high mitochondrial activity (AIH); dead, intact acrosome, and high mitochondrial activity (DIH); alive, intact acrosome, and poor mitochondrial activity (AIP); or dead, intact acrosome, and poor mitochondrial activity (DIP). Statistical analysis was performed by Fisher test at a 5% level. The results are presented in Table 1. There was no significant difference between groups except for the AIH category which was reduced during incubation in either DPBS or SP for 15 or 30 min. These last data allow the conclusion that ram sperm is highly susceptible to cryogenic damage. Nevertheless, the high percentage of the AIH category suggests that the spermatozoa that can resist the freezing protocol remain alive and intact. Hence, we can infer that ram frozen semen has approximately half the fertilizing potential of fresh semen. In addition, we observed a negative effect of the incubation period as the decreased percentage of the AIH category was accompained by an increase of the DDP and DIP ones. We conclude that seminal plasma incubation after thawing does not benefit sperm quality. It is necessary to study the seminal plasma constitution to conclude why this experiment differed from other published data.

Table 1.Spermatozoa percentages in the categories during incubation periods with or without seminal plasma

This work was supported financially by FAPESP 05/55256-3
331 DIRECT EFFECTS OF ERGOT ALKALOIDS ON BOVINE SPERM MOTILITY IN VITRO
Authors: Wang H. , Johnson Z. , Rorie R. , Rosenkrans, Jr C.
Published: December 2006

Ergot alkaloids have been associated with decreased livestock reproductive rates. Concentration of alkaloids in the reproductive tract after consumption of toxic forage is unknown. In addition, the direct effects of alkaloids on bovine spermatozoa have not been determined. We investigated the direct and interactive effects of 3 ergot alkaloids (ergotamine, dihydroergotamine, and ergonovine) on motility of frozen–thawed bovine spermatozoa. Thawed spermatozoa from 3 bulls were pooled and washed using a Percoll density gradient. Sperm motility was visually estimated by counting at least 100 spermatozoa in each triplicate well (> 300 per treatment per replicate). The cell counting was conducted using phase contrast (400�) on an inverted bright field microscope. Spermatozoa were considered motile if they exhibited free progressive forward or other movement and were not attached to the well surface. Motile spermatozoa were exposed to alkaloids ranging in concentration from 0 to 100 �M. Assays were conducted in modified sperm-TL (mSPTL) medium at 39�C in moist air without CO2 for 6 to 12 h. The results showed that both ergotamine (ET) and dihydroergotamine (DHET) inhibited (P < 0.05) sperm motility at concentrations greater than 50 �M and 33.3 �M, respectively. Ergonovine (EN) did not inhibit sperm motility at the test concentrations. Inhibitory effects of alkaloids on sperm motility were concentration-dependent for ET and DHET incubations and time-dependent for DHET incubations. Sperm motility also was inhibited by an interaction (P < 0.05) between ET and DHET at concentrations of 16.7 �M or above. The medium pH affected the toxic effects of both ET and DHET, whereas the medium osmolarity affected only the toxic effect of ET on relative sperm motility (P < 0.05). Medium osmolarity of 358 mOsm and/or pH higher than 7.1 exacerbated the toxic effects of the alkaloids. These results demonstrate that ergot alkaloids can directly interact with spermatozoa and impair sperm motility. Herbivores consuming toxic tall fescue are exposed to a cocktail of ergot alkaloids. Alkaloid interactive effects coupled with altered cell chemistry, due to increased respiration rates and frequent urination, on spermatozoa may indicate the mechanism by which reproduction is impaired in animals consuming toxic forage.
333 DYNAMICS OF MITOGEN-ACTIVATED PROTEIN KINASE ACTIVITY IN PIG OOCYTES FOLLOWING PARTHENOGENETIC ACTIVATION BY DIFFERENT METHODS
Authors: Nanassy L. , Lee K. , Javor A. , Machaty Z.
Published: December 2006

Cell cycle progression during mitosis and meiosis is known to be regulated by the M-phase promoting factor (MPF). However, recent findings revealed that mitogen-activated protein kinase (MAPK) also plays an important regulatory role during transition through the cell cycle. At fertilization the activity of MAPK drops shortly after MPF inactivation; the objective of this study was to investigate the dynamics of MAPK activity in pig oocytes after different activation methods. In vitro-matured oocytes were allocated to 3 groups. In group 1 (EP), the oocytes were activated by 2 DC pulses of 1.2 kV cm-1, 60 �s each. In the second group (EP + BU), the oocytes were electroporated and incubated for 4 h in 100 �M butyrolactone I (BU, an inhibitor of cdc2 kinase). In group 3 (EP + CHX), the oocytes were electroporated and treated for 5 h with 10 �g mL-1 cycloheximide (CHX, a protein synthesis inhibitor). After electroporation all oocytes were incubated in 7.5 �g mL-1 cytochalasin B for 4 h. Some oocytes were used to determine MAPK activity at 0, 1, 2, 3, 4, 5, and 6 h after electroporation using a MAPK assay kit. The assay measures MAPK activity by determining the phosphorylation of myelin basic protein by MAPK using the transfer of the γ-phosphate of [γ-32P] ATP. Pronuclear formation was evaluated at 6 h after electroporation; blastocyst formation and total cell numbers per embryo were determined after a 7-day culture in PZM-3 medium. Pronuclear formation was compared by the chi-square test, blastocyst formation was assessed using ANOVA, and the kinase activity was evaluated using the Student t-test. Pronuclear formation was highest in the combined methods [69.39% (EP) vs. 86.32% (EP + BU) and 87.56 % (EP + CHX); P < 0.05]. Similarly, the combined methods supported better development to the blastocyst stage [25.06 � 7.96% (EP), 58.32 � 7.62% (EP + BU), and 63.91 � 6.35% (EP + CHX); P < 0.05], whereas the average cell numbers of the blastocysts did not differ (47.11 � 3.12, 46.56 � 2.33, and 44.04 � 1.86, respectively). The initial MAPK activity was 0.123 � 0.017 pmol/min/oocyte which, after 1 h, dropped in all cases to values of 0.069 � 0.009 (EP), 0.072 � 0.007 (EP + BU), and 0.077 � 0.012 (EP + CHX) pmol/min/oocyte (P < 0.05). The MAPK activity in the EP group reached its lowest level at 3 h (0.057 � 0.007 pmol/min/oocyte); however, at 4 h it started to recover and by 6 h the activity (0.079 � 0.022 pmol/min/oocyte) did not differ from that of the non-activated oocytes. In the other groups, MAPK activity stayed low, and by the end of the experimental period it was significantly lower than that in the nontreated metaphase II oocytes (P < 0.05). The results indicate that electroporation followed by protein kinase inhibition or protein synthesis inhibition leads to the efficient inactivation of MAPK activity, and confirm our earlier findings that these combined treatments support superior embryo development after oocyte activation.
336 DEVELOPMENT OF PORCINE NUCLEAR TRANSFER EMBRYOS PRODUCED USING SPERM CYTOSOLIC FACTOR (SCF) AT FUSION-ACTIVATION
Authors: Shim J. H. , Hwang I. S. , Moon H. J. , Park M. R. , Kim D. H. , Yang B. C. , Ko Y. K. , Kim S. W. , Yang B. S. , Jin D. I. , Im G. S.
Published: December 2006

At fertilization, the sperm activates the developmental program of the oocyte by inducing an elevation in the intracellular free Ca2+ concentration ([Ca2+]i). One possible explanation is that at sperm–oocyte fusion the fertilizing spermatozoon introduces a factor into the cytoplasm of the oocyte which opens the Ca2+ release channels from the intracellular stores through a yet unidentified mechanism. This study investigated the development of porcine nuclear transfer embryos fused and activated in the presence of sperm cytosolic factor (SCF) isolated from porcine sperm. Ovaries were collected at a local slaughterhouse and transported to the laboratory within 2 h at 35–39�C, and rinsed in 0.9% NaCl. Fetal fibroblast cells were prepared from a 35-day-old porcine fetus for use as donor cells. For parthenogenesis, matured oocytes were activated using 2 DC pulses of 1.2 kV cm-1 for 30 �s in fusion medium (0.1 mM CaCl2) supplemented with 100, 200, or 300 �g mL-1 SCF. For NT, matured oocytes were enucleated, reconstructed, and fused. Reconstructed embryos were divided into 2 groups. The embryos in one group were fused with fusion medium (1.0 mM CaCl2), and the embryos in the other group were fused with fusion medium (0.1 mM CaCl2) supplemented with 100 �g mL-1 SCF. After fusion, the embryos were cultured in PZM-3 under 5% CO2 in air at 38.5�C. A TUNEL assay was used to assess the presence of apoptotic cells (In Situ Cell Death Detection Kit, TMR red; Roche, Mannheim, Germany). Data were subjected to a generalized linear model procedure (PROC-GLM) of the Statistical Analysis System (SAS Institute, Inc., Cary, NC, USA). Oocytes activated parthenogenetically in the presence of SCF showed significantly higher developmental rate to the blastocyst stage compared to that of controls (21.3–27.6% vs. 12.5%; P < 0.05). For NT, there was no difference between treatments in developmental rate to the blastocyst stage (18.2% vs. 17.1%). However, the apoptosis rate was slightly lower in blastocysts produced in the presence of SCF than that in controls. These results indicate that the presence of SCF in fusion medium can support a higher quality of porcine nuclear transfer embryos.
347 EFFECTS OF NEWBORN CANINE SERUM ON IN VITRO MATURATION OF CANINE OOCYTES
Authors: Lee H. S. , Choi D. S. , Jung D. G. , Bae I. H. , Lee S. S. , Yin X. Y. , Kong I. K.
Published: December 2006

Research on in vitro maturation (IVM) of canine oocytes has been scant, due to the fact that canine oocytes are at an immature stage (geminal vesicle stage) during ovulation. The most commonly used protein source for IVM of canine oocytes is fetal bovine serum (FBS), estrus bitch serum (EBS), or bovine serum albumin (BSA). However, the IVM rate of canine oocytes is still low. One of the reasons may be that the culture medium is not appropriate enough for canine oocytes in IVM. Therefore, we evaluated the effects of four different protein sources, BSA, FBS, EBS, and newborn canine serum (NBCS), on IVM of canine oocytes. The NBCS was collected from 3 puppies that had induced euthanasia within 30 min after delivery. The entire volume of blood was collected by heart puncture, through which an 18 G needle was attached to a 10-mL sterile plastic syringe (without anticoagulant), and then transferred into a 10-mL plastic conical tube. The samples were stored overnight at 4�C to allow clotting to occur. To separate the serum, the sample was centrifuged for 10 min at 3000 rpm; the NBCS was heat inactivated at 56�C for 30 min and stored at -70�C for later use. Ovaries were collected from various stages of the estrous cycle by ovariohysterectomy, and the oocytes collected were cultured in NCSU37 supplemented with 1 mg mL-1 cysteine, 0.2 mM pyruvic acid, 20 �g mL-1 E2, and various protein sources (0.3% BSA, 10% FBS, 10% EBS, and 10% NBCS) at 38.5�C, 5% CO2 in air for 72 h. Cumulus cells were removed from around oocytes using a small glass pipette. Denuded oocytes were then fixed in 4% paraformaldehyde supplemented with 10 �g mL-1 Hoechst 33342 at room temperature for 40 min. Nuclear status was observed using fluorescence under a UV light. The proportion of oocytes at the MII stage was significantly higher in the NBCS group than in the EBS, FBS, and BSA groups (14.6 vs. 10.4, 8.8, and 3.3%; P < 0.05). These results indicate that the supplementation of NBCS in IVM medium can enhanced the development of canine oocytes to the MII stage.
This work was supported by KOSEF (Grant M10525010003-05N2501-00310) and ARPC (Grant 203121-03-2-SB010).
348 INACTIVATION OF MAPK AFFECTS CENTROSOME ASSEMBLY, BUT NOT ACTIN FILAMENT ASSEMBLY, IN MOUSE OOCYTES MATURING IN VITRO
Authors: Lee S. E. , Kim J. H. , Ji Y. K. , Kim N. H.
Published: December 2006

Mitogen-activated protein kinase (MAPK) plays a crucial role in meiotic maturation of mouse oocytes. In order to understand the mechanism by which MAPK regulates meiotic maturation, we examined the effects of the MAPK pathway inhibitor U0126 on microtubule organization, γ-tubulin and NuMA distribution, and actin filament assembly in mouse oocytes maturing in vitro. Significant differences between control and treatment were determined using Tukey's multiple range test; P < 0.05 was considered significant. Western blotting with antibodies that detect active, phosphorylated MAPK revealed that MAPK was inactive in fully grown germinal vesicle (GV) oocytes. Phosphorylated MAPK was first detected 3 h after the initiation of maturation cultures, was fully active at 6 h, and remained active until metaphase II (MII). When GV oocytes were cultured in medium containing 20 �M U0126 for 9 h, the oocytes underwent geminal vesicle breakdown (GVBD), but were significantly inhibited in progression to the MI stage as compared to controls (control: 76.7 � 5.66%; 10 �M U0126: 29.0 � 7.35%; 20 �M U0126: 26.4 � 5.74%). Polar body extrusion was observed in most oocytes when cultured in the presence of 20 �M U0126 from 4 h after initiation of in vitro oocyte maturation to 14 h into the in vitro maturation incubation (control: 76.6 � 4.21%; 20 �M U0126: 70.7 � 4.61%). However, there was a decrease in normal MII-stage oocytes when cultured for 4 to 14 h (control: 87.7 � 5.12%; 20 �M U0126: 32.8 � 3.79%); abnormality included, for example, a polar body lacking a nucleus, as well as an abnormal spindle or metaphase plate. The inhibition of MAPK resulted in abnormal spindles and abnormal distributions of γ-tubulin and NuMA, but did not affect actin filament assembly. In conclusion, the meiotic abnormalities caused by U0126, a specific inhibitor of MAPK signaling, indicate that MAPK plays an important regulatory role in microtubule and centrosome assembly, but not actin filament assembly.