bluuebunny/crossref_metadata_2025 huggingface dataset:
307 ADDITION OF HIGH CONCENTRATIONS OF HEPARIN DURING IN VITRO FERTILIZATION IMPROVES PERCENTAGES OF CLEAVAGE AND BLASTOCYST FORMATION WHEN BOVINE OOCYTES ARE INSEMINATED IN VITRO WITH SEX-SORTED SPERM
Authors: Nedambale T. L. , Xu J. , Chaubal S. A. , Tian X. C. , Yang X. , Du F.
Published: December 2006

The developmental potential of oocytes fertilized in vitro by sexed–sorted frozen–thawed sperm remains low. The aim of this study was to find an optimal concentration of heparin during IVF using frozen–thawed sex-sorted sperm to improve the percentages of fertilization and blastocyst formation. A total of 1708 matured bovine oocytes were randomly allocated among different concentrations of heparin (0, 2.5, 5, 10, 20, and 40 µg mL−1) in Bracket-Olifant medium and co-incubated with non-sexed or sex-sorted sperm for 6 h (Day 0). The sperm (sorted or not) used for IVF was from one bull of proven fertility. Presumptive zygotes then were cultured in CR1aa + 6 mg mL−1 of BSA in 5% O2, 5% CO2, and 90% N2 at 39°C until Day 8. Percentages of cleavage were recorded on Day 2. Data (3 replicates) were analyzed by ANOVA. The results (Table 1) demonstrated that addition of different concentrations of heparin in IVF medium for non-sexed sperm groups did not alter cleavage and blastocyst formation. However, the lowest percentages of cleavage were observed with 0, 2.5, and 5 µg mL−1 of heparin in sexed sperm groups, demonstrating that high concentrations of heparin (20 and 40 µg mL−1) positively affected the percentages of cleavage and blastocyst formation. A greater proportion of zygotes in 20 and 40 µg mL−1 sexed sperm groups developed into grade 1 blastocysts on Day 7, and subsequently formed more blastocysts on Day 8 compared with 0, 2.5, and 5 µg mL−1 of heparin in sexed sperm groups. In conclusion, addition of heparin was not necessary when frozen–thawed non-sexed sperm were used for IVF with this particlar bull. However, addition of higher concentrations of heparin during IVF improved fertilization and blastocyst formation in vitro (40 µg mL−1 of heparin could be used as an alternative concentration for frozen–thawed sex-sorted sperm with this particular bull). The incidence of polyspermy as well as polyploidy in blastocysts is currently being studied.

Table 1.Effect of heparin concentrations during IVF on cleavage and blastocyst formation
316 DEVELOPMENTAL COMPETENCE OF OOCYTES SELECTED BY THE BRILLIANT CRESYL BLUE STAINING IN PREPUBERTAL AND ADULT CATTLE
Authors: Tagawa M. , Matoba S. , Okada M. , Metoki K. , Imai K.
Published: December 2006

Transvaginal ultrasound guided ovum pickup (OPU) can be carried out repeatedly not only in adult cows but also in prepubertal calves. Moreover, recently the brilliant cresyl blue (BCB) staining has been used successfully to select oocytes homologous stained with BCB as an indirect measure of oocyte growth for in vitro fertilization (IVF) in several farm animals. The aim of this study was to evaluate the effectiveness of BCB staining on the selection of developmentally competent oocytes, collected from cows and calves by OPU, before in vitro maturation by assessing the embryonic development to the blastocyst stage after IVM/IVF. OPU was performed once weekly for a period of 7 weeks on Japanese Black prepubertal calves (9 months old, n = 4) and adult cows (n = 4) without gonadotropin stimulation. Calf and cow oocytes with homogeneous cytoplasm were exposed to 26 �M BCB for 90 min and classified according to their cytoplasmic coloration: blue coloration (BCB+) or colorless (BCB-). Classified oocytes were then matured for 20 h in TCM-199 supplemented with 5% calf serum (CS). Matured oocytes were inseminated with Percoll-separated spermatozoa (3 � 106 mL) for 6 h in BO solution supplemented with 5 mM hypotaurine and 2 U mL-1 heparin. Presumptive zygotes were cultured in CR1aa supplemented with 5% CS for 8 days. Data were analyzed by Student's t-test. The mean (± SEM) percentage of oocytes classified as BCB+ in calves was significantly lower than that in cows (34.4 ± 2.9% and 69.2 ± 2.1%, respectively; P < 0.01). In cows, BCB+ oocytes showed significantly higher cleavage and blastocyst formation percentages (72.5% and 42.4%, respectively) than those of BCB− oocytes (47.0% and 13.0%, respectively). In contrast, in calves there were no significant differences in cleavage and blastocyst formation percentages between BCB+ oocytes (56.9% and 25.3%, respectively) and BCB− oocytes (65.4% and 22.4%, respectively). The mean (± SEM) numbers of usable oocytes and blastocysts obtained per calf (19.0 ± 1.5 and 4.5 ± 0.6, respectively) were similar to those obtained per cow (16.4 ± 1.1 and 5.2 ± 0.6, respectively). No significant difference was observed in the numbers between calves and cows. These results indicate that the selection of developmentally competent oocytes before IVM/IVF, using the BCB staining, was effective for cow oocytes but not for calf oocytes, and that blastocysts could be produced by OPU-IVF of oocytes from 9-month-old prepubertal calves at an efficiency equivalent to that achieved from adult cows.
319 PROTEIN GENE PRODUCT 9.5 REGULATES SPERM PENETRATION DURING PORCINE FERTILIZATION IN VITRO
Authors: Yi Y. J. , Manandhar G. , Sutovsky M. , Park C. S. , Sutovsky P.
Published: December 2006

The protein gene product 9.5 (PGP9.5) belongs to a family of ubiquitin C-terminal hydrolases (UCHs), which regenerate monoubiquitin from ubiquitin–protein complexes or polyubiquitin chains by cleaving the amide linkage next to the C-terminal glycine of ubiquitin. Identified in the acrosome of boar spermatozoa, we hypothesized that PGP9.5 might regulate sperm–zona pellucida interactions during porcine IVF. The cumulus–oocyte complexes isolated from slaughterhouse ovaries were cultured in TCM-199 media for 44 h at 38.5�C, 5% CO2 in air. After completion of in vitro maturation (IVM), cumulus cells were removed by 0.1% hyaluronidase, and metaphase II (MII) oocytes were used for IVF. In Experiment 1, oocytes were co-incubated with different sperm concentrations (1 � 106, 5 � 105, and 1 � 105 sperm mL-1) in TBM medium with or without anti-PGP9.5 antibody (1 : 50 dilution). In Experiment 2, oocytes were inseminated with 1 � 106 sperm mL-1 in TBM medium containing different concentrations of extracted oviductal fluids (0, 0.1, 0.5, 1, 2, and 3 �g mL-1) for 6 h. After IVF, oocytes were transferred into NCSU23 medium containing 0.4% BSA for further culture. The fertilization rates were evaluated by DAPI staining at 13 to 19 h. Data were analyzed by ANOVA and Duncan's multiple range test using the SAS program. Polyspermy was increased by the addition of anti-PGP9.5 antibody to the IVF medium (56.5–60.2% at polyspermy). This PGP9.5-antibody-induced polyspermy increase was sustained even with decreasing sperm concentrations. The polyspermy rates were reduced by the addition of isolated porcine oviductal fluid to IVF medium (50.4, 44.8, 28.0, 31.1, 1.6, and 0.0% at oviductal fluid concentrations of 0, 0.1, 0.5, 1, 2, and 3 µg mL−1, respectively). Biochemical analysis by Western blotting detected the appropriate 24-kDa PGP9.5 band in porcine oviductal fluid used for these experiments. Enzymatic UCH activity comparable to activity of recombinant UCH-L3 was detected in sperm extract, whole spermatozoa, and isolated oviductal fluid by fluorometric assay using fluorogenic UCH-substrate ubiquitin-AMC. This UCH activity was not reduced by the general protease inhibitor phenyl methyl sylfonyl fluoride, but it was reduced in a statistically significant manner (P < 0.05) by the specific UCH-inhibitor ubiquitin aldehyde. In conclusion, the polyspermy increased with different concentrations of sperm in the anti-PGP9.5 antibody, and PGP9.5 was detected in oviductal fluids, suggesting that PGP9.5 is involved in the sperm–zona pellucida interaction during porcine fertilization.
This work was supported by the US Department of Agriculture (USDA-NRI grant #2002-35203-12237 to P.S.), the F21C Program of The University of Missouri–Columbia (P.S.), and the ERC Program of the Korea Science and Engineering Foundation (KOSEF grant no. R11-2002-100-00000-0 to Y.J. Yi and C.S. Park).
325 PROLONGING THE LIFE OF RAM SPERMATOZOA IN VITRO USING OVIDUCTAL EPITHELIAL CELLS
Authors: Lloyd R. E. , Badia E. , Watson P. F. , Holt W. V.
Published: December 2006

Although binding to oviductal epithelial cells (OEC) enhances both bull (Ellington et al. 1991 Theriogenology 35, 970–977) and boar (Fazeli et al. 1997 J. Reprod. Fertil. 20, 49 abst) sperm survival in vitro, the ram sperm-OEC interaction is largely uninvestigated. Consequently, this study aimed to determine (1) whether ram spermatozoa bind to OEC, and (2) whether this binding prolongs ram sperm survival in vitro. Four types of OEC (follicular isthmus, follicular ampulla, luteal isthmus, and luteal ampulla), isolated from the oviducts of ewes slaughtered locally, and sheep kidney epithelial (MDOK)cells, purchased commercially, were used in this study. Each cell type was grown in separate wells of chamber slides in a humidified incubator (39�C and 5 % CO2 in air) overnight. The next day, the growth medium was removed and the cells were washed once with PBS. Ficoll-washed ram spermatozoa, resuspended in Tyrode's medium, were added to the cells; as controls, spermatozoa were added to medium only (no OEC) in some wells. The viability of spermatozoa, unbound and bound to the cells, and in medium alone, was determined at 0, 1, 6, 24, and 48 h by incubating with the live and dead stains, Sybr-14 (100 nm) and ethidium homodimer (1 �m), respectively, for 15 min. In each case, 200 spermatozoa were counted and classified as
live or dead using a fluorescence microscope. The experiment was replicated 4 times using an ejaculate from a different ram each time. The mean percentage (%) of viable spermatozoa was determined in each case by normalizing to the viability determined for each ram ejaculate at 0 h. Data were log transformed and analyzed using factorial ANOVA. Statistical significancewas defined as P < 0.05. Following 1, 6, 24, and 48 h of co-culture, the % of viable spermatozoa bound to the OEC for each of the culture periods (mean � SEM: 129.87 � 7.34, 101.21 � 7.34, 126.72 � 7.34, and 135.97 � 7.34, respectively) was significantly greater (P < 0.001) than the % unbound to the OEC (20.94 � 7.34, 9.55 � 7.34, 14.47 � 7.34, and 31.38 � 7.34, respectively), and in medium alone (35.44 � 14.67, 31.38 � 14.67, 24.45 � 14.67, and 28.49 � 14.67, respectively). This effect was irrespective of the oviductal region and the reproductive cycle of the ewe used to derive the OEC. Interestingly, the MDOK cells, like the OEC, selectively bound viable spermatozoa following 1 and 6 h of co-culture (136.75 � 39.41 and 153.31 � 39.41, respectively). However, the % of viable spermatozoa bound to the MDOK was significantly less than to the OEC following 24 h (P = 0.05) and 48 h (P < 0.01) of co-culture (93.52 � 39.41 and 72.85 � 39.41, respectively). These data show that epithelial cells originating from reproductive and non-reproductive tissues both are capable of binding and prolonging sperm viability over the first 6 h of co-culture, but only OEC are capable of prolonging sperm viability for longer periods of time.
This work was funded by DEFRA (UK), Innovis (UK), and IMV (France).
330 CORRELATION BETWEEN TWO APOPTOTIC MARKERS IN FRESH BOAR SPERMATOZOA
Authors: Trzcinska M. , Bryla M. , Smorag Z.
Published: December 2006

Apoptosis is a carefully regulated process of cell death that occurs as a normal part of development. It is a well-characterized mechanism that allows eukaryotes to eliminate unneeded, senescent, or aberrant cells, but the significance of apoptosis in ejaculated animal sperm is still unresolved. In this experiment, we designed 2 methods to detect early changes in the membranes of boar spermatozoa based on the slight increase in membrane permeability (Vybrant Apoptosis Assay Kit #4; Molecular Probes, Eugene, OR, USA) and on translocation of the phospholipid phosphatidyserine (PS) from the inner to the outer leaflet of the plasma membrane (Annexin-V-FLUOS Staining Kit; Roche, Mannheim, Germany). Detection of early changes in the sperm plasma is very important when designing storage protocols. Three ejaculates from 3 boars were used in the experiment. After collection and separation of the gel, the semen was analyzed using the following assays: (1) Annexin-V-FITC/PI assay: Sperm (2 � 106) were washed and diluted in 100 �L of HEPES buffer; 6 �L of Annexin-V-FITC and 4 �L of PI were added to the sample. The tubes were incubated for 15 min at room temperature in the dark. (2) YO-PRO-1/PI assay: YO-PRO-1 stock solution (1 �L) and 1 �L of PI stock solution were added to the samples. The tubes were gently mixed and incubated for 20 min at room temperature in the dark. After the incubation period, the sperm cell suspensions were analyzed under a fluorescence microscope at 40� magnification. At least 200 spermatozoa per sample were evaluated. Using the YO-PRO-1/PI assay, we observed 3 groups of sperm: apoptotic sperm showed green fluorescence (2–8%), necrotic sperm showed red and green fluorescence (9–37%), and live sperm showed no fluorescence (58–89%). Using the Annexin V-PI assay, 4 sperm subpopulations were easily detectable: apoptotic sperm showed green fluorescence (0.5–7%), early necrotic sperm showed red and green fluorescence (10–35%), necrotic sperm showed only red fluorescence (2–6%), and live sperm showed no fluorescence (57–87%). The results were compared by a chi-squared test. Significant differences (P < 0.01) in the percentage of all sperm subpopulations (apoptotic, necrotic, and live sperm) among boars and among ejaculates from the same boar were observed. We also observed a strong correlation between these 2 methods. Using the Annexin-V-FITC/PI assay, we detected more sperm subpopulations, and this method seems to be more sensitive than the YO-PRO-1/PI assay. However, these 2 methods detect changes in membrane spermatozoa but in different aspects of apoptosis, and this may also cause differences in the frequencies of apoptotic cells found by the different assays.
This study was supported by the Polish Research Committee, grant no. 2 P06D 023 30.
337 GENE EXPRESSION IN BOVINE OOCYTES AFTER MEIOSIS BLOCK WITH BUTYROLACTONE I
Authors: Adona P. R. , Biase F. H. , de Bem T. H. C. , Leal C. L. V.
Published: December 2006

The present study aimed to assess the effect of meiosis block on the expression of genes involved in apoptosis. Slaughterhouse bovine ovaries were collected soon after slaughter and transported to the laboratory in saline. Follicles with a diameter of 2–6 mm were aspirated, and oocytes were selected and distributed to the different treatments. For meiosis block (MB), oocytes were cultured for 24 h in TCM-199 supplemented with 100 �M butyrolactone I (BLI) and 3 mg mL-1 BSA (B100) or with 10 �M BLI, but without BSA (B10). After the 24-h MB culture, the oocytes were subjected to IVM in TCM-199 supplemented with 10% fetal calf serum (FCS), 5.0 �g mL-1 LH, 0.5 �g mL-1 FSH, 0.2 mM pyruvate, and 10 �g mL-1 gentamicin for 22 h. All cultures were at 38.5�C under an atmosphere of 5% CO2 in air. As controls (C), a group of oocytes was collected immediately after aspiration (immature oocytes) and after IVM without prior meiosis block (mature oocytes). In the treated groups, oocytes were collected at the end of MB culture (immature oocytes) and at the end of IVM post-MB (mature oocytes). After removal of cumulus cells and zonae pelucidae, oocytes were frozen at -80�C in PBS with 0.1% PVA and 1 IU �L-1 RNase inhibitor. Reverse transcription was performed using SuperScript II Reverse Transcriptase (Invitrogen Brasil, Ltda., Sao Paulo, Brazil) according to the manufacturer's recommendations in an Applied Biosystems 7500 fast real-time PCR system (Applied Biosystems, Sao Paulo, Brazil). Efficiency of the reactions was assessed by the software LinRegPCR (Ramakers et al. 2003 Neurosci. Lett. 339, 62–66) and submitted to REST Beta 2005 V1.9.9 analysis (Relative Expression Software Tool). A level of significance of 5% was used. Relative expression of BAX and BCL2 genes did not differ (P > 0.05) when the expression of the constitutive gene GAPDH was used to normalize the data. However, without normalization, a significant reduction in expression was observed after IVM (P < 0.05). For immature oocytes, GAPDH, BAX, and BCL2 expression was 1.0% (for the three genes) for control; 0.26, 0.36, and 0.56% for B100; and 0.6, 0.44, and 0.26% for B10, respectively. After IVM, expressions were 0.06, 0.33, and 0.26% for control; 0.06, 0.19, and 0.25% for B100; and 0.02, 0.15, and 0.3% for B10, respectively. The results show a reduction in transcripts (GAPDH, BAX, and BCL2) after meiosis block, and this reduction was more pronounced in matured oocytes, irrespective of treatment. The reduction in transcripts during MB could influence oocyte competence, but the reduction of these transcripts during maturation seems to be inherent to the oocytes.
This work was supported by Fapesp, Brazil, grant # 03/01479-6.
339 OXYGEN CONCENTRATION DURING IN VITRO MATURATION OF MURINE OOCYTES INFLUENCES SUBSEQUENT FETAL AND PLACENTAL OUTCOMES
Authors: Banwell K. M. , Lane M. , Russell D. L. , Kind K. L. , Thompson J. G.
Published: December 2006

Although the oxygen environment of the ovarian follicle is thought to influence oocyte developmental competence, little is known of the optimal oxygen environment for oocyte in vitro maturation (IVM). Previously, we found that oxygen concentration (either 2, 5, 10, or 20% O2; 6% CO2; and balance of N2) during IVM of murine oocytes had no effect on maturation rate or subsequent fertilization, cleavage, and blastocyst development rates. However, 2% O2 results in blastocysts with a higher (P < 0.05) trophectoderm cell number (mean � SEM, 35.1 � 2.3) when compared to 20% (19.4 � 1.7), with 5 and 10% O2 yielding similar but intermediate cell numbers. When examined for cell apoptosis by TUNEL labelling, the 2% O2 IVM-derived embryos were also found to have a significantly higher percentage of cells undergoing apoptosis compared to the 5% O2 IVM-derived embryos and embryos derived from in vivo matured oocytes (unpublished data). Although the blastocyst development rate is not affected by varying oxygen environment during oocyte maturation, the resultant blastocysts exhibited signs of differing quality. The aim of this study was to investigate the effect of varying oxygen during IVM on post-transfer outcomes. Immature cumulus-oocyte complexes (COCs) were collected from the ovaries of eCG-stimulated CBAB6F1 females (21 days) and cultured for 17–18 h under 2, 5, or 20% O2, whereas in vivo-matured COCs were also collected post-hCG. After IVF/C (both under 5% O2), 6 blastocysts were transferred to each uterine horn of pseudopregnant Swiss recipients. Fetal and placental parameters were measured on Day 18 of pregnancy. The ability of the embryos to implant or develop was not altered by IVM oxygen concentration. However, the average weight of fetuses derived from 5% O2 matured oocytes was reduced (823.3 � 28.1 mg, P < 0.05) compared to those in the 20% O2 group (928.5 � 26.1 mg). The average weight of the placenta in the 5% O2 group was also reduced (87.4 � 4.0 mg) compared to those derived from in vivo-matured oocytes (104.5 � 5.4 mg). In contrast, the fetal:placental weight ratio was unchanged in the 5% O2 treatment, suggesting these placentae, although small, are still efficient. This is the first evidence that programming of fetal/placental growth occurs from treatments applied during oocyte maturation.
351 EFFECTS OF FETAL CALF SERUM, GROWTH FACTOR, AND HORMONE SUPPLEMENTATION DURING IN VITRO MATURATION ON PARTHENOGENETIC ACTIVATION AND EMBRYO DEVELOPMENT OF FOLLICULAR RABBIT OOCYTES
Authors: Polgar Z. , Somfai T. , Angeli V. , Tang X. H. , Ji W. , Dinnyes A.
Published: December 2006

Improvement of the in vitro maturation (IVM) system for rabbit oocytes could play a role in rabbit biotechnology. Our goal was to improve IVM to have an efficient source of rabbit oocytes for further studies on nuclear transfer. The effects of FCS, growth factors, and hormone supplementation on oocyte maturation, activation, and embryo development rates were evaluated. Rabbit ovaries were transferred from the slaughterhouse to the laboratory in PBS. Oocytes were collected by aspiration and subjected to IVM in 3 types of media based on TCM-199. In one group, growth factors (50 ng mL−1 of insulin-like growth factor-I and 10 ng mL−1 of epidermal growth factor) and hormones (5 IU mL−1 of hCG and 5 IU mL−1 of pregnant mare serum gonadotropin) and BSA were added to the IVM medium (IVM+); in the other groups, IVM medium was supplemented with either 10% (IVM + 10% FCS) or 20% FCS (IVM + 20% FCS). Maturation was assessed by the presence of a polar body after 16 h. Matured oocytes were activated twice by electric stimuli (3 DC pulses, 1.6 kV cm−1, 60 µs) and twice by chemical activation (incubation with 2.5 mM 6-DMAP for 30 min, second time for 2 h) and cultured in vitro in Earle's balanced salt solution complete medium at 38.5°C under 5% CO2 in air. Cleavage rates were recorded 16 h after activation and the blastocyst rates were recorded at Day 5 of in vitro culture. Data were analyzed by ANOVA. Maturation rates did not differ between the treatment groups (Table 1). There was no significant difference in cleavage rates between the IVM+ and the IVM + 10% FCS groups; however, the cleavage rate of the IVM + 20% FCS group was significantly lower compared with the others (Table 1; P < 0.05). Development to the blastocyst did not differ significantly between the treatment groups (Table 1; P < 0.05). The results showed that high (20%) FCS supplementation during IVM had a detrimental effect on oocyte cleavage. Hormonal and growth factor supplementation had no beneficial effects on maturation, activation rates, or blastocyst formation, and in fact were not essential for in vitro embryo production in the rabbit.

Table 1.Effect of hormonal and growth factor supplementation on embryo development in rabbit oocytes

The project was supported by RABIOTECH OMFB-00330/2004, EU FP6 (MEXT-CT-2003-509582, and 518240), Wellcome Trust (Grant No. 070246), and TET CH-28/04.
354 IN VITRO DEVELOPMENT OF BOVINE EMBRYOS AFTER NITRIC OXIDE INHIBITION
Authors: Schwarz K. R. L. , de Bem T. H. C. , Zampieri T. T. , Adona P. R. , Leal C. L. V.
Published: December 2006

Nitric oxide (NO) is a chemical messenger detected in several cell types such as endothelial cells, neurons, and macrophages, exerting varied functions including vasodilatation, neurotransmission, and cell death induction. NO is generated by the activity of the enzyme nitric oxide synthase (NOS), which has been detected in several organs and tissues including the reproductive system. The aim of the present study was to assess the dose-response effect of N-omega-nitro-l-arginine-methyl ester (l-NAME), an NOS inhibitor, on in vitro nuclear and cytoplasmic maturation of bovine oocytes. Slaughterhouse ovaries were collected and their follicles (2–6 mm) were aspirated to obtain cumulus–oocyte complexes (COCs). Increasing l-NAME concentrations (0, 10-7, 10-5, 10-4, and 10-3 M) were added to IVM medium (TCM-199, supplemented with 10% fetal calf serum, 0.5 �g mL-1 FSH, 5.0 �g mL-1 LH, 0.2 mM pyruvate, and 10 mg mL-1 gentamicin); oocytes were cultured for 22 h. Nuclear maturation was assessed by propidium iodide staining (10 �g mL-1). For IVF, frozen–thawed semen prepared by Percoll gradient was used. Sperm cells were co-cultured with the oocytes at a final concentration of 2 � 106 sperm cells mL-1 in TALP-IVF medium supplemented with 2 �M penicillamine, 1 �M hypotaurine, 250 �M epinephrine, and 20 �g mL-1 heparin. After 20 h, presumptive zygotes were partially denuded and transferred to IVC medium (TCM-199 supplemented with 10% fetal calf serum, 2.0 mM pyruvate, and 10 mg mL-1 gentamicin). All cultures were at 38.5�C under 5% CO2 in air and maximum humidity. Cytoplasmic maturation was assessed by blastocyst development rates on Day 7. DNA fragmentation was assessed on Day 8 embryos by TUNEL (In Situ–Cell Death Detection kit, fluorescein; Roche Diagnostica Brasil, Sao Paulo, Brazil). Data were analyzed by ANOVA using the GLM procedure (SAS Institute, Inc., Cary, NC, USA), and means were compared by Duncan test at a 5% level. After IVM, the control group (0 M l-NAME) showed a greater number of oocytes in metaphase II (MII: 95.8 � 3.7%; P < 0.05), whereas the groups cultured with l-NAME had lower MII rates (78–82%; P < 0.05), irrespective of concentration (P > 0.05). Many oocytes remained in metaphase I (MI: 18–22%). Cleavage rates at 48 h IVC was not affected (77–88%; P > 0.05). Blastocyst rates (34.0 � 7.2% to 41.5 � 4.8%; P > 0.05) and total cell numbers (151 to 174) were also unaffected by NO inhibition by l-NAME. However, the number of TUNEL-positive cells was lower in the control group (1.4 � 4.7; P < 0.05) than in the treated groups (2.7 � 4.8 to 4.4 � 6.4; P > 0.05). In conclusion, NO synthesis inhibition in oocytes during IVM reduces nuclear maturation, particularly during MI–MII transition, and increases apoptosis in blastocysts, suggesting that NO may be involved in oocyte maturation and apoptosis protection.
373 OOCYTE-ACTIVATING CAPACITY OF BOVINE SPERMATOGENIC CELLS AND ACTIVATION PROTOCOLS FOR INTRACYTOPLASMIC INJECTION OF BOVINE ROUND SPERMATIDS
Authors: Kani C. , Takenaka M. , Muneto T. , Yamamoto M. , Horiuchi T.
Published: December 2006

In vitro spermatogenesis can be applied to generate spermatids or spermatozoa and produce a genetically modified male germ line. Intracytoplasmic injection of the spermatids or spermatozoa is an important technique for effective production of offspring. The objective of this study is to evaluate oocyte-activation capacity of bovine spermatids or spermatozoa and to determine the effective activation treatment for in vitro development of bovine oocytes injected with round spermatids. Cryopreserved testicular spermatogenic cells and cauda epididymal spermatozoa obtained from a 1-year-old Japanese bull were used. In the first experiment, we injected bovine round (ROS) and elongated (ELS) spermatids, or testicular (TES) and cauda epididymal (CES) spermatozoa into mouse oocytes to examine their oocyte-activating capacity. The presence of pronuclei within whole-mounted oocytes was observed 4 h after injection. In the second experiment, we injected similar spermatids and spermatozoa into bovine oocytes without additional activation, and examined cleavage and blastocyst development. In the third experiment, bovine oocytes injected with ROS were activated with 7% ethanol or 5 �M ionomycin for 5 min (1 � Et or 1 � Iono) immediately after injection; some were further activated repeatedly at 3 h after injection (2 � Et or 2 � Iono), and some of these were subjected to 1.9 mM 6-dimethylaminopurine (DMAP) for 3 h after the second activation (2 � Et + DMAP or 2 � Iono + DMAP). Data were analyzed by the chi-square test in all experiments. The vast majority of bovine ROS failed to activate mouse oocytes (activation rate 10%). Activation rates of mouse oocytes injected with bovine ELS, TES, and CES were 61%, 75%, and 91%, respectively. The results suggest that oocyte-activation capacity is acquired during transformation from ROS to ELS. Cleavage and blastocyst rates of bovine oocytes injected with CES (59% and 19%, respectively) were significantly higher (P < 0.05) than the rates obtained with injections of TES (37% and 9%) and ROS (5% and 0%) without additional activation. However, cleavage and blastocyst rates of bovine oocytes injected with ROS in the groups of 2 � Et + DMAP (80% and 19%) and 2 � Iono + DMAP (76% and 19%) were significantly higher (P < 0.05) than those in the groups of 1 � and 2 � Et (37% and 2%, 59% and 4%), 1 � and 2 � Iono (10% and 7%, 22% and 4%), or those receiving a sham injection and activated with 2 � Iono + DMAP (43% and 4%). These results demonstrate that intracytoplasmic injection of ROS with repeated Et or Iono activation followed by DMAP treatment is more efficient than single or double Et or Iono activation.
376 EFFECT OF SPERM TREATMENT ON THE ABILITY TO ACTIVATE OOCYTES AFTER ICSI IN PIGS
Authors: Nakai M. , Kashiwazaki N. , Maedomari N. , Ozawa M. , Noguchi J. , Kaneko H. , Kikuchi K.
Published: December 2006

During fertilization, sperm penetration (gamete membrane fusion and exposure of sperm cytoplasm) allows oocyte activation (resumption of oocyte meiosis, pronuclear formation, etc.) by inducing an elevation of the intracellular free Ca2+ concentration. So a spermatozoon ought to be able to fully activate an oocyte. However, in pig ICSI oocytes, although a spermatozoon is injected successfully into ooplasm, complete activation is deficient in some of the oocytes. A variety of sperm pre-treatments before ICSI have been reported; however, there is a possibility that the treatment affects the ability to activate oocytes after the injection. We examined the effect of sperm treatments (freezing, freeze-drying, and sonication) on the ability to activate oocytes. Ejaculated boar semen was centrifuged (10 min, 600g) and the supernatant was discarded. The sperm pellet was resuspended in Modena solution (Weitze 1991 Reprod. Domest. Anim. (Suppl. 1), 231–253). The sperm were then treated with or without sonication for 10 s (fresh whole and sonicated sperm, respectively). The freezing of sperm was carried out as was described (Kikuchi et al. 1998 Theriogenology 50, 615–623). Frozen–thawed spermatozoa were then treated with or without sonication (frozen–thawed sonicated and whole sperm, respectively). The fresh whole and sonicated sperm were subjected to a freeze-drying system and the sperm were then re-hydrated (freeze-dried whole and sonicated sperm, respectively). A whole sperm or 1 or 3 sonicated sperm heads were then injected into in vitro-matured oocytes, as described previously (Nakai et al. 2003 Biol. Reprod. 68, 1003–1008; 2006 Reproduction 131, 603–611). Sham injection was also performed. No artificial stimulation was added to the injected oocytes. The oocytes with more than one pronucleus(i) at 10 h after the injection were defined as being activated. As shown in Table 1, the rates of activated oocytes after injection of one sonicated head or sham injection were significantly lower than those of the oocytes injected with whole sperm or 3 sonicated sperm heads in each sperm source (P < 0.05 by ANOVA and Duncan's multiple range test). Furthermore, the rates of activated oocytes for each injection category were not different among the 3 sperm sources. These results suggest that sonication before ICSI may reduce the quantity of activation-inducing sperm factor. It is also suggested that sperm pre-treatment such as freezing or freeze-drying does not affect the ability for oocyte activation.

Table 1.
Effect of sperm treatment on oocyte activation after ICSI
377 SPERMATOZOA RETRIEVED FROM MALE MICE FROZEN FOR 15 YEARS CAN PRODUCE NORMAL OFFSPRING
Authors: Ogonuki N. , Mochida K. , Miki H. , Inoue K. , Iwaki T. , Morozumi K. , Yanagimachi R. , Ogura A.
Published: December 2006

Cryopreservation of male germ cells is a strategy to conserve animal species and strains of animals valuable to biomedical research. However, to minimize damage that may occur during freezing and thawing, complex cryopreservation protocols that have been optimized for the stage and species of male germ cells are usually employed. Recently, we have found that mouse male germ cells can be cryopreserved at -80�C by freezing the whole epididymides and testes without cryoprotectant for at least one year (Ogonuki et al. 2006 Reprod. Fertil. Dev. 18, 286 abst). This study was undertaken to determine whether mouse male germ cells retrieved from the bodies of mice frozen at -20�C for 15 years could produce normal offspring by microinsemination. Mature males of BALB/c-nude and C3H/He (8 weeks of age) were euthanized by overdose of pentobarbital on February 20 and March 8, 1991, respectively, and kept in a -20�C freezer. The frozen body was thawed about 15 years after freezing (February 2006) by putting it in a water bath until the outer surface of the body was softened. The body was then removed from the water, and the testes were isolated through an abdominal incision. Testicular spermatozoa were collected from the testes and microinseminated into B6D2F1 oocytes. Within 24 h after sperm injection, over 80% of oocytes developed into 2-cell embryos. Apparently normal pups were born after embryo transfer in both strains of mice at rates of 21% (17/81) and 12% (12/97) per transfer, respectively. Two pups from the BALB/c-nude group died shortly after Caesarian section due to respiratory failure, but others grew normally and were proven to be fertile when they matured (at least 19 mice out of 20 mice tested). We further mated these F1 offspring and confirmed that the nude gene was safely propagated. The present study demonstrates that spermatozoa can retain their fertilizing ability in frozen whole bodies for longer than we anticipated. If spermatozoa of extinct mammalian species (e.g. woolly mammoth) can be retrieved from animal bodies that were kept frozen in permanent frost, live animals might be restored by injecting them into oocytes from females of closely related species.
378 INTRACYTOPLASMIC SPERM INJECTION AND ESTABLISHMENT OF EMBRYONIC STEM CELLS IN AFRICAN GREEN MONKEYS (CERCOPITHECUS AETHIOPS)
Authors: Shimozawa N. , Okada H. , Hatori M. , Yoshida T. , Sankai T.
Published: December 2006

There has been little embryological research in African green monkeys (Cercopithecus aethiops), although Vero and Cos7 cell lines derived from the kidney of the green monkey are used as valuable resources for testing viral infections and introducing foreign genes. Embryological research would not only contribute to medical science but would also help to maintain and propagate other monkeys, including those in the wild. We examined intracytoplasmic sperm injection (ICSI) and in vitro culture of green monkey embryos and attempted to establish embryonic stem (ES) cells, which would be precious resources. We collected mature oocytes from females treated with eCG + hCG or human FSH + hCG to induce follicular growth stimulation and oocyte maturation. Of 61 oocytes from eCG-treated females and of 44 oocytes from FSH-treated females, 41 (67.2%) and 27 (61.4%) fertilized oocytes with 2 pronuclei and a second polar body were produced with ICSI, respectively. We cultured fertilized oocytes from eCG-treated and FSH-treated females in CMRL-1066 medium containing 10% fetal bovine serum (FBS), with or without a buffalo rat liver (BRL) cell monolayer. Of 28 zygotes from eCG-treated females, 9 were cultured without a monolayer, and 3 (33.3%) developed into blastocysts; none of 19 cultured with a monolayer did. Of 22 zygotes from FSH-treated females, 18 were cultured without a monolayer, and 5 (27.8%) developed into blastocysts; none of 4 cultured with a monolayer did. Thus, there were no differences in the fertilization rates and development into blastocysts between the oocytes from eCG- and FSH-treated females. On the other hand, BRL cells did not support development into blastocysts [0/23 (0%) with a monolayer vs. 8/27 (29.6%) without a monolayer; P < 0.01]. The other embryos were used for experiments such as embryo transfer. Parts of the inner cell mass (ICM), isolated by removing trophectoderm and the zona pellucida (ZP) with a 27-gauge needle (5 blastocysts) or by dissolving the ZP with an enzyme (3 blastocysts) were transferred to DMEM/F12 (1 : 1) medium containing 20% FBS or knockout serum replacement with a mitomycin C-treated STO cell or mouse embryonic fibroblast cell monolayer. Only 3 ICMs formed extended colonies; the others, including the 3 blastocysts from which the ZP was dissolved, did not. When the 3 colonies were subcultured with collagenase or divided into small clusters with a needle, 2 flat colonies, as in primate ES cells, appeared. At the next subculture, the flat colonies disappeared. However, we confirmed the formation of ES-like cell colonies. These results show that it is possible to produce blastocysts with ICSI and that ES cells may be established in African green monkeys.
379 EFFECT OF SUPPLEMENTARY INOSITOL 1,4,5-TRIPHOSPHATE INJECTION ON FERTILIZATION AND EMBRYO DEVELOPMENT AFTER BOVINE ICSI
Authors: Suh T. K. , Seidel, Jr G.E.
Published: December 2006

During fertilization in mammals, sperm membrane-bound phospholipase C zeta induces breakdown of ooplasmic membrane-bound phosphatidylinositol-4,5-bisphosphate (PIP2), which leads to the production of diacylglycerol (DAG) and inositol 1,4,5-trisphosphate (IP3). The IP3 induces intracellular Ca2+ oscillations ([Ca2+]i), which trigger inactivation of maturation-promoting factor (MPF) and mitogen-activated kinase (MAPK), resulting in decondensation of the sperm head, resumption of meiosis, extrusion of a second polar body, cortical granule exocytosis, formation of pronuclei (PN), and entry into the first cell cycle. In bovine ICSI, injection of a single spermatozoon into an oocyte does not consistently induce [Ca2+]i oscillations, as was observed in IVF, and this may, at least in part, explain the low rates of fertilization and embryo development. Although IP3 has been used as a powerful activator for nuclear transferred zygotes or parthenogenetic oocytes, few studies have evaluated the effect of IP3 injection on normal fertilization and embryo development after ICSI. The objective of this study was to determine the effect of injecting IP3 during bovine ICSI on fertilization and embryo development in vitro. Chemically defined media (CDM) were used throughout (De La Torre-Sanchez et al. 2006 Reprod. Fertil. Dev. 18, 585–596). The injection volume of IP3, which was dissolved in calcium- and magnesium-free PBS, was approximately 0.01 nL, and the total dose for injection along with a spermatozoon was about 250 �M, which was determined by the 2PN formation rate in preliminary experiments. Semen from 3 bulls was used to produce embryos in 5 replicates. Oocytes obtained from slaughterhouse ovaries were matured in vitro in CDM-M for 22–23 h under 5% CO2 in air at 38.5�C, and oocytes with a first polar body were used for ICSI. Motile sperm from frozen–thawed semen were used for sperm injection, with or without IP3 in a 50-�L drop of GMOPS medium, with a piezo-driven pipet of 7–8 �m inner diameter. After ICSI, presumptive zygotes were cultured in CDM-1 for 3 days, and then in CDM-2 for 5 days at 39�C under 5% CO2/5% O2/90% N2. Cleavage and blastocyst development were evaluated at the end of each culture period. Data were subjected to Fisher's exact test. Cleavage in control and IP3 groups was 36.4 and 50.0%, respectively (P < 0.05). Respective blastocyst rates per oocyte were 5.5 and 13.0% (P > 0.05). This study showed that injection of IP3 during the sperm injection process improved cleavage of bovine oocytes after ICSI.
380 FULL-TERM DEVELOPMENT OF MOUSE EMBRYOS PRODUCED FROM RECONSTRUCTED GIANT OOCYTES BY INTRACYTOPLASMIC SPERM HEAD INJECTION
Authors: Wakayama S. , Kishigami S. , Nguyen V. T. , Ohta H. , Hikichi T. , Mizutani E. , Sakaide Y. , Thuy B. H. , Suetsugu R. , Wakayama T.
Published: December 2006

In mammals, unusually large diploid giant oocytes are ovulated occasionally, but as any fertilized embryos would be triploid, the potential of the cytoplasm to support normal development is unknown. However, if such giant oocytes possess normal cytoplasm, the oocytes or their excessive cytoplasm would have potential applications in the treatment of human infertility and would enhance the study of basic biology, such as the influence of cytoplasmic factors on genomic reprogramming. We reconstructed oocytes 2 to 9 times normal volume by electrofusion or mechanical fusion between intact and enucleated oocytes. First, we examined the in vitro developmental potential of 2- to 9-times giant oocytes after parthenogenetic activation. Second, 2-times giant oocytes were reconstructed by fusing with intact and enucleated oocytes; then we examined the in vitro and in vivo developmental potential of these giant oocytes after intracytoplasmic sperm injection (ICSI). Third, somatic cell nuclear transfer (SCNT) was carried out using enucleated giant oocytes. When the giant oocytes were activated parthenogenetically, most developed into morulae or blastocysts, irrespective of the original size. When sperm heads were injected into 2-times giant oocytes, these were fertilized and developed normally in vitro; after embryo transfer, we obtained 12 healthy offspring (3 female, 3 male, and 6 cannibalized the next day) by Caesarian section. All survivors grew to adulthood and demonstrated normal fertility. However, we failed to generate cloned mice by SCNT. In conclusion, reconstructed giant oocytes have normal potential for development after activation and fertilization. The reconstructed giant oocytes will provide us with interesting tools for basic biology, such as providing new material for the study of nuclear reprogramming and genomic imprinting, and may be important in widening the treatment options in human assisted reproductive technology.
381 FERTILIZABILITY AND CHROMOSOMAL INTEGRITY OF FROZEN - THAWED BRYDE's WHALE (BALAENOPTERA EDENI) SPERMATOZOA INTRACYTOPLASMICALLY INJECTED INTO MOUSE OOCYTES
Authors: Watanabe H. , Tateno H. , Kusakabe H. , Kamiguchi Y. , Fujise Y. , Ishikawa H. , Ohsumi S. , Fukui Y.
Published: December 2006

In this study, we applied intracytoplasmic sperm injection (ICSI) to mouse oocytes to evaluate the fertilizability and chromosomal integrity of the three types of frozen–thawed Bryde's whale spermatozoa. B6D2F1 female mice (7–11 weeks of age) were superovulated by injections of PMSG followed by hCG 48 h later. The oocytes recovered from oviducts between 14 and 16 h after hCG injection were denuded of their cumulus cells. Sperm samples were obtained from a Bryde's whale (Balaenoptera edeni) captured under the Japanese Whale Research Program with Special Permit in the Western North Pacific between May and August 2003 (presumptive feeding season). The whale was killed by an explosive harpoon which has been recognized as the best humane method for whales by the International Whaling Commission (IWC) and stipulated by Schedule III (Capture) of the International Convention for the Regulation of Whaling. Spermatozoa collected from vasa deferentia were cryopreserved. Frozen Bryde's whale spermatozoa were thawed at 37�C and washed with HEPES-TYH by centrifugation at 500g for 5 min. Motile and immotile spermatozoa were obtained, and some spermatozoa in HEPES-TYH were refrozen without cryoprotectant at -20�C to be completely killed. Within 24 h, they were thawed at 37�C and prepared for ICSI. Comparison of group values was performed by either Fisher's exact probability test or chi-square test where necessary. Differences at P d 0.05 were considered significant. Chromosomal normality was determined by analyses of karyotyped haploid chromosomes (n = 22) of the whale sperm. Regardless of motility and viability, whale spermatozoa efficiently led to the activation of mouse oocytes (90.3–97.4%), and sperm nuclei successfully transformed into male pronuclei within activated ooplasm (87.2–93.6%). Chromosome analysis at the first cleavage metaphase of the hybrid zygotes revealed that a majority (95.2%) of motile spermatozoa had the normal chromosome complement, whereas the percentage of chromosomal normality was significantly (P ≤ 0.001) reduced to 63.5% in immotile spermatozoa and 50.0% in dead spermatozoa, due to the increase in structural chromosome aberrations such as chromosome fragments. This is the first report showing that motile Bryde's whale spermatozoa are competent to support embryonic development. Furthermore, we have shown that chromosomal analysis of whale spermatozoa is a useful technique for measuring the influences of marine pollution on reproduction in cetacean species that occupy the top niche in the marine ecosystem.
384 EFFECTS OF CONDITION SCORE ON OVARIAN RESPONSE AND TRANSFERABLE EMBRYOS IN SUPERSTIMULATED COWS IN ARGENTINA
Authors: Garcia Guerra A. , Rodriguez D. , Villareal J. , Albrecht A. , Brogliatti G.
Published: December 2006

The success of an embryo transfer program is influenced by ovarian response, number of ovulations, fertilization, and embryo viability (Armstrong 1993). It has been observed that over-conditioned, overweight donor females tend to produce fewer viable embryos per collection (Stroud and Hasler 2006 Theriogenology 65, 65–76). The condition score is one of the many intrinsic factors that affects cow reproduction. A retrospective analysis was done to compare the ovarian response, number of ovulations, fertilization, and total transferable embryos recovered of cows with different condition scores in Argentina. This research was carried out using different breed donors (n = 640; 82% Angus; 18% other) during the last 4 years in Buenos Aires province, Argentina. Donors at random stages of follicular wave with CL received an intravaginal progesterone device (DIB; Syntex, Buenos Aires, Argentina), 2 mg of estradiol benzoate, and 50 mg of progesterone (Syntex, Buenos Aires, Argentina) IM on the same day. On Day 4 after DIB insertion, superestimulatory treatment was initiated on a decreasing dose regimen of FSH (Pluset®; Callier, São Paulo, Brazil, or Folltropin®; Bioniche Animal Health, Inc., Belleville, Ontario, Canada) as IM injections every 12 h during 4 days. On Day 6, DIBs were removed, and cows received a double dose of 2 mL of cloprostenol within 12 h. At heat detection, all donors received a dose of 2 mL of GnRH (Dalmarelin; Von Franken, Buenos Aires, Argentina) by IM injection and were inseminated 12 and 24 h thereafter. Seven days later, embryo collection was performed and ovarian response was evaluated as number of CL + anovulatory follicles by transrectal ultrasound using a 7.5-MHz transducer (Pie Medical, Maastricht, The Netherlands). The condition score was determined at this time using a 1 to 5 scale. Embryos recovered were evaluated and classified according to the IETS manual. Kruskal–Wallis test was used to compare variables among score groups, and results are shown in Table 1. Ovarian response as the number of CL and anovulatory follicles was significantly different (P = 0.0001) among groups. There is a negative correlation (−0.2194) between condition score and ovarian response. At higher condition scores, ovarian response decreased. There is a significant difference (P = 0.0001) for the number of ovulations and total number of fertilized ova among groups. Finally, the total number of transferable embryos was no different for either score; however, there is a trend toward decrease when the condition score is higher. In conclusion, these results indicate that using donors with condition scores of 2 to 3 improves the results of a superstimulatory treatment. Controlling donors' condition score by nutrition management gives us the possibility to produce more embryos per donor treatment.

Table 1.
Condition score, ovarian response, fertilization, and transferable embryos in donor cows in Argentina