Vivo-matured Oocytes Research Articles

Bovine Nuclear Transfer Using Fresh Cumulus Cell Nuclei andIn Vivo- orIn Vitro-Matured Cytoplasts

We examined the effects of the source of recipient oocytes and timing of fusion and activation on the development competence of bovine nuclear transferred (NT) embryos derived from fresh cumulus cells isolated immediately after collection by ovum pickup (OPU). As recipient cytoplasts, we used in vivo-matured oocytes collected from hormone-treated heifers by OPU, or in vitro-matured oocytes from slaughterhouse-derived ovaries. NT embryos were chemically activated immediately (simultaneous fusion and activation, FA) or 2 h (delayed activation, DA) after fusion. When in vitro-matured oocytes were used as recipient cytoplasts, the development rate to the blastocyst stage of NT embryos produced by the DA method (23%) tended to be higher than those by the FA method (15%), but the difference was not significant. NT embryos derived from in vivo-matured cytoplasts have a high blastocyst yield (46%). Pregnancy rate at day 35 did not differ with the timing of fusion and activation (FA vs. DA; 50% vs. 44%) or oocyte source (in vivo- vs. in vitro-matured; 50% vs. 44%). Subsequently, the high fetal losses (88% of pregnancies) were observed with in vitro-matured cytoplasts, whereas no abortions were observed in NT fetuses from in vivo-matured cytoplasts. A total of three embryos derived from fresh cumulus cells developed to term. However, all three cloned calves were stillborn. These results indicate that improvement of development competence after NT is possible by using in vivo-matured oocytes as recipient cytoplasts in bovine NT.

Developmental potential of oocytes fertilized by conventional in vitro fertilization (IVF) or intracytoplasmic sperm injection (ICSI) after cryopreservation and mesometrial autotransplantation of rabbit ovarian tissue

Objective: To evaluate mesometrial transplantation of frozen-thawed ovarian tissue in rabbit and to choose the optimized fertilization method for oocytes retrieved from grafts by investigating the capability of oocyte fertilization and further development. Forty rabbits were divided into three groups randomly: control group, fresh tissues transplantation group and frozen-thawed tissues transplantation group. Three months after the transplantation, rabbits were stimulated with FSH and oocytes were retrieved 13 h after human chorionic gonadotropin (HCG) injection. Oocytes matured in vivo or in vitro were then fertilized by conventional in vitro fertilization (IVF) or intracytoplasmic sperm injection (ICSI), followed by observation and evaluation of fertilization rate and blastocyst formation rate. Blastocytes embryos were transferred to pseudopregnancy rabbits to observe pregnancy rate and birth rate. There were no significant differences in the percentage of oocytes matured either in vivo or in vitro among the three groups. The fertilization rate, cleavage rate and blastocyst formation rate of in vivo-matured oocytes had no difference among the three groups, whether they were fertilized by IVF or ICSI. Significantly higher fertilization rates of in vitro-matured oocytes were observed with ICSI compared with IVF in each group. The blastocyst formation rate of in vitro-matured oocytes was significantly lower than that of in vivo-matured oocytes in each group. The birth rate of in vivo-matured oocytes was significantly higher than that of in vitro-matured oocytes, although the pregnancy rate was similar between them. Mesometrial transplantation of frozen-thawed ovarian tissue may provide favorable conditions for follicle development. Oocytes retrieved from mesometrial grafts can develop to the blastocyst stage and produce live offspring. ICSI can optimize the fertilization rate of in vitro-matured oocytes retrieved from grafts.

Normal developmental competence to the blastocyst stage is preserved in rabbit ovarian tissue following cryopreservation and autografting to the mesometrium

The aim of the present study was to evaluate mesometrial autotransplantation of frozen-thawed ovarian tissue in the adult rabbit and investigate the developmental competence of oocytes retrieved from grafts by in vitro maturation, fertilisation and blastocyst formation. Twenty-five rabbits were divided into control, fresh tissue transplantation and frozen-thawed tissue transplantation groups. Rabbits were stimulated with follicle-stimulating hormone (FSH) and oocytes were retrieved 3 months after transplantation. Oocytes matured in vivo or in vitro were then fertilised by conventional in vitro fertilisation (IVF) or intracytoplasmic sperm injection (ICSI), followed by observation and evaluation of fertilisation and blastocyst formation rates. No significant differences were found in the percentage of oocytes, maturation, fertilisation, cleavage and blastocyst formation among the three groups. Significantly higher fertilisation rates of in vitro-matured (IVM) oocytes were observed with ICSI compared with IVF in each group (81.1% v. 58.5%, 79.2% v. 59.6% and 80.4% v. 56.0% in the control, fresh tissue transplantation and frozen-thawed tissue transplantation groups, respectively). The blastocyst formation rate of IVM oocytes was significantly lower than that of in vivo-matured oocytes in each group (25.5% v. 65.7%, 22.4% v. 61.8% and 28.9% v. 63.0% in the control, fresh tissue transplantation and frozen-thawed tissue transplantation groups, respectively). In conclusion, the mesometrium is a promising site for ovarian autografts in the rabbit. Oocytes retrieved from mesometrial grafts can develop to the blastocyst stage.

Comparison of spindle and chromosome configuration in in vitro- and in vivo-matured mouse oocytes after vitrification

To compare the cytogenetic changes in in vitro- and in vivo-matured oocytes after vitrification. In vitro experiments using murine model. Animal model study in university laboratory. CD-1 mice. In vitro maturation and vitrification of oocytes. Post-warming survival, analysis of spindle and chromosome configurations, aneuploidy screening of parthenogenetically activated oocytes, extent of DNA fragmentation, and early embryonic development after IVF. Eighty percent of germinal vesicle-stage oocytes matured after in vitro maturation and were cryopreserved by vitrification (n = 354). There was no significant difference in the post-warming survival of in vitro- and in vivo-matured oocytes (94.1% vs. 91.8%, respectively). The majority of in vitro- and in vivo-matured oocytes maintained normal meiotic spindle morphology and chromosome alignment (88.2% vs. 86.9%, respectively) after vitrification and the incidence of aneuploidy was not increased (11.5% vs. 9.3%). However, in vitro-matured oocytes showed a higher rate of DNA fragmentation after vitrification compared to in vivo-matured oocytes. After vitrification, the cleavage and blastocyst formation rates of in vitro-matured oocytes were significantly lower than those of in vivo-matured oocytes (37.0% vs. 60.0% and 5.4% vs. 18.9%, respectively). Vitrification of in vitro-matured mouse oocytes results in high survival rates, normal meiotic spindle and chromosome alignment, and no increased incidence of aneuploidy. A possible cause of the reduced developmental competence of in vitro-matured and vitrified oocytes may be due to DNA fragmentation.

Prognostic value of meiotic spindle imaging on fertilization rate and embryo development in in vitro-matured human oocytes

To evaluate the relationship between spindle visualization and intracytoplasmic sperm injection (ICSI) outcomes in controlled ovarian stimulation (COS) cycles. Prospective study. Assisted reproduction center. Thirty patients undergoing ICSI cycles. Meiotic spindle was evaluated before ICSI in 234 in vivo- and 101 in vitro-matured oocytes Meiotic spindle imaging, fertilization rate, and embryo development. Spindle was present in 74.3% and 73.8% of the in vivo- and in vitro-matured oocytes, respectively. Spindle detection rate in oocytes derived from germinal vesicle and metaphase-I stage was, respectively, 50% and 86%. The fertilization rate achieved by the in vivo-matured oocytes was 71.8%, and spindle was detected in 75.6% of the fertilized oocytes and only 34.8.% of the nonfertilized oocytes. In the in vitro-matured oocytes, the fertilization rate was 66.1%, and spindle was detected in 81.4% of the fertilized oocytes and in 59.1% of the nonfertilized oocytes. Ten out of 43 (23.2%) in vitro-matured derived embryos were considered to be high quality, all derived from spindle-detected oocytes, which represents an increase of 13.0% on the overall number of high-quality embryos. Meiotic spindle imaging may be useful to predict in vitro-matured oocyte development. However, other factors may contribute to the decreased developmental competence of in vitro-matured oocytes.

Improvement of canine somatic cell nuclear transfer procedure

The purpose of the present study on canine somatic cell nuclear transfer (SCNT) was to evaluate the effects of fusion strength, type of activation, culture media and site of transfer on developmental potential of SCNT embryos. We also examined the potential of enucleated bovine oocytes to serve as cytoplast recipients of canine somatic cells. Firstly, we evaluated the morphological characteristics of in vivo-matured canine oocytes collected by retrograde flushing of the oviducts 72 h after ovulation. Secondly, the effectiveness of three electrical strengths (1.8, 2.3 and 3.3 kV/cm), used twice for 20 micros, on fusion of canine cytoplasts with somatic cells were compared. Then, we compared: (1) chemical versus electrical activation (a) after parthenogenetic activation or (b) after reconstruction of canine oocytes with somatic cells; (2) culture of resulting intergeneric (IG) embryos in either (a) mSOF or (b) TCM-199. The exposure time to 6-DMAP was standardized by using bovine oocytes reconstructed with canine somatic cells. Bovine oocytes were used for SCNT after a 22 h in vitro maturation interval. The fusion rate was significantly higher in the 3.3 kV/cm group than in the 1.8 and 2.3 kV/cm treatment groups. After parthenogenesis or SCNT with chemical activation, 3.4 and 5.8%, respectively, of the embryos developed to the morula stage, as compared to none of the embryos produced using electrical activation. Later developmental stages (8-16 cells) were transferred to the uterine horn of eight recipients, but no pregnancy was detected. However, IG cloned embryos (bovine cytoplast/canine somatic cell) were capable of in vitro blastocyst development. In vitro developmental competence of IG cloned embryos was improved after exposure to 6-DMAP for 4 h as compared to 0, 2 or 6h exposure, although the increase was not significantly different among culture media. In summary, for production of canine SCNT embryos, we recommend fusion at 3.3 kV/cm, chemical activation, culture in mSOF medium and transfer of presumptive zygotes to the oviduct of recipient animals. The feasibility of IG production of cloned canine embryos using bovine cytoplasts as recipient of canine somatic cells was demonstrated.

Effects of ovarian stimulation, with and without human chorionic gonadotrophin, on oocyte meiotic and developmental competence in the marmoset monkey ( Callithrix jacchus)

A reliable ovarian stimulation protocol for marmosets is needed to enhance their use as a model for studying human and non-human primate oocyte biology. In this species, a standard dose of hCG did not effectively induce oocyte maturation in vivo. The objectives of this study were to characterize ovarian response to an FSH priming regimen in marmosets, given without or with a high dose of hCG, and to determine the meiotic and developmental competence of the oocytes isolated. Ovaries were removed from synchronized marmosets treated with FSH alone (50 IU/d for 6 d) or the same FSH treatment combined with a single injection of hCG (500 IU). Cumulus–oocyte complexes (COCs) were isolated from large (>1.5 mm) and small (0.7–1.5 mm) antral follicles. In vivo-matured oocytes were subsequently activated parthenogenetically or fertilized in vitro. Immature oocytes were subjected to in vitro maturation and then activated parthenogenetically. Treatment with FSH and hCG combined increased the number of expanded COCs from large antral follicles compared with FSH alone (23.5 ± 9.3 versus 6.4 ± 2.7, mean ± S.E.M.). Approximately 90% of oocytes surrounded by expanded cumulus cells at the time of isolation were meiotically mature. A blastocyst formation rate of 47% was achieved following fertilization of in vivo-matured oocytes, whereas parthenogenetic activation failed to induce development to the blastocyst stage. The capacity of oocytes to complete meiosis in vitro and cleave was positively correlated with follicle diameter. A dramatic effect of follicle size on spindle formation was observed in oocytes that failed to complete meiosis in vitro. Using the combined FSH and hCG regimen described in this study, large numbers of in vivo matured marmoset oocytes could be reliably collected in a single cycle, making the marmoset a valuable model for studying oocyte maturation in human and non-human primates.

Oxygen concentration during mouse oocyte in vitro maturation affects embryo and fetal development

Little is known of how the oxygen environment in the ovarian follicle affects oocyte and embryo development, but this has an important impact on the conditions used for in vitro maturation (IVM) of oocytes. We investigated the effect of varying oxygen concentrations during IVM on subsequent pre and post-implantation development. IVM of mouse cumulus-oocyte complexes (COCs) was performed under 2, 5, 10 or 20% O(2) (6% CO(2), balance N(2)). In vivo-matured COCs were collected post ovulation. Embryos were generated by IVF and culture. Blastocyst development, cell number and apoptosis were assessed, and fetal and placental outcomes analysed following embryo transfer at day 18 of pregnancy. Oxygen concentration during IVM did not affect oocyte maturation or subsequent fertilization, cleavage and blastocyst development rates. Maturation of oocytes under 2% O(2) increased blastocyst trophectoderm cell number compared with all groups and numbers at 5% were higher than 20% (both P < 0.05). Percentage of apoptotic cells was increased in blastocysts developed from 2% O(2)-matured oocytes, compared with maturation at 5% O(2) or in vivo (P < 0.05). Rates of embryo implantation and development into a viable fetus were not altered by IVM oxygen. However, fetal weight was reduced following oocyte maturation at 5% O(2) compared wiht 20% O(2) and maturation at 5% O(2) also reduced placental weight, when compared with in vivo-matured oocytes (both P < 0.05). Level of O(2) exposure during oocyte maturation can alter the cellular composition of blastocysts, but these changes in cell number do not correlate with the altered fetal and placental outcomes after transfer.

Toward a Feline-Optimized Culture Medium: Impact of Ions, Carbohydrates, Essential Amino Acids, Vitamins, and Serum on Development and Metabolism of In Vitro Fertilization-Derived Feline Embryos Relative to Embryos Grown In Vivo1

The objective of this study was to define the physiologic needs of domestic cat embryos to facilitate development of a feline-specific culture medium. In a series of factorial experiments, in vivo-matured oocytes (n = 2040) from gonadotropin-treated domestic cats were inseminated in vitro to generate embryos (n = 1464) for culture. In the initial study, concentrations of NaCl (100.0 vs. 120.0 mM), KCl (4.0 vs. 8.0 mM), KH(2)PO(4) (0.25 vs. 1.0 mM), and the ratio of CaCl(2) to MgSO(4)-7H(2)O (1.0:2.0 mM vs. 2.0:1.0 mM) in the medium were evaluated during Days 1-6 (Day 0: oocyte recovery and in vitro fertilization [IVF]) of culture. Subsequent experiments assessed the effects of varying concentrations of carbohydrate (glucose, 1.5, 3.0, or 6.0 mM; l-lactate, 3.0, 6.0, or 12.0 mM; and pyruvate, 0.1 or 1.0 mM) and essential amino acids (EAAs; 0, 0.5, or 1.0x) in the medium during Days 1-3 and Days 3-6 of culture. Inclusion of vitamins (0 vs. 1.0x) and fetal calf serum (FCS; 0 vs. 5% [v/v]) in the medium also was evaluated during Days 3-6. Development and metabolism of IVF embryos on Day 3 or Day 6 were compared to age-matched in vivo embryos recovered from naturally mated queens. A feline-optimized culture medium (FOCM) was formulated based on these results (100.0 mM NaCl, 8.0 mM KCl, 1.0 mM KH(2)PO(4), 2.0 mM CaCl(2), 1.0 mM MgSO(4), 1.5 mM glucose, 6.0 mM L-lactate, 0.1 mM pyruvate, and 0x EAAs with 25.0 mM NaHCO(3), 1.0 mM alanyl-glutamine, 0.1 mM taurine, and 1.0x nonessential amino acids) with 0.4% (w/v) BSA from Days 0-3 and 5% FCS from Days 3-6. Using this medium, ~70% of cleaved embryos developed into blastocysts with profiles of carbohydrate metabolism similar to in vivo embryos. Our results suggest that feline embryos have stage-specific responses to carbohydrates and are sensitive to EAAs but are still reliant on one or more unidentified components of FCS for optimal blastocyst development.

59 IN VITRO DEVELOPMENT OF FELINE CLONED EMBRYOS RECONSTRUCTED WITH PREADIPOCYTES

The technique of somatic cell nuclear transfer (NT) in domestic cats is expected to contribute to the conservation of wildcats, for which extinction is a concern. In this study, we examined in vitro developmental ability of cloned embryos produced using the preadipocytes of domestic cats as nuclear donors. Primary cultures of preadipocytes were established as reported previously (Yagi et al. 2004 Biochem. Biophys. Res. Commun. 321, 967–974). Briefly, fat tissue (2–3 g) was excised from an adult female cat and digested using 0.1% collagenase for 1 h at 37�C followed by centrifugation. Only mature adipocytes that were floating near the surface of the supernatant were collected and placed in a 12.5-cm2 culture flask filled with DMEM containing 20% FBS. The flask was filled with medium, tightly capped, and cultured upside down for 7–10 days, so that the floating adipocytes attached to the inner ceiling surface of the flask. When firm attachment of the cells to the ceiling surface of the flask was confirmed, the flask was then inverted and culture was continued using the routine cell culture technique for adherent cells. In vivo-matured oocytes were collected from the ovaries of domestic cats superovulated by eCG and hCG. IVM oocytes were obtained by culturing cumulus–oocyte complexes from the ovaries collected at local veterinary clinics in TCM199-based medium for 24 to 30 h. In vivo-matured and IVM oocytes were enucleated by aspirating the first polar body and adjacent cytoplasm using a bevelled pipette in the presence of 7.5 µg mL–1 cytochalasin B. The nuclei of the donor cells were transferred to enucleated in vivo-matured and IVM oocytes by means of electric fusion (300 V mm–1, 30 µs, twice). The reconstructed embryos were activated electrically (125 V mm–1, 60 µs, twice), followed by treatment with 10 µg mL–1 cycloheximide and 5 µg mL–1 cytochalasin B. The cloned embryos were cultured in vitro for 7 days in MK-1 so that their developmental ability might be examined. The fusion rate of donor cells was similar between in vivo-matured and IVM oocytes (56.8%, 21/37 v. 54.5%, 42/77). The developmental ability of NT embryos reconstructed with in vivo-matured oocytes was similar to that of NT embryos reconstructed with IVM oocytes (cleavage: 52.4%, 11/21 v. 42.9%, 18/42; development to blastocysts: 9.5%, 2/21 v. 11.9%, 5/42). The results indicate that cloned feline embryos reconstructed using preadipocytes can develop in vitro into blastocysts.

3 IN VITRO MATURATION ALTERS GENE EXPRESSION IN MOUSE OOCYTES

The in vitro culture environment significantly impacts nuclear maturation, fertilization, embryonic development, and epigenetic competence; however, our knowledge of the effects of in vitro maturation on oocyte developmental competence, and specifically cytoplasmic maturation, is limited. The objective of this experiment was to identify alterations in the transcriptome of oocytes matured in vitro compared to those matured in vivo that correlate to developmental competence. Immature oocytes were collected from Day 26 and 7-8-week-old B6D2F1 mice 48 h post-pregnant mare serum gonadotropin (PMSG) administration and matured for 16 h in Gmat supplemented with 0.5 mm citric acid, 0.5 mm cysteamine, 100 ng mL–1 epidermal growth factor (EGF), 0.05% insulin-transferrin-selenium (ITS; v/v), 0.01% recombumin (v/v) and 2 mg mL–1 fetuin. In vivo-matured oocytes from females of the same ages were collected from the oviducts 62 h post-PMSG and 14 h post-hCG and mating to vasectomized males. In vivo- and in vitro-matured oocytes were identified visually by the presence of the first polar body. Mature oocytes were pooled into three groups of 150 oocytes per treatment and lysed; poly A+ RNA was extracted. Samples were processed through two cycles of linear amplification and hybridized to the GeneChip� Mouse Genome 430 2.0 Array (Affymetrix, Inc., Santa Clara, CA, USA), with three arrays per treatment. Microarray data were sorted and filtered to include genes that were classified as having two present calls per treatment. The data were then normalized to the chip median and analyzed using a one-way analysis of variance; the level of significance was calculated at P &lt; 0.01. In total, 2.17% (482/22170) and 1.61% (358/22170) of genes were differentially expressed between in vitro- and in vivo-matured oocytes in Day 26 and 7–8-week-old mice, respectively. However, 72.82% (351/482) and 67.87% (243/358) of differentially expressed genes had increased abundance in the in vitro- and in vivo-matured oocytes, respectively. Transcripts involved in gene expression, cellular growth and proliferation, and cellular development were increased in in vivo-matured oocytes from both age groups compared to those matured in vitro. Cell death was one of the higher ranking functional groups increased in the 7–8-week-old in vitro-matured oocytes compared to the 7–8-week-old in vivo-matured oocytes. Specific genes altered by in vitro maturation conditions in Day 26 oocytes were DNA methyltransferase 1 (&gt;7-fold increase in vivo), caspase 8 (&gt;4-fold increase in vivo), and eukaryotic translation initiation factor 1B (&gt;4-fold increase in vivo). DNA methyltransferase 1 and ubiquitin-conjugating enzyme E2T were significantly increased in in vivo-matured 7–8-week-old oocytes (&gt;3-fold and &gt;5-fold, respectively). These results indicate that gene expression is altered in oocytes matured in vitro compared to those matured in vivo. Based on the functional annotations of genes differentially expressed, dysregulation of gene expression in the oocyte resulting in altered DNA methylation and an up-regulation in cell death pathways are potential developmental mechanisms influenced by in vitro culture conditions that correlate to reduced embryonic developmental potential.

267 BIRTH OF DOMESTIC CAT KITTENS OF PREDETERMINED SEX AFTER TRANSFER OF EMBRYOS PRODUCED BY IN VITRO FERTILIZATION OF OOCYTES WITH FLOW-SORTED SPERMATOZOA

In cats, sex selection by fertilization of oocytes with sperm sorted into X- andY-chromosome-bearing populations has credible biomedical, commercial, and conservation connotations. Our objectives were (1) to evaluate the efficiency with which embryos could be produced by IVF of in vivo- and in vitro-matured oocytes with cooled sex-sorted sperm after overnight shipment to the sorting facility and overnight return delivery to an IVF laboratory, and (2) to determine if live kittens of predetermined sex (female) could be produced after transfer of embryos derived by IVF of in vivo-matured oocytes with X-chromosome-bearing sperm to recipient females. Semen samples (n = 5) collected from a single male using an artificial vagina were extended in electrolyte-free solution (glucose–BSA) and shipped overnight in an Equitainer (4�C) to the sorting facility. Upon arrival, sperm were stained (9 µm Hoechst 33 342; 75 � 106 sperm mL–1), adjusted to 50 � 106 sperm mL–1 with 4% egg yolk TALP containing 0.002% food dye, and sorted on an SX MoFlo� flow cytometer (Dako, Fort Collins, CO, USA). Purities from resort analysis averaged 94% (X) and 83% (Y). After sorting, sperm were concentrated by centrifugation, suspended in TEST-yolk buffer (Irvine Scientific, Santa Ana, CA, USA), and return-shipped to the IVF lab where they were received 48 h after collection. In vivo-matured oocytes recovered from gonadotropin-treated donors were inseminated in vitro with X-chromosome-bearing sperm. In vitro-matured oocytes obtained from ovaries donated by local clinics were inseminated in vitro with control (cooled/shipped/non-sorted) or X- orY-chromosome-bearing sperm. At 5 h or 18 h post-insemination, in vivo- and in vitro-matured oocytes, respectively, were rinsed and placed in IVC-1 medium (Pope et al. 2006 Theriogenology 66, 59–71). Embryos produced from in vitro-matured oocytes were allowed to develop until Day 8 in a three-step culture system (Pope et al. 2006). Cleavage frequency of in vitro-matured oocytes after insemination with X-, Y-, and control sperm was 33% (40/120), 35% (52/150), and 42% (48/115), and blastocyst development was 50% (11/22), 55% (23/42), and 53% (21/40), respectively (P &gt; 0.05). Incidence of cleavage after insemination of in vivo-matured oocytes with X-sperm was 62% (54/87). On Day 2, 45 embryos (9–16 per recipient) produced by in vitro insemination of in vivo-matured oocytes with X-sperm were transferred by laparoscopy to the oviducts of four Day 1 gonadotropin-treated recipients. Three recipients established pregnancies and delivered litters of one, four, and seven female kittens between Day 62 and Day 66 of gestation. We have demonstrated that sperm-sorting technology can be applied and used effectively in domestic cats and, potentially, should be relevant to the selective breeding of endangered cats.

243 NUCLEOSIDES REDUCE THE POTENTIAL OF THE MITOCHONDRIAL INNER MEMBRANE AND THE DEVELOPMENTAL COMPETENCE OF MOUSE OOCYTES DURING IN VITRO MATURATION

Although nucleosides such as adenosine exist in follicular fluid and decrease during oocyte maturation (Eppig et al. 1985 Biol. Reprod. 33, 1041–1049), the role of nucleosides is still unclear. The present study was undertaken to determine the effect of nucleoside on nuclear and cytoplasmic maturation of mouse oocytes. Oocyte–cumulus complexes (OCCs)were collected from the large antral follicles of C57BL/6J female mice (3–5 weeks old) 4 h after a combination injection of equine chorionic gonadotropin (eCG) and hCG with a 48-h interval, and cultured in maturation medium (αMEM containing 3 mg mL–1 BSA, 0.23 mm Na pyruvate and antibiotics) with or without ribo- and deoxyribo-nucleosides (10–11 µg mL–1) for 12 h. Statistical analyses in this study were carried out by ANOVA and Bonferroni/Dunn's post hoc test. Regardless of the presence of nucleosides, a majority of oocytes developed to the metaphase-II stage in vitro, and the incidence was not different with in vivo-matured oocytes. However, the mitochondrial membrane potential (MMP) of oocytes was significantly lower when the oocytes were matured in the presence of nucleosides, as compared with nucleoside-free controls. In the scanning experiment of MMP level during in vitro maturation (IVM), the MMP of oocytes maturing in vivo increased between 8 and 12 h after hCG injection, whereas no rises of MMP was observed in oocytes matured in vitro in the presence of nucleosides. To examine the affecting period of nucleosides, OCCs were cultured for the first 4 h or the latter 8 h of IVM in the presence of nucleosides. MMP of the oocytes was significantly lower only when the OCCs were exposed to nucleosides for the latter 8 h of IVM. To determine if adenosine in the nucleosides (10 µg mL–1) affects the MMP of oocytes, OCCs were exposed to adenosine or a mixture of guanosine, cytidine, and uridine during the latter 8 h of IVM. The MMP of oocytes exposed to adenosine was lower than that of in vivo-matured oocytes and of oocytes exposed to a mixture of guanosine, cytidine, and uridine. To determine the effect of nucleosides on the developmental competence, oocytes exposed to adenosine during the latter 8 h of IVM were cultured in kSOMaa containing 1 mg mL–1 BSA after activation in the presence of 10 mm SrCl2 and 5 µg mL–1 cytochalasin B for 6 h. Pronuclear formation and early development of those were compared with artificially activated oocytes matured in vivo or in the absence of nucleosides. The incidences of pronuclear formation and cleavage of oocytes matured in the presence of adenosine following activation were extremely decreased, as compared with control oocytes matured in vivo or in the absence of adenosine. These observations indicate that nucleosides, at least adenosine, inhibit an increase of MMP of oocytes during the latter half of meiotic maturation, and detrimentally affect the developmental competence of murine oocytes.

74 IN VIVO SURVIVAL OF DOMESTIC CAT OOCYTES AFTER VITRIFICATION, INTRACYTOPLASMIC SPERM INJECTION, AND TRANSFER TO RECIPIENTS

The ability to cryopreserve female gametes efficiently holds immense economic and genetic implications. The purpose of the present project was to determine if domestic cat oocytes could be cryopreserved successfully by use of the Cryotop method. We evaluated (a) cleavage frequency after in vitro fertilization (IVF) v. intracytoplasmic sperm injection (ICSI) of in vivo- and in vitro-matured oocytes after vitrification, and (b) fetal development after transfer of resultant embryos into recipients. In vivo-matured cumulus–oocyte complexes (COCs) were recovered from gonadotropin-treated donors at 24 h after LH treatment, denuded of cumulus cells, and examined for the presence of the first polar body (PB). In vitro-matured COCs were obtained from ovaries donated by local clinics and placed into maturation medium for 24 h before cumulus cells were removed and PB status was determined. Oocytes were cryopreserved by the Cryotop method (Kuwayama et al. 2005 Reprod. Biomed. Online 11, 608–614) in a vitrification solution consisting of 15% DMSO, 15% ethylene glycol, and 18% sucrose. For IVF, oocytes were co-incubated with 1 � 106 motile spermatozoa mL–1 in droplets of modified Tyrode's medium in 5% CO2/air at 38�C (Pope et al. 2006 Theriogenology 66, 59–71). For ICSI, an immobilized spermatozoon was loaded into the injection pipette, which was then pushed through the zona pellucida into the ooplasm. After a minimal amount of ooplasm was aspirated into the pipette, the spermatozoon was carefully expelled, along with the aspirated ooplasm. After ICSI, or at 5 or 18 h post-insemination, in vivo- and in vitro-matured oocytes, respectively, were rinsed and placed in IVC-1 medium (Pope et al. 2006). As assessed by normal morphological appearance after liquefaction, the survival rate of both in vivo- and in vitro-matured oocytes was &gt;90% (93–97%). For in vitro-matured oocytes, cleavage frequencies after IVF of control and vitrified oocytes were 73% (16/22) and 53% (30/57), respectively, as compared to 68% (19/28) after ICSI of vitrified oocytes (P &gt; 0.05). For in vivo-matured oocytes, cleavage frequencies after IVF of control and vitrified oocytes were 55% (18/33) and 35% (6/17), respectively, compared to 50% (10/20) after ICSI of vitrified oocytes (P &gt; 0.05). At 18–20 h after ICSI, 18 presumptive zygotes and four 2-cell embryos derived from vitrified in vitro-matured oocytes and 19 presumptive zygotes produced from seven in vivo-matured and 12 in vitro-matured vitrified oocytes were transferred by laparoscopy into the oviducts of two recipients at 24–26 h after oocyte retrieval. The two recipients were 9-month-old IVF/ET-derived females produced with X-sperm sorted by flow cytometry. At ultrasonography on Day 22, both recipients were pregnant, with three live fetuses observed in one recipient and one live fetus seen in the second recipient. On Day 63 and Day 66 of gestation, four live kittens were born, without assistance, to the two recipients. The one male and three female kittens weighed an average of 131 g. In summary, in vivo viability of zygotes/embryos produced by ICSI of cat oocytes vitrified by the Cryotop method was demonstrated by the birth of live kittens following transfer to recipients.

7 EFFECT OF DENSITY GRADIENT CENTRIFUGATION WITH TRYPSIN ON THE FERTILIZING CAPABILITY OF BOVINE SPERM

The goal of this research was to investigate the effect of a novel density gradient centrifugation (DGC) treatment on the fertilizing capability of bovine sperm as compared to a standard method. Domestic bull (Bos taurus) semen was used for AI and the production of embryos from in vivo-matured bovine oocytes. In 2004, a preliminary study compared the novel semen treatment using a trypsinized PVP-coated silica particle suspension (Percoll; Sigma, St. Louis, MO, USA) to a standard method (4� dilution with an egg yolk diluent) on the fertilizing capacity in vivo of bovine sperm (de la Rey et al. 2005 J. Reprod. Fertil. 17, 242 abst). Although not statistically significant (P = 0.69), there were more transferable quality embryos recovered from cows inseminated using the treated sperm method v. control (58.9 v. 43%, respectively). In this report we provide the results of two additional trials utilizing the novel semen treatment and substituting Percoll with a silane-coated silica particle medium containing a recombinant trypsin (r-protease). In the second trial (2005), semen samples collected from three bulls were processed by DGC: 2 mL of 40% PureSperm (NidaCon International AB, M�lndal, Sweden) containing recombinant trypsin (TrypLE Select, Gibco/Invitrogen, Carlsbad, CA, USA), which overlaid 2 mL of 80% PureSperm containing 10 µg mL–1 soy-based protease inhibitor (Sigma), was overlaid with the semen sample using a novel centrifuge tube insert (ProInsert, Nidacon) and then centrifuged at 300g for 20 min. The sperm pellets were recovered and washed (500g for 10 min) in 10 mL pre-warmed TL-HEPES medium (Cambrex Corp., East Rutherford, NJ, USA). The washed sperm pellets were then resuspended in the same total volume of pre-warmed Biladyl� A (Minit�b, Tiefenbach, Germany) as the standard method and used to AI a total of 42 (control) and 47 (treatment) superovulated cows three times at 12 h intervals. Day 7 embryos were recovered and assessed for stage and morphological quality. In Trial 3 (2006), semen samples collected from three bulls were processed by DGC containing r-protease and a soybean protease inhibitor (BoviPure Pro, NidaCon), media specifically formulated for domestic bull semen. Sperm pellets were washed in 10 mL BoviWash medium (NidaCon). The washed sperm pellet was resuspended in the same total volume of pre-warmed Biladyl A as the standard method and used to AI a total of 23 (control) and 25 (treatment) superovulated cows and embryos evaluated as in Trial 2. The results between control and treated groups were compared using the Mann-Whitney (Wixcoxon rank sum) test. Trial 2 using PureSperm tended to result in higher fertilization rates than for cows inseminated using the standard method (75.2% v. 67%, respectively) but the results were not statistically significant (P = 0.63). Results for Trial 3 indicated that cows inseminated with BoviPure Pro-treated sperm had significantly increased fertilization rates as compared to the standard method (88.4% v. 63.1%, respectively; P = 0.02) and had higher numbers of transferable quality embryos (70.3% v. 51.8%, respectively; P = 0.38). In summary, BoviPure Pro sperm treatment before AI significantly increases fertilization rates and can result in as much as an 18.5% increase in transferable quality embryos as compared to standard methods.

36 CLONED BEAGLE DOGS DERIVED FROM FETAL FIBROBLASTS BY NUCLEAR TRANSFER

Somatic cell nuclear transfer (SCNT) has been successfully performed in various mammals including sheep, cow, pig, and mouse using a variety of somatic cell types as nuclear donors. Several reports of livestock SCNT indicate that fetal fibroblasts are superior to adult fibroblasts as donor cells. In canine SCNT, however, only adult ear fibroblasts have been used as donor cells (Lee et al. 2005 Nature 436, 641; Jang et al. 2007 Theriogenology 67, 941–947). Accordingly, in the present study, we evaluated the ability of canine fetal fibroblasts to support fetal development to term after nuclear transfer. For SCNT, in vivo-matured oocytes flushed (approximately 72 h after ovulation) from the oviducts of six estrus females were used. Donor cells (fetal fibroblasts) were isolated from the fetus of a beagle bitch obtained at 28 days after artificial insemination. Before using fetal fibroblasts as donor cells, sex was determined by SRY gene detection using PCR. Oocytes were enucleated, microinjected with a female fetal fibroblast, fused by electrical stimulation, and activated chemically (Jang et al. 2007). A total of 50 cloned presumptive embryos were transferred (Day 0) into the oviducts of two naturally synchronous recipient bitches. One pregnancy, detected by ultrasonography on Day 23, was maintained to term and two healthy female puppies weighing 250 and 260 g were born by natural delivery on Day 60. They were genotypically identical to the donor cells, and had phenotypically similar black and white coat color patterns. Analysis of their mtDNA distribution showed that mtDNA in the two cloned beagles originated from one of the six oocyte donor dogs. In conclusion, our results demonstrate the potential of using fetal fibroblasts to facilitate nuclear transfer in the dog. The cloned beagle dogs, which had identical nucleus and mitochondrial DNA, will be provided for biomedical research as bioresources. This study was financially supported by KOSEF (grant # M10625030005-07N250300510) and the Korean MOE, through the BK21 program for Veterinary Science.

Present Status and Prospects for Bovine Intracytoplasmic Sperm Injection with In Vitro-Matured Oocytes and Frozen Semen

In general, in vitro-matured oocytes and commercially available frozen semen are used for bovine intracytoplasmic sperm injection (ICSI), unlike ICSI in humans and experimental animals, which uses in vivo-matured oocytes and fresh semen. Bovine ICSI is also characterized by difficulties in pronuclear formation. Therefore, increasing in vitro development of produced ICSI embryos is considered to necessitate use of an artificial activation treatment after injection of motile sperm. However, because parthenogenetic embryos are found amongst produced ICSI embryos, it is necessary to establish a bovine ICSI protocol that leads to normal karyomorphism.

The use of transmission electron microscopy and oocyte transfer to evaluate in vitro maturation of equine oocytes in different culture conditions

The current study evaluates the ability of equine oocytes matured in different conditions to undergo nuclear and cytoplasmic maturation. After oocyte transfer, embryonic development was diagnosed at 15 and 90 days of gestation. For each group, immature oocytes obtained from slaughterhouse ovaries were matured in vitro (5 replicates). In experiment I, three different media were tested, HTF:BME, SOFaa, and TCM 199. In experiment II, the HTF:BME was chosen as maturation medium containing pFSH, eFSH, or eFSH + eGH. Nuclear maturation was estimated after stripping the oocytes and staining with Hoechst 33342. The evaluation of cytoplasmic maturation was performed by transmission electron microscopy. For oocyte transfer, six non-cycling recipient mares were used, and 8 to 15 oocytes were transferred in each mare. In experiment I, the results showed no differences ( P > .05) in nuclear maturation (MII) among experimental groups. The percentage of MII was 29.3 (±9.6), 23.4 (±8.4), and 13.5 (±12.4) for HTF:BME, SOF, and TCM, respectively. In experiment II, all media tested were efficient in inducing metaphase II. Also, no statistical differences ( P > .05) were observed in percentages of nuclear maturation rates when porcine (37.1 ± 22.4) or equine (25.8 ± 8.2) FSH were used, or when eFSH + eGH was added to HTF:BME (29.4 ± 12.3). The analysis of cytoplasmic morphology of oocytes cultured in TCM 199 and SOFaa showed signs of incomplete cytoplasmic maturation and premature cortical reaction. Meanwhile, oocytes cultured in HTF:BME medium presented cytoplasmic characteristics similar to those described by others for in vivo-matured oocytes. The addition of eFSH to the HTF:BME medium resulted in an improvement of cytoplasmic morphology. After oocyte transfer, two mares became pregnant, one from pFSH group and one from eFSH+eGH group. These results indicate that although in vitro matured equine oocytes are capable of fertilization and embryonic development, the percentage of competent oocytes is still low.

372 SPERM ASTER FORMATION AND BLASTOCYST DEVELOPMENT OF IN VIVO-MATURED BOVINE OOCYTES FERTILIZED BY INTRACYTOPLASMIC SPERM INJECTION

In cattle, activation treatment after intracytoplasmic sperm injection (ICSI) is required to improve cleavage and blastocyst rates (Horiuchi et al. 2002 Theriogenology 57, 1013–1024). The reason why the exogenous activation treatment in bovine ICSI is needed to promote cleavage and blastocyst development is not clear. The objective of this study was to examine the effect of activation treatment on sperm aster formation, cleavage, and blastocyst development of in vivo- and in vitro-matured bovine oocytes following ICSI. In vivo-matured oocytes were collected using transvaginal devices under ultrasound guide at about 29 h after GnRH injection from Japanese Black cows superstimulated with a total 19 mg FSH (Antrin�; Denka Pharmaceutical Co., Kanagawa, Japan) divided into twice daily over 3 days, and treated with 750 �g cloprostenol (Estramate�; Sumitomo Chemical Co., Tokyo, Japan). In a total of 8 aspiration sessions, 131 oocytes were collected; of 116 oocytes with expanded cumulus cells, 84 (72%) had a first polar body and were used for ICSI. On the other hand, in vitro-matured bovine oocytes were prepared by culturing immature follicular oocytes derived from abattoir ovaries. Bull spermatozoa, immobilized by scoring their tails, were injected into in vivo- or in vitro-matured oocytes. At 4 h after ICSI, the oocytes were treated with or without 7% ethanol for 5 min for activation. The injected oocytes were fixed at 8 h after ICSI, and sperm aster formation was examined by using specific antibodies and immunofluorescence microscopy. Data were analyzed by the chi-square test in all experiments. The rate of sperm aster formation in in vivo-matured oocytes was similar regardless of activation treatment (71% vs. 65%), but the rate in in vitro-matured oocytes was significantly (P &amp;lt; 0.05) higher in the group receiving activation treatment than in the non-activation group (57% vs. 19%). Cleavage (88% vs. 88%) and blastocyst rates (59% vs. 47%) of in vivo-matured oocytes after ICSI were also similar, regardless of activation treatment, but cleavage (72% and 20%) and blastocyst rates (19% and 7%) of in vitro-matured oocytes were significantly (P &amp;lt; 0.05) higher in the group receiving activation treatment than in the non-activation group. Moreover, the blastocyst rate of in vivo-matured oocytes was significantly (P &amp;lt; 0.05) higher than the rate in in vitro-matured oocytes. These results show that activation treatment after ICSI of in vivo-matured bovine oocytes is not necessary for cleavage and blastocyst development, and suggest that the necessity of activation treatment in bovine ICSI has relevance to in vitro maturation of bovine oocytes.

270 IN VITRO MATURATION ALTERS THE EXPRESSION OF SPECIFIC PUTATIVELY IMPRINTED GENES IN BOVINE MII OOCYTES

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 &amp;lt; 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.