Comparative evaluation of the cost and efficiency of four types of sexing methods for the production of dairy female calves.

This study was conducted to evaluate and compare the economic benefits of different embryo sexing methods, based on the cost per female dairy calf produced. Female calves were produced from four kinds of female embryos: (1) those collected from superstimulated donors at 7-8 days after artificial insemination (AI) with X-sorted semen; (2) those sex-determined by loop-mediated isothermal amplification assay of a biopsy sample of embryos collected from superstimulated donors after AI with conventional unsorted semen; (3) those obtained by invitro embryo production (IVEP), using X-sorted semen and in vitro-matured oocytes collected from donors by ovum pick-up (OPU); and (4) those obtained by IVEP, using X-sorted semen and oocytes collected by OPU after dominant follicle ablation and follicle growth stimulation of the donors. The respective productivities of female calves per technical service and the total production cost per female calf of each sexing method were compared. The production cost per female calf (66,537 JPY), as calculated from the number of female calves per service (1.30), pregnancy rate of transfer (42.9%), rate of female calves obtained (92.9%), and total cost of the method (56,643 JPY plus embryo transfer fee), was less for IVEP with X-sorted semen and follicular growth-stimulated (FGS) oocytes than for the other groups (P < 0.05). The results demonstrate that embryo production with X-sorted semen and FGS oocytes provides a more efficient method for producing female calves than the other embryo sexing methods.

Microtubule stabilisers docetaxel and paclitaxel reduce spindle damage and maintain the developmental competence of in vitro-mature bovine oocytes during vitrification.

This study compared the efficacy of docetaxel (DT) and paclitaxel (PT) in reducing spindle damage during vitrification and maintaining the developmental competence of in vitro-matured (IVM) bovine oocytes after vitrification and warming. Pretreatment of IVM oocytes with 0.05µM DT for 30min before vitrification resulted in significantly higher (P<0.05) rates of oocyte survival and cleavage after IVF, as well as subsequent blastocyst rates on Days 7-9 and hatching on Days 8-9, compared with oocytes pretreated with 1.0µM PT before vitrification or those vitrified without pretreatment. When nuclear status and spindle morphology of vitrified oocytes were assess after warming by immunostaining, DT pretreatment before vitrification resulted in a significantly higher (P<0.05) percentage of oocytes at the MII stage with a normal, intact spindle compared with PT pretreatment or no pretreatment, but the percentage of MII oocytes was still significantly lower (P<0.05) than in the control group. Pretreatment of IVM bovine oocytes with 0.05µM DT or 1.0µM PT for 30min before vitrification reduces spindle damage to the same extent, without side effects on fertilisation and development. Pretreatment with 0.05µM DT improved the developmental competence of vitrified-warmed oocytes to a greater degree than 1.0µM PT pretreatment.

Effect of storage tube material and resveratrol during liquid storage of matured bovine oocytes on subsequent development.

This study determined the optimum storage vessel and the effects of resveratrol for the storage of in vitro matured (IVM) bovine oocytes. After IVM, the oocytes were kept in a Hepes-buffered medium at 25 °C for 20 h in different containers including Eppendorf tubes (ET) made of polypropylene (PP) and polystyrene (PS), and tissue culture tubes (TCT) made of PP, PS, and glass. Then oocytes were subjected to IVF and subsequent in vitro embryo development was compared among the groups and to that of a control group without storage. The percentage of blastocyst development in the control group was significantly higher than in the stored groups (P < 0.05). Among oocytes stored in TCT, the percentage of blastocyst development of oocytes stored in glass TCT was significantly higher than that of oocytes stored in PP and PS TCT (P < 0.05); however, it did not differ from that of oocytes stored in ET. The quality of blastocysts did not differ among the control and stored groups. Embryo development was not affected when 0.1, 1 or 10 µM resveratrol was added to the medium during oocyte storage. In conclusion, glass tubes were optimal for oocyte storage and resveratrol did not improve the development of stored oocytes.

Cryopreservation method affects DNA fragmentation in trophectoderm and the speed of re-expansion in bovine blastocysts.

This study investigated re-expansion dynamics during culture of bovine blastocysts cryopreserved either by slow-freezing or vitrification. Also, the extent and localization of membrane damage and DNA fragmentation in re-expanded embryos were studied. Frozen-thawed embryos showed a significantly lower re-expansion rate during 24 h of post-thawing culture compared to vitrified embryos. Vitrified embryos reached the maximum level of re-expansion rate by 12 h of culture whereas frozen embryos showed a gradual increase in re-expansion rate by 24 h of culture. When assayed by Hoechst/propidium iodide staining there was no difference in the numbers and ratio of membrane damaged cells between re-expanded frozen and vitrified embryos; however, the extent of membrane damage in blastomeres was significantly higher in both groups compared with non-cryopreserved embryos (control). TUNEL assay combined with differential ICM and TE staining revealed a significantly higher number and ratio of TE cells showing DNA-fragmentation in frozen-thawed re-expanded blastocysts compared to vitrified ones; however, vitrification also resulted in an increased extent of DNA fragmentation in TE cells compared with control blastocysts. In frozen-thawed blastocysts increased extent of DNA fragmentation was associated with reduced numbers and proportion of TE cells compared with vitrified and control embryos. The number and ratio of ICM cells and the extent of DNA fragmentation in ICM did not differ among control, frozen and vitrified groups. In conclusion, compared with vitrified embryos, blastocysts preserved by slow-freezing showed a delayed timing of re-expansion which was associated with an increased frequency of DNA fragmentation in TE cells.

Sex-sorting of spermatozoa affects developmental competence of in vitro fertilized oocytes in a bull-dependent manner.

The aim of the present study was to clarify if flow-cytometric sex-sorting of bovine sperm affected in vitro blastocyst production in different bulls, either in terms of its ability to fertilize the oocyte or by interfering with post-fertilization embryo development. We performed in vitro fertilization (IVF) using both commercially available frozen-thawed X-sorted and non-sorted sperm of 4 Holstein bulls at 3 concentrations (1 × 106, 2 × 106, and 5 × 106 sperm/ml). When fertilization rates were compared, a variation in fertilization rates among different sperm concentrations was detected in 2 bulls, with similar results for X-sorted and non-sorted sperm. However, we found no evidence that the fertilization rates were affected by the sorting process. To investigate effects on embryo development, we determined the optimum sperm concentration for IVF in each bull, which resulted in similar fertilization rates among bulls. We next performed IVF using both X-sorted and non-sorted sperm of the 4 bulls at their optimum sperm concentration and compared in vitro embryo development. Cleavage rates with X-sorted sperm were similar to their non-sorted counterparts. However, significantly reduced blastocyst development was associated with the use of X-sorted sperm in one bull, whereas in the other three bulls, blastocyst development after IVF with X-sorted and non-sorted sperm was similar. In conclusion, in our system, X-sorting affects in vitro blastocyst production by reducing the developmental competence of fertilized oocytes rather than affecting the fertilization ability of the sperm. However, the occurrence of this phenomenon varies among bulls.

Pretreatment of bovine sperm with dithiobutylamine (DTBA) significantly improves embryo development after ICSI.

We assessed the effect of pretreating sperm with dithiobutylamine (DTBA) to improve embryo development by intracytoplasmic sperm injection (ICSI) in cows. Acridine Orange staining revealed that when applied at different concentrations (2.5, 5, and 10 mM) and exposure times (5 min, 20 min, 1 h, and 2 h), DTBA reduced disulfide bonds in spermatozoa with the highest efficacy at 5 mM for 5 min. DTBA enhanced the percentage of spermatozoa with free protamine thiol groups compared with untreated spermatozoa (control) (P < 0.05); however, this result did not differ from that of dithiothreitol (DTT) treatment. The percentage of live spermatozoa after DTBA treatment was identical to that in the control, but significantly higher than that after DTT treatment (P < 0.05). After ICSI, DTBA treatment tended to improve male pronuclear formation rate (P = 0.071) compared with non-treated sperm injection. Blastocyst formation rate was significantly improved by DTBA treatment compared with that in DTT, control, and sham injection groups (P < 0.05). Blastocyst quality in terms of cell numbers and ploidy was not different among these groups. In conclusion, DTBA increases the efficacy of blastocyst production by ICSI even if DTT treatment does not work.

Abnormal chromosome segregation at early cleavage is a major cause of the full-term developmental failure of mouse clones.

To clarify the causes of the poor success rate of somatic cell nuclear transfer (SCNT), we addressed the impact of abnormalities observed at early cleavage stages of development on further full-term development using 'less-damage' imaging technology. To visualize the cellular and nuclear division processes, SCNT embryos were injected with a mixture of mRNAs encoding enhanced green fluorescent protein coupled with α-tubulin (EGFP-α-tubulin) and monomeric red fluorescent protein 1 coupled with histone H2B (H2B-mRFP1) and monitored until the morula/blastocyst stage three-dimensionally. First, the rate of development of SCNT embryos and its effect on the full-term developmental ability were analyzed. The speed of development was retarded and varied in SCNT embryos. Despite the rate of development, SCNT morulae having more than eight cells at 70h after activation could develop to term. Next, chromosomal segregation was investigated in SCNT embryos during early embryogenesis. To our surprise, more than 90% of SCNT embryos showed abnormal chromosomal segregation (ACS) before they developed to morula stage. Importantly, ACS per se did not affect the rate of development, morphology or cellular differentiation in preimplantation development. However, ACS occurring before the 8-cell stage severely inhibited postimplantation development. Thus, the morphology and/or rate of development are not significant predictive markers for the full-term development of SCNT embryos. Moreover, the low efficiency of animal cloning may be caused primarily by genetic abnormalities such as ACS, in addition to the epigenetic errors described previously.

Supplementation of culture medium with L-carnitine improves development and cryotolerance of bovine embryos produced in vitro.

High lipid content in embryos is associated with low freezing tolerance. This study assessed the effects of exogenous L-carnitine, an enhancer of lipid metabolism, on the in vitro development and freezing survival of bovine embryos. Also, effects on metabolic activity, reactive oxygen species (ROS) and apoptosis were investigated. Supplementation of embryo culture medium with 1.518 mM or 3.030 mM L-carnitine significantly increased the rates of zygote development to the blastocyst stage and blastocyst cell numbers whereas 6.072 mM of this compound did not improve embryo development. Survival rates after slow freezing of blastocysts were significantly higher when embryos were cultured in the presence of 1.518 mM or 3.030 mM L-carnitine compared with the control. A lower density of lipid droplets was detected in L-carnitine-treated blastocysts compared with the control. L-carnitine significantly reduced ROS levels in 2-cell embryos but did not reduce ROS levels at later stages. The apoptotic cell rate was not different between control and L-carnitine-treated blastocysts. L-carnitine significantly increased ATP levels in 2-cell embryos but not at the 8-cell or blastocyst stages. L-carnitine increased the expression of metabolism-related ATP6 and COX1 genes in blastocysts. In conclusion, L-carnitine supplementation enhanced lipid metabolism in embryos resulting in improved development and cryotolerance of bovine blastocysts produced in vitro.

Follicular fluid supplementation during in vitro maturation promotes sperm penetration in bovine oocytes by enhancing cumulus expansion and increasing mitochondrial activity in oocytes.

The aim of this study was to examine the effects of bovine follicular fluid (bFF) on mitochondrial activity in in vitro-matured (IVM) oocytes and to assess its importance for fertilisation and embryo development. Bovine follicular oocytes were subjected to IVM in medium supplemented either with polyvinylpyrrolidone, bovine serum albumin, calf serum or bFF. Nuclear maturation, cumulus expansion, mitochondrial distribution and ATP content in oocytes were compared between groups along with subsequent in vitro fertilisation (IVF) and embryo development. Compared with other supplements, bFF generated significantly enhanced re-distribution of active mitochondria in oocytes and this effect was associated with elevated intracellular ATP content. Furthermore, bFF significantly improved cumulus expansion, which was associated with improved fertilisation rates when cumulus-enclosed oocytes were subjected to IVF; however, its promoting effect was neutralised when denuded oocytes were inseminated. Elevating ATP content in oocytes by bFF did not affect maturation or embryo development but promoted fertilisation when mitochondrial electron transport was blocked in oocytes before IVF by Rotenone. In conclusion, supplementation of IVM medium with bFF promotes sperm penetration both by the improvement of cumulus expansion and by enhancing ATP levels in oocytes, which maintains their ability to be fertilised after mitochondrial stress.

Evidence of melatonin synthesis in the cumulus oocyte complexes and its role in enhancing oocyte maturation in vitro in cattle.

Melatonin is a multifunctional molecule that mediates several circadian and seasonal reproductive processes. The exact role of melatonin in modulating reproduction, however, is not fully understood-especially its effects on the ovarian follicles and oocytes. This study was conducted to investigate the expressions of the ASMT and melatonin-receptor MTNR1A and MTNR1B genes in bovine oocytes and their cumulus cells, as well as the effects of melatonin on oocyte nuclear and cytoplasmic maturation in vitro. Cumulus-oocyte complexes (COCs) from abattoir ovaries were cultured in TCM-199 supplemented with melatonin at concentrations of 0, 10, 50, and 100 ng/ml. The expression of ASMT, MTNR1A, and MTNR1B genes was evaluated by RT-PCR. Moreover, the effects of melatonin on cumulus cell expansion, nuclear maturation, mitochondrial characteristics and COCs steroidogenesis were investigated. Furthermore, the level of reactive oxygen species (ROS) was evaluated in denuded oocytes. Our study revealed that ASMT and MTNR1A genes were expressed in COCs, while the MTNR1B gene was expressed only in oocytes. Additionally, melatonin supplementation at 10 and 50 ng/ml to in vitro maturation medium significantly enhanced oocyte nuclear maturation, cumulus cell expansion and altered the mitochondrial distribution patterns, but had no effects on oocyte mitochondrial activity and COCs steroidogenesis. Melatonin-treated oocytes had a significantly lower level of ROS than controls. The presence of melatonin receptors in COCs and its promoting effects on oocyte nuclear and cytoplasmic events, indicate the potentially important roles of this hormone in regulating bovine oocyte maturation. Moreover, the presence of ASMT transcript in COCs suggests the possible involvement of these cells in melatonin biosynthesis.

Enhancement of lipid metabolism with L-carnitine during in vitro maturation improves nuclear maturation and cleavage ability of follicular porcine oocytes.

The aim of the present study was to assess the effects of L-carnitine, an enhancer of lipid metabolism and mitochondrial activity, during in vitro maturation (IVM) on nuclear maturation and in vitro fertilisation of porcine follicular oocytes and subsequent embryo development. Mitochondrial functions, intracellular lipid content and reactive oxygen species (ROS) levels in oocytes were also investigated. L-carnitine supplementation in 0.6-5mgmL(-1) concentration during IVM significantly improved (P<0.05) the rates of metaphase-II (MII) stage oocytes compared with the control; however, fertilisation rates and monospermy were not improved. Although supplementation of IVM medium with L-carnitine significantly increased oocyte cleavage (P<0.05), further development to the blastocyst stage was not improved. The density of active mitochondria was significantly higher and the density of lipid droplets was significantly lower (P<0.05) in L-carnitine-treated oocytes compared with the control. Furthermore, the ROS levels in L-carnitine-treated oocytes were significantly lower than those in the control. In conclusion, enhancing mitochondrial functions by L-carnitine improved oocyte maturation and cleavage underlining the importance of lipid metabolism for nuclear and cytoplasmic maturation of porcine oocytes.

Improvement of the developmental ability of nuclear transfer embryos by using blastomeres from in vitro fertilized embryos selected according to the early developmental stage and cell division status as donor cells in cattle.

This study was conducted to improve the developmental ability of nuclear transfer (NT) embryos by using blastomeres from in vitro fertilized (IVF) embryos with high quality as donor cells. The IVF embryos selected at the 2-cell stage at 24-h postinsemination (hpi) and again at the ≥8-cell stage at 48 hpi (Selected-IVF-embryos) showed the highest blastocyst formation rate among embryos. When blastomeres from the Selected-IVF-embryos (Selected-NT group) or Nonselected-IVF-embryos (Non-selected-NT group) were used as donor cells for NT, the blastocyst formation rate in the Selected-NT group (25.6%) was significantly higher than that in the Non-selected-NT group (13.5%). When blastomeres from the Selected-IVF-embryos at 108 (contained many cells before cell division) and 126 hpi (contained many cells immediately after cell division) were used as donor cells for NT (108- and 126-NT groups, respectively), the 126-NT group showed a significantly higher blastocyst formation rate (32.1%) than the 108-NT group (16.8%). Embryo transfer of blastocysts in the 126-NT group showed that 11 of 23 recipients became pregnant; nine calves were obtained. For the NT embryos reconstructed using in vivo derived embryos, 9 of 20 recipients became pregnant; seven calves were obtained. These results indicate that the blastocyst formation rate of NT embryos can be improved by using blastomeres from IVF embryos selected at the early developmental stage, especially immediately after cell division, and that the resultant NT embryos have a high developmental ability to progress to term that is comparable to NT embryos reconstructed using in vivo derived embryos.

The effect of ovary storage and in vitro maturation on mRNA levels in bovine oocytes; a possible impact of maternal ATP1A1 on blastocyst development in slaughterhouse-derived oocytes.

Since BSE testing of slaughtered cattle is obligatory in Japan, storage of ovaries at 15-20 C overnight in phosphate buffered saline has become a routine protocol in in vitro production (IVP) of cattle embryos. Ovary storage is known to reduce developmental competence of oocytes; however, its effects on oocyte gene expression have not been clarified yet. This study compared oocytes collected from stored slaughterhouse-derived ovaries with those collected by Ovum Pick-Up (OPU) in terms of the expression of 20 selected genes to determine if ovary storage affects cellular processes at the molecular level. Expression of mRNA in oocytes was assayed before and after in vitro maturation (IVM) by real-time quantitative PCR. Maternal mRNA levels of genes were investigated in 2-cell stage embryos obtained from slaughterhouse oocytes to assess their roles for blastocyst formation. In immature OPU oocytes, genes related to metabolism (GAPDH), transporters (GLUT8, ATP1A1) and stress resistance protein (HSP70) showed significantly higher expression compared with oocytes derived from stored ovaries. During IVM, the expression of GDF9, GLUT8, CTNNB1 and PMSB1 was significantly decreased irrespective of oocyte source. Two-cell stage embryos cleaving at 22-25 h after in vitro fertilization (IVF) showed a significantly higher blastocyst formation rate and ATP1A1 gene expression level compared with those cleaving at 27-30 h after IVF. Our results reveal that storage of ovaries alters mRNA levels in oocytes. Correlation of Na/K ATPase ATP1A1 expression in IVP embryos at the 2-cell and 8-cell stages with their developmental ability to the blastocyst stage may suggest the importance of maternal mRNA of this gene during blastulation in embryos derived from slaughterhouse oocytes.

Proper reprogramming of imprinted and non-imprinted genes in cloned cattle gametogenesis.

Epigenetic abnormalities in cloned animals are caused by incomplete reprogramming of the donor nucleus during the nuclear transfer step (first reprogramming). However, during the second reprogramming step that occurs only in the germline cells, epigenetic errors not corrected during the first step are repaired. Consequently, epigenetic abnormalities in the somatic cells of cloned animals should be erased in their spermatozoa or oocytes. This is supported by the fact that offspring from cloned animals do not exhibit defects at birth or during postnatal development. To test this hypothesis in cloned cattle, we compared the DNA methylation level of two imprinted genes (H19 and PEG3) and three non-imprinted genes (XIST, OCT4 and NANOG) and two repetitive elements (Satellite I and Satellite II) in blood and sperm DNAs from cloned and non-cloned bulls. We found no differences between cloned and non-cloned bulls. We also analyzed the DNA methylation levels of four repetitive elements (Satellite I, Satellite II, Alpha-satellite and Art2) in oocytes recovered from cloned and non-cloned cows. Again, no significant differences were observed between clones and non-clones. These results suggested that imprinted and non-imprinted genes and repetitive elements were properly reprogramed during gametogenesis in cloned cattle; therefore, they contributed to the soundness of cloned cattle offspring.

Effect of medium additives during liquid storage on developmental competence of in vitro matured bovine oocytes.

Our aim was to improve the developmental competence of bovine oocytes during their liquid storage by using additives. In vitro matured oocytes were stored for 20 h at 25°C in HEPES buffered TCM 199 medium (base medium). After storage, in vitro embryo development after in vitro fertilization was compared to those of non-stored (control) ones. Addition of 10% (v/v) newborn calf serum or 10.27 mmol/L pyruvate alone to the base medium did not improve blastocyst formation rates in stored oocytes; however, their simultaneous addition significantly improved the rate compared with those stored in base medium (P < 0.05). Supplementation of the holding medium with dithiothreitol (DTT) at any concentrations did not improve embryo development from stored oocytes. Although supplementation with cyclosporine A (CsA) significantly reduced apoptosis and membrane damage rates during storage, it did not improve the developmental competence of oocytes. 1,2-bis(2-aminophenoxy) ethane N,N,N',N'-tetraacetic acid tetrakis-acetoxymethyl ester and ruthenium red had no effect on oocyte apoptotic rates. Blastocyst formation rates in all stored groups remained significantly lower than that of the control. In conclusion, pyruvate and serum had a synergic effect to moderate the reduction of oocyte quality during storage, whereas mitochondrial membrane pore inhibitor CsA and the antioxidant DTT did not affect their developmental competence.

The effect of temperature during liquid storage of in vitro-matured bovine oocytes on subsequent embryo development.

The aim of the present study was to optimize the temperature for the temporal storage of matured bovine oocytes. In vitro-matured bovine oocytes were preserved in HEPES-buffered TCM199 medium supplemented with 10% newborn calf serum at different temperatures (4 °C, 15 °C, 25 °C, and 38.5 °C) for 20 hours. Embryo development and blastocyst quality after in vitro fertilization, cytoplasmic ATP and glutathione levels in oocytes, and the frequency of apoptotic oocytes were compared among storage groups and a control group without storage. Among the storage groups, those at 25 °C and 38.5 °C showed the highest rates of blastocyst development (19.3% and 24.5%, respectively) compared with those stored at 4 °C and 15 °C (8.5% and 14.9%, respectively); however, blastocyst formation rates in all storage groups were lower than that in the control group (39.8%; P < 0.05). Storage at 38.5 °C and 15 °C was associated with reduced cell numbers in resultant blastocysts compared with the control and the 25 °C storage groups. Storage at 4 °C reduced metabolic activity of oocytes characterized by their lower ATP levels compared with the other groups. Storage for 20 hours significantly reduced the glutathione content in oocytes in all groups in a similar manner, irrespective of the temperature. Storage at 4 °C or 15 °C but not at 25 °C and 38.5 °C significantly increased the percentage of apoptotic oocytes compared with the control group. In conclusion, 25 °C was found to be the most suitable temperature for the temporal storage of matured bovine oocytes regarding both the developmental competence of oocytes and the quality of resultant blastocysts.

Establishment of a specific radioimmunoassay for bovine interferon tau.

A radioimmunoassay (RIA) was developed for quantification of bovine interferon (bIFN) tau, conceptus secretory protein, which allows for the maintenance of the corpus luteum during early pregnancy. A cDNA coding bIFN tau was derived from cultured trophoblast cells (TBs). Recombinant (r) bIFN tau was produced in a baculovirus expression system with two different viruses. The RIA was a double-antibody competitive binding assay that used anti-bIFN tau antiserum (raised in rabbits) as the primary antibody, a radioiodinated derivative of bIFNtau as the radioactive tracer, and goat anti-rabbit IgG as the secondary antibody. The antibody did not cross-react with rbIFN alpha, recombinant human IFN beta or recombinant ovine IFN tau. The correct recovery of amounts of rbIFN tau indicated good accuracy. Serially concentrated TB conditioned media, paralleled the standard curve for bIFN tau. The intra-assay and inter-assay coefficients of variation at bIFN tau levels of 7.8 and 15.6 ng/mL were 7.1 and 8.1%, and 11.0 and 8.5%, respectively. bIFN tau was directly detected in uterine flushings obtained from cows at Day 16 of pregnancy. In summary, this assay was suitable for the measurement of bIFN tau.

Cytoskeletal and mitochondrial properties of bovine oocytes obtained by Ovum Pick-Up: the effects of follicle stimulation and in vitro maturation.

Follicle stimulation by follicular stimulating hormone (FSH) is known to improve developmental competence of bovine oocytes obtained by Ovum Pick-Up (OPU); however, the exact factors in oocytes affected by this treatment have remained unclear. We compared in vitro matured (IVM) oocytes obtained at the immature stage from cows by OPU either without or with stimulation with FSH (non-stimulated and stimulated OPU, respectively) to those obtained by superstimulation and in vivo maturation in terms of cytoskeleton morphology, mitochondrial distribution, intracellular adenosine triphosphate (ATP) content and H2 O2 levels at the metaphase-II stage and intracellular Ca(2+) levels after in vitro fertilization (IVF). Confocal microscopy after immunostaining revealed reduced size of the meiotic spindle, associated with increased tendencies of microfilament degradation and insufficient mitochondrial re-distribution in non-stimulated OPU-derived IVM oocytes compared with those collected by stimulated OPU, which in turn resembled in vivo matured oocytes. However, there was no difference in mitochondrial functions between oocytes obtained by stimulated or non-stimulated OPU in terms of ATP content, cytoplasmic H2 O2 levels, base Ca(2+) levels and the frequencies and amplitudes of Ca(2+) oscillations after IVF. Larger size of metaphase spindles in oocytes obtained by stimulated OPU may reflect and potentially contribute to their high developmental competence.

Enhancement of maternal recognition of pregnancy with parthenogenetic embryos in bovine embryo transfer.

This study was performed to elucidate the changes in IFNT messenger RNA (mRNA) levels in in vivo-fertilized and parthenogenetic bovine embryos and their interferon-τ (IFNT) secretion amounts during the elongation phase. We assessed the induction capability of maternal recognition of pregnancy by parthenogenetic embryos and attempted cotransfer of in vivo-fertilized and parthenogenetic embryos. The expression level of IFNT mRNA in in vivo-fertilized embryos peaked on Day 18 after estrus, and the highest amount of uterine IFNT was observed on Day 20. Transfer of 10 parthenogenetic embryos produced a detectable amount of uterine IFNT. Transfer of one or three parthenogenetic embryos inhibited luteolysis. An increase in ISG15 mRNA levels in peripheral granulocytes was induced by the transfer of three parthenogenetic embryos. Cotransfer of three parthenogenetic embryos significantly improved the pregnancy rate on Day 40 in code 3 in vivo-fertilized embryos compared with single transfer without parthenogenetic embryos (65% vs. 35%). However, the pregnancy rate on Day 90 (35%) in cotransfer of code 3 in vivo-fertilized embryos did not differ from that upon single transfer (29%), because the cotransfer group had a higher incidence of pregnancy loss than with single transfer (47% vs. 17%) after Day 40. Cotransfer did not affect the pregnancy rate of code 2 in vivo-fertilized embryos. The incidence of pregnancy loss was higher in cotransfer of code 2 in vivo-fertilized embryos than in single transfer (30% vs. 7%). In conclusion, parthenogenetic embryos in the elongation phase secreted IFNT, enabling induction of maternal recognition of pregnancy. The present study revealed that enhancement of the maternal recognition of pregnancy using parthenogenetic embryos promoted the viability of poor-quality embryos until Day 40 of gestation. However, the incidence of pregnancy loss increased after Day 40 in the cotransfer of parthenogenetic embryos. A technique for promoting the full-term survival of poor-quality embryos is needed.

Biological activity of recombinant bovine interferon τ produced by a silkworm-baculovirus gene expression system.

Bovine interferon (bIFN) τ plays a crucial role in maternal-fetal recognition and was expressed using a Bombyx mori (Bm) nuclear polyhedrosis virus (silkworm baculovirus) gene expression system. The biological effects of Bm-recombinant bIFNτ (rbIFNτ) on prostaglandin (PG) F2α synthesis were investigated in cultured bovine endometrial epithelial cells with oxytocin (OT, 100 nM) and on the in vitro development of bovine embryos. Bm-rbIFNτ and OT were shown to suppress PGF2α production in a dose-dependent manner. When in vitro produced morula stage embryos were cultured for 72 hr in modified CR1aa medium supplemented with or without rbIFNτ, Bm-rbIFNτ (10 ng/ml) significantly promoted development to the expanded blastocyst stage. In conclusion, Bm-rbIFNτ was suggested to have the same bioactivity as native IFNτ.