谷原 史倫

This study was conducted to determine suitable conditions for an experimental method in which the CRISPR/Cas9 system is introduced into in vitro-produced porcine zygotes by electroporation. In the first experiment, when putative zygotes derived from in vitro fertilization (IVF) were electroporated by either unipolar or bipolar pulses, keeping the voltage, pulse duration and pulse number fixed at 30 V/mm, 1 msec and five repeats, respectively, the rate of blastocyst formation from zygotes electroporated by bipolar pulses decreased compared to zygotes electroporated by unipolar pulses. In the second experiment, the putative zygotes were electroporated by electroporation voltages ranging from 20 V/mm–40 V/mm with five 1-msec unipolar pulses. The rate of cleavage and blastocyst formation of zygotes electroporated at 40 V/mm was significantly lower (p &lt .05) than that of zygotes electroporated at less than 30 V/mm. Moreover, the apoptotic nuclei indices of blastocysts derived from zygotes electroporated by voltages greater than 30 V/mm significantly increased compared with those from zygotes electroporated by voltages less than 25 V/mm (p &lt .05). When zygotes were electroporated with Cas9 mRNA and single-guide RNA (sgRNA) targeting site in the FGF10 exon 3, the proportions of blastocysts with targeted genomic sequences were 7.7% (2/26) and 3.6% (1/28) in the embryos derived from zygotes electroporated at 25 V/mm and 30 V/mm, respectively. Our results indicate that electroporation at 25 V/mm may be an acceptable condition for introducing Cas9 mRNA and sgRNA into pig IVF zygotes under which the viability of the embryos is not significantly affected.

© CryoLetters. BACKGROUND: Short-term storage is valuable method to reuse manipulated embryos. OBJECTIVE: The present study evaluated the effects of antifreeze protein (AFP) supplementation on the quality and development of in vitro-produced porcine morulae after short-term storage (24 h). MATERIALS AND METHODS: The morulae were stored with various concentrations of AFP type III for 24 h at 5, 15 and 25 C. RESULTS: Supplementation of AFP type III (1.0 µg/ml) improved the developmental competence of embryos stored at 25°C. The proportions of DNA-fragmented nuclei in the blastocysts did not differ between the embryos stored at 25°C and the control embryos without storage treatment. However, the developmental competence of embryos stored at hypothermic temperatures decreased relative to that of the control embryos. CONCLUSION: Supplementation of AFP type III (1.0 µg/ml) maintained the quality of embryos stored at 25°C, but did not have beneficial effects on the development of embryos stored at hypothermic temperatures.

Here, we investigated a suitable peripheral site for ovarian grafting that would facilitate monitoring of graft and oocyte recovery. Canine hemi-ovaries were autografted to three subcutaneous sites (under the fascia of the quadriceps femoris muscle and the thoracolumbar muscle and in the scapular deltoid muscle). All grafted ovaries were recovered from 10 bitches at 4 weeks post-transplantation and bisected for histological assessment and evaluation of oocyte viability. The longest ovarian diameters gradually decreased with the increase in the duration of grafting, irrespective of the grafting site. The mean number of total follicles was lower in the scapular deltoid muscle than in the quadriceps femoris fascia. Most follicles were classified as atretic follicles. Sixteen oocytes were recovered from 30 quartered ovaries; morphological analysis revealed three of these oocytes from the ovaries grafted under the fascia of quadriceps femoris and thoracolumbar muscles to be of good quality. Of these three oocytes, two reached metaphase I after 72 h of maturation culture. These results indicate that the environment in the fascia may be better suited for ovarian grafting than that in the intramuscular site. However, improvement of follicle survival and meiotic competence of oocytes in the grafts is necessary.

Refreezing of spermatozoa is necessary for flow cytometric sex sorting when using semen from proven bulls that have already been frozen-stored. This study evaluated the effects of Orvus ES Paste (OEP) on the post-thaw viability of refrozen bull spermatozoa. Semen samples that had been frozen by the standard procedures were thawed and then refrozen in freezing extender supplemented with 0%, 0.375%, 0.75%, 1.5%, or 3% of OEP. The post-thaw indicators (motility, viability, and plasma membrane integrity) were higher (P < 0.05) in the spermatozoa refrozen with 0.375% or 0.75% OEP than in the spermatozoa refrozen without OEP. Moreover, the addition of 0.375% OEP increased the percentages of viability and plasma membrane integrity of the post-refrozen spermatozoa compared with 0.75% OEP. When the effect of the 0.375% OEP supplementation on the viability of the refrozen-thawed spermatozoa was assessed in six bulls, the presence of OEP significantly increased the percentages of the total and progressive motility in three bulls and the percentages of the viability and plasma membrane integrity in five bulls (P < 0.05). Our findings indicate that the addition of 0.375% OEP to freezing extender improves the motility, viability, and plasma membrane integrity of refrozen-thawed spermatozoa.

Chlorogenic acid (CGA) is a quinic acid conjugate of caffeic acid, and a phytochemical found in many fruits and beverages that acts as an antioxidant. The present study investigated the effects of CGA supplementation during in vitro maturation (IVM), on in vitro development of porcine oocytes, to improve the porcine in vitro production (IVP) system. Oocytes were matured either without (control) or with CGA (10, 50, 100 and 200 mu M). Subsequently, the matured oocytes were fertilized and cultured in vitro for 7 day. The rates of maturation, fertilization and blastocyst formation of oocytes matured with 50 mu M CGA were significantly (p &lt; .05) higher than those of the control oocytes. Hydrogen peroxide (H2O2) is one of the reactive oxygen species and induces DNA damage in porcine oocytes. When oocytes were matured with 1 mM H2O2 to assess the protective effect of CGA, 50 mu M CGA supplementation improved the maturation rate and the proportion of DNA--fragmented nuclei in oocytes compared with control oocytes matured without CGA. Moreover, when oocytes were matured with either 50 mu M CGA (control) or caffeic acid (10, 50 and 100 mu M), the rates of maturation, fertilization and the blastocyst formation of oocytes matured with 50 mu M CGA were similar to those of oocytes matured with 10 and 50 mu M caffeic acid. Our results suggest that CGA has comparable effects to caffeic acid, and IVM with 50 mu M CGA is particularly beneficial to IVP of porcine embryos and protects oocytes from DNA damage induced by oxidative stress. Supplementation of CGA to the maturation medium has a potential to improve porcine IVP system.

The present study was conducted to clarify whether the meiotic stage of porcine oocytes has the highest sensitivity to hyperthermia during in vitro maturation by evaluating meiotic competence and DNA damage. Oocytes were exposed to 41 degrees C for 12 h at various intervals during 48 h of maturation culture. When the oocytes were exposed to 41 degrees C from 12 to 24 h of the maturation culture, the proportion of oocytes reaching metaphase II (MII) decreased as compared to the control oocytes cultured at 38.5 degrees C (P &lt; 0.05). Moreover, the proportions of DNA fragmentation in all oocytes exposed to 41 degrees C in each culture period after 12 h from the start of maturation culture were significantly higher (P &lt; 0.05) than for the control oocytes. When the meiotic stage of oocytes cultured at 38.5 degrees C between 12 and 24 h was examined, the majority of oocytes remained at the germinal vesicle (GV) stage at 12 h and approximately half of the oocytes reached metaphase I (MI) at 24 h. These results indicate that the meiotic stage of porcine oocytes having the highest sensitivity to hyperthermia during in vitro maturation is a transition period from the GV stage to the MI stage.

The current applications for cat cloning include production of models for the study of human and animal diseases. This study was conducted to investigate the optimal fusion protocol on in vitro development of transgenic cloned cat embryos by comparing duration of electric pulse. Cat fibroblast cells containing a human artificial chromosome (HAC) vector were used as genetically modified nuclear donor cells. Couplets were fused and activated simultaneously with a single DC pulse of 3.0 kV/cm for either 30 or 60 mu s. Low rates of fusion and embryo development to the blastocyst stage were observed in the reconstructed HAC-transchromosomic embryos, when the duration of fusion was prolonged to 60 mu s. In contrast, the prolongation of electric pulse duration improved the embryo development and quality in the reconstructed control embryos without HAC vector. Our results suggested that the optimal parameters of electric pulses for fusion in cat somatic cell nuclear transfer vary among the types used for donor cells.

Genetically modified pigs for biomedical applications have been mainly generated using the somatic cell nuclear transfer technique; however, this approach requires complex micromanipulation techniques and sometimes increases the risks of both prenatal and postnatal death by faulty epigenetic reprogramming of a donor somatic cell nucleus. As a result, the production of genetically modified pigs has not been widely applied. We provide a simple method for CRISPR (clustered regularly interspaced short palindromic repeats)/Cas9 gene editing in pigs that involves the introduction of Cas9 protein and single-guide RNA into in vitro fertilized zygotes by electroporation. The use of gene editing by electroporation of Cas9 protein (GEEP) resulted in highly efficient targeted gene disruption andwas validated by the efficient production of Myostatin mutant pigs. Because GEEP does not require the complex methods associated with micromanipulation for somatic reprogramming, it has the potential for facilitating the genetic modification of pigs.

BACKGROUND: Cryopreservation of semen is one of the most important methods for the preservation of endangered tigers. OBJECTIVE: This study evaluated the effects of sugar supplementation on the cryosurvival of spermatozoa from Sumatran tigers (Panthera tigris sumatrae). MATERIALS AND METHODS: The post-thaw characteristics and fertility of spermatozoa cryopreserved with different sugars (glucose, lactose, and trehalose) were evaluated using heterologous in-vitro fertilisation with cat oocytes. RESULTS: All parameters of post-thaw spermatozoa significantly decreased as compared with those of fresh spermatozoa. The index of sperm motility for semen cryopreserved with lactose was significantly higher than that for semen cryopreserved with trehalose. The percentage of total fertilisation for tiger spermatozoa cryopreserved with trehalose was significantly lower than that for control cat spermatozoa. CONCLUSION: Our findings indicated that supplementation with lactose or glycerol as the main sugar in the egg yolk extender resulted in a better motility and fertility potential for post-thawed spermatozoa.

The changes triggered by sperm-induced activation of oocytes, which are required for normal oocyte development, can be mediated by other agents, thereby inducing the parthenogenesis. In this study, we exposed porcine oocytes to 1 mM Ca-EDTA, a metal-ion chelator, at various intervals during 48 hr of in vitro maturation to determine the optimum period of Ca-EDTA treatment for parthenogenetic activation. When the oocytes were cultured with or without Ca-EDTA from 36 hr (post-12), 24 hr (post-24), 12 hr (post-36) and 0 hr (post-48) after the start of maturation culture, the blastocyst formation rates were significantly higher (P&lt;0.05) in the post-24, post-36 and post-48 groups (3.3%, 4.0% and 2.6%, respectively) than those in the control group without treatment (0%). Furthermore, when the oocytes were cultured with Ca-EDTA for 0 hr (control), 12 hr (pre-12), 24 hr (pre-24), 36 hr (pre-36) and 48 hr (pre-48) from the start of maturation culture, the oocytes formed blastocysts only in the pre-36 and pre-48 groups (0.4% or 0.8%, respectively). Pronuclei (&lt;66.7%) were observed only when the periods of Ca-EDTA treatment were more than 12 hr during maturation culture. In the control group, no pronuclei were detected. Our findings demonstrate that porcine immature oocytes can be parthenogenetically activated by Ca-EDTA treatment for at least 24 hr to 36 hr during maturation culture, leading to pronucleus formation followed by the formation of blastocysts.

To assist the process of oocyte activation, which is essential for promotion of fertilization events, i.e., resumption of meiosis, extrusion of the second polar body and formation of the pronucleus (PN), artificial stimuli such as an electrical pulse have been applied to porcine oocytes after injection of sperm. However, the efficiency of fertilization and embryonic development remains low. It is well known that in vertebrates, inactivation of mitogen-activated protein (MAP) kinase is required for oocyte activation. We have hypothesized that even after electrical stimulation of sperm-injected oocytes, MAP kinase may not be inactivated. As it has been reported that MAP kinase activity is regulated by protein kinase C, we examined the effectiveness of phorbol 12-myristate 13-acetate (PMA), a protein kinase C activator, for improvement of fertilization and embryonic development of sperm injected porcine oocytes. First, we examined the concentrations (0, 0.01, 0.1, 1, and 10 mu M) and durations (0, 1, 3, 5 hours) of PMA treatment that were efficient for the extrusion of two polar bodies and formation of two PNs (2PB+2PN) and embryonic development. When the sperm-injected oocytes were treated with 0.01-mu M PMA for 3 hours after electrical stimulation, the rates of 2PB+2PN and embryonic development were higher than those in the other treatment groups. We then examined the effect of PMA treatment (0.01 mu M, 3 hours) on MAP kinase activity. Unexpectedly, after electrical stimulation, the activity remained low until PN formation, irrespective of whether or not the oocytes had been treated with PMA. On the other hand, transformation of the injected sperm nucleus into the male PN was accelerated after the PMA treatment. Our present results suggest that the low efficiency of fertilization and embryonic development in sperm-injected oocytes is not due to high activity of MAP kinase but due to poor transformation of the injected sperm nucleus into the male PN. Furthermore, a combination of electrical stimulation and PMA is a fairly effective artificial protocol for promoting 2PB+2PN and embryonic development in sperm-injected porcine oocytes. (C) 2016 Elsevier Inc. All rights reserved.

Repeat-breeder (RB) cows are a major source of economic waste due to their decreased fertility. Embryo transfer (ET) is an alternative tool to improve the fertility of RB cows. The aims of the present study were to evaluate the effects of recipient parity and the season on pregnancy rates following ET in RB Japanese Black beef cattle. Embryos were transferred nonsurgically to recipients, consisting of 155 heifers (&lt; 2 years old) and 172 cows (&lt; 8 years old), which were defined as RB cattle. Of the recipients that were presented for ET, 57 recipients received a fresh embryo and 270 recipients received a frozen embryo. There were no differences in the pregnancy rates between cattle that received fresh embryos or frozen embryos. The rates of recipients with pregnancy, abortion, stillbirth, and normal calving were similar between heifers and cows. In cows, the pregnancy rates were lower (P &lt; 0.05) in summer (June to August) than in spring (March to May) and winter (December to February). In heifers, however, there were no differences in the pregnancy rates among the seasons. Our findings indicate that in RB Japanese Black beef cattle, the parity of the recipients does not have an effect on the pregnancy rates following the transfer of fresh and frozen embryos. However, heat stress may affect reproductive performance in RB Japanese Black cows.

This study evaluated the effect of dibutyryl cyclic adenosine monophosphate (dbcAMP) and human chorionic gonadotropin (hCG) on the formation of antral follicle-like structures (AFLSs) and on the meiotic status of bovine cumulus-oocyte complexes (COCs) embedded in collagen gel. Supplementation with dbcAMP increased the mean diameter of AFLSs during days 4-8 of culture compared with that of control COCs, irrespective of the concentration of dbcAMP used (0.5-2.0 mM). When the embedded COCs were cultured for 8 days with hCG, the diameters of AFLSs after 4 days of culture tended to be lower in the supplemented COCs than in the control COCs without hCG, irrespective of the concentration used (1-100 IU/mL). Supplementation with 10 IU/mL hCG increased the concentrations of anti-Mullerian hormone but not progesterone and oestradiol in the culture medium after 4 days of culture. Almost all oocytes collected from AFLSs had resumed meiosis by the end of culture, irrespective of supplementation of dbcAMP and hCG. These results indicate that although dbcAMP had a positive effect on AFLS formation and development, supplementation with hCG was detrimental. Moreover, hCG supplementation did not influence the luteinisation of granulosa cells in the AFLS for 4 days after the start of culture.

Melatonin has been reported to improve the in vitro development of embryos in some species. This study was conducted to investigate the effect of melatonin supplementation during in vitro maturation (IVM) and development culture on the development and quality of porcine embryos. In the first experiment, when the in vitro fertilized embryos were cultured with different concentrations of melatonin (0, 10, 25 and 50 ng/ml) for 8 days, the blastocyst formation rate of embryos cultured with 25 ng/ml melatonin (10.7%) was significantly increased (p &lt; 0.05) compared to the control embryos cultured without melatonin (4.2%). The proportion of DNA-fragmented nuclei in blastocysts derived from embryos cultured with 50 ng/ml melatonin was significantly lower (p &lt; 0.05) than that of embryos cultured without melatonin (2.1% vs 7.2%). In the second experiment, when oocytes were cultured in the maturation medium supplemented with different concentrations of melatonin (0, 10, 25 and 50 ng/ml), fertilized and then cultured with 25 ng/ml melatonin for 8 days, there were no significant differences in the rates of cleavage and blastocyst formation among the groups. However, the proportions (2.7-5.4%) of DNA-fragmented nuclei in blastocysts derived from oocytes matured with melatonin were significantly decreased (p &lt; 0.05) compared to those (8.9%) from oocytes matured without melatonin, irrespective of the concentration of melatonin. Our results suggest that supplementation of the culture media with melatonin (25 ng/ml) during IVM and development has beneficial effects on the developmental competence and quality of porcine embryos.

The optimization of single-embryo culture conditions is very important, particularly in the in vitro production of bovine embryos using the ovum pick-up (OPU) procedure. The purpose of this study was to examine the development of embryos derived from oocytes obtained by OPU that were cultured either individually or in groups in medium supplemented with or without sericin and to investigate the viability of the frozen-thawed embryos after a direct transfer. When two-cell-stage embryos were cultured either individually or in groups for 7 days in CR1aa medium supplemented with or without 0.5% sericin, the rates of development to blastocysts and freezable blastocysts were significantly lower for the embryos cultured individually without sericin than for the embryos cultured in groups with or without sericin. Moreover, the rate of development to freezable blastocysts of the embryos cultured individually with sericin was significantly higher than that of the embryos cultured without sericin. When the frozen-thawed embryos were transferred directly to recipients, the rates of pregnancy, abortion, stillbirth and normal calving in the recipients were similar among the groups, irrespective of the culture conditions and sericin supplementation. Our findings indicate that supplementation with sericin during embryo culture improves the quality of the embryos cultured individually but not the viability of the frozen-thawed embryos after transfer to recipients.

BACKGROUND: The addition of the detergent Orvus ES Paste (OEP) to semen freezing extenders has been observed to improve the post-thaw survival and longevity of spermatozoa from various species but has never been evaluated for yak spermatozoa. OBJECTIVE: This study evaluated the effects of OEP on the post-thaw motility and viability of epididymal and ejaculated yak spermatozoa. MATERIALS AND METHODS: Semen samples were frozen and thawed in semen freezing extender supplemented with 0%, 0.375%, 0.75% or 1.5% OEP. The motility and viability of frozen-thawed spermatozoa were evaluated before and after 3 h of incubation. RESULTS: The addition of 0.75% OEP to the freezing extender significantly improved the mean motility and viability values of both the epididymal and ejaculated spermatozoa immediately after thawing, but the beneficial effects on motility disappeared after 3 h of incubation. CONCLUSION: Our findings indicate that the addition of 0.75% OEP is effective for the preservation of yak spermatozoa.

Fragmin/protamine microparticles (F/P MPs) approximately 0.5-1 mu M in diameter were prepared by the simple mixing of fragmin with protamine. This study investigated the effects of F/P MP-containing collagen gels as a hormone carrier on the formation of antral follicle-like structures and on the development of growing bovine oocytes. The supplementation of F/P MPs in collagen gels contributed to the beneficial effects of follicle stimulating hormone (FSH) on the formation and size of antral follicle-like structures. The F/P MPs may serve as potential hormone carriers for the growth of cultured bovine oocytes from early antral follicles.

Xenografting of immature testicular tissue combined with cryopreservation can preserve and use genetic information of prepubertal animals. For establishment of this new approach, it is essential to clarify whether offspring derived from sperm grown in host mice harboring cryopreserved xenografts show normal reproductive development. This study examined serum profiles of gonadal hormones during sexual maturation in pigs generated by intracytoplasmic sperm injection using sperm derived from cryopreserved xenografts (CryoXeno pigs; three males and three females). We also assessed the reproductive abilities of the male CryoXeno pigs by mating them with conventionally produced (conventional) pigs, and by examining the in vitro fertilizing ability of their sperm. For female CryoXeno pigs, reproductive ability was evaluated by artificial insemination with semen from a conventional boar. During the growth of male CryoXeno pigs, the serum concentrations of inhibin and testosterone showed similar changes (P &gt; 0.17) to those in conventional pigs (n = 4). Histologic analyses of the testes revealed no differences (P &gt; 0.2) in the growth and differentiation of seminiferous tubules between CryoXeno and conventional pigs. Three conventional sows delivered 13.0 +/- 1.0 (mean standard error of the mean) live piglets after being mated with the three CryoXeno males. Sperm obtained from all CryoXeno pigs had the ability to penetrate oocytes, and these fertilized oocytes reached the blastocyst stage in vitro. During the growth of female CryoXeno pigs, the serum inhibin profile was similar (P &gt; 0.17) to that observed in conventional pigs (n = 5). The first rise in serum progesterone concentration to more than 2 ng/mL was noted at 32.0 +/- 2.3 weeks of age in the CryoXeno pigs and at 32.0 +/- 3.3 weeks in the conventional pigs, suggesting that both pigs reached puberty at a similar age. After puberty, female CryoXeno pigs farrowed 8.3 +/- 1.7 (mean +/- standard error of the mean; n = 3) live piglets after artificial insemination with semen from a conventional boar. In conclusion, these findings demonstrate that both male and female CryoXeno pigs have normal reproductive abilities. (C) 2014 Elsevier Inc. All rights reserved.

This study investigated the effect of chemical inhibitors on the cell-cycle synchronisation in cat fibroblast cells and evaluated the development of interspecies embryos reconstructed from cat donor cells and enucleated bovine oocytes. Cat fibroblast cells were treated with 15 mu g/mL roscovitine or 0.05 mu g/mL demecolcine prior to cell cycle analysis and nuclear transfer. The percentage of cat fibroblast cells arrested at the G0/G1 phase in the roscovitine group was similar to that in the control group without any treatment. The percentage of cells arrested at the G2/M phase was significantly higher in the demecolcine group than in the control group. The fusion rate of interspecies couplets was significantly greater in the roscovitine group than in the control group. Most embryos stopped the development at the 2- or 4-cell stage, and none developed into blastocysts. Chemical inhibitor-induced donor cell cycle synchronisation did not overcome developmental arrest in interspecies cloned embryos.

We report the successful piglet production from cryopreserved oocytes for the first time by using a simple, high capacity vitrification protocol for preservation and a defined system for in vitro embryo production. Immature cumulus-oocyte complexes (COCs) from prepubertal gilts were vitrified in microdrops and stored in liquid nitrogen. After warming, COCs were subjected to in vitro maturation (IVM), fertilization (IVF), and subsequent culture (IVC). Adjusting warmplate temperature to 42 degrees C during warming prevented temperature drops in a medium below 34.0 degrees C and significantly increased the percentage of oocyte survival and thus blastocyst yields obtained from total vitrified oocytes compared with that of warming at 38 degrees C (87.1% vs 66.9% and 4.4% vs 2.7%, respectively). Nuclear maturation and fertilization of oocytes were not affected by vitrification and warming temperature. Blastocyst development on day 7 (day 0 = IVF) of the surviving oocytes after warming at 38 degrees C and 42 degrees C was not different but lower (P&lt;0.05) than those of non-vitrified control oocytes (4.6%, 5.2% and 17.9%, respectively). However, blastocyst cell numbers in the control and vitrified groups were similar irrespective of warming temperature. Omitting porcine follicular fluid (pFF) from IVM medium (POM) did not affect maturation, fertilization and embryo development of vitrified-warmed oocytes. Transfer of blastocysts obtained on day 5 from vitrified oocytes matured either with or without pFF into 4 recipients (2 for each group) resulted in 4 pregnancies and the delivery of a total of 18 piglets. In conclusion, optimization of warming temperature was a key factor for achieving high survival rates, and surviving oocytes could be utilized in vitro using defined media. Using these modifications, live piglets could be obtained from cryopreserved oocytes for the first time.

The zona pellucida (ZP) is considered to play important roles in the prevention of polyspermy in mammalian oocytes. However, in pigs we have shown that the presence of the ZP accelerates sperm penetration into the ooplasm during in vitro fertilization (IVF). In the present study, we investigated the effects of the ZP on sperm binding, acrosomal status, and functional exposure of IZUMO, a critical factor involved in sperm-egg fusion, during IVF in pigs. We evaluated the numbers and acrosomal statuses of sperm binding to the ZP and oolemma, and being present in the ZP and perivitelline space (PVS) using ZP-intact and ZP-free oocytes. More sperm bound to the ZP than to the oolemma. The average number of sperm present in the PVS was 0.44-0.51 per oocyte, and all sperm had lost their acrosomes. The proportion of sperm that were immunopositive for anti-IZUMO antibody was significantly higher after they were passing or had passed through the ZP. Furthermore, addition of anti-IZUMO antibody to the fertilization medium significantly inhibited the penetration of sperm into ZP-free oocytes. These results suggest that, in pigs, the ZP induces the acrosome reaction, which is associated with the functional exposure of IZUMO, resulting in completion of fertilization.

Aim of the study was to investigate the effect of vitrification on viability, cytoskeletal integrity and in vitro developmental competence after in vitro fertilization (IVF) of oocytes vitrified before or after in vitro maturation (IVM) using a pig model. Oocytes from abattoir-derived porcine ovaries were vitrified at either the germinal vesicle (GV) or metaphase II (MII) stage by modified solid surface vitrification (SSV). Oocyte viability was evaluated by stereomicroscopic observation whereas their nuclear stage and morphology of microtubules and F-actin were observed by confocal microscopy after immunostaining. Fertilization was assessed by orcein staining. The survival rate after vitrification was higher for MII-stage than for GV-stage oocytes. However, the ability of surviving oocytes to reach the MII stage after vitrification at the GV stage (GV-vitrified oocytes) was similar to that of control oocytes. Furthermore, after IVM, GV-vitrified oocytes had better spindle and F-actin integrity than oocytes vitrified at the NI stage (MII-vitrified oocytes). In accordance with this result, GV-vitrified oocytes had better ability to extrude the second polar body and support male pronucleus formation after in vitro fertilization (IVF), in comparison to MII-vitrified oocytes. Fertilization rates did not differ among groups. Finally, the ability of GV-vitrified oocytes to develop into embryos was superior to that of MII-vitrified oocytes. However, both vitrified groups showed reduced blastocyst development compared with the control group. In conclusion vitrification of porcine oocytes at the GV stage is advantageous in conferring better cytoskeletal organization and competence to develop to the blastocyst stage in comparison with vitrification at the MII stage. (C) 2013 Elsevier Inc. All rights reserved.

Freeze-drying (FD) medium containing ethylene glycol-bis(2-aminoethylether)-N,N,N',N'-tetraacetic acid (EGTA) is reported to be beneficial for maintenance of sperm DNA integrity after FD. Recently, trehalose has also been reported to have notable ability to stabilize the protein structure and biomembranes of sperm in a dry state. In this study, we examined the effect of a combination of EGTA and different concentrations of trehalose in FD medium on sperm DNA integrity and the in vitro development of IVM porcine oocytes after intracytoplasmic sperm injection (ICSI) using freeze-dried boar sperm. Ejaculated sperm from a boar were suspended in basic FD medium supplemented with 0, 3.75, 7.5, 15, 30, 60, or 90 mM trehalose and freeze-dried. After rehydration, the sperm in all groups were subjected to DNA damage detection using a Halomax kit. It was found that the level of DNA damage in 15-mM group was significantly lower than that in 0-mM group, and no difference was observed between the 15-, 7.5-, and 3.75-mM groups. Moreover, there were no significant differences in the DNA damage level among 0, 3.75 mM, and other groups treated with trehalose. When freeze-dried sperm were used for ICSI, the fertilization rates and blastocyst formation rates (observed at 10 hours and 6 days of IVC after ICSI, respectively) in the 7.5- and 15-mM groups were not different from those in 0-mM group. These results suggest that FD medium supplemented with trehalose at appropriate concentrations improves sperm DNA integrity, but does not improve fertilization and preimplantation embryo development of IVM oocytes following ICSI. (C) 2013 Elsevier Inc. All rights reserved.

Mitochondria are reported to be critical in in vitro maturation of oocytes and subsequent embryo development after fertilization, but their contribution for fertilization has not been investigated in detail. In the present study, we investigate the contribution of mitochondria to fertilization using reconstructed porcine oocytes by fusion of ooplasmic fragments produced by serial centrifugations (centri-fusion). Firstly, we evaluated the characteristics of ooplasmic fragments. Three types of fragments were obtained by centrifugation of porcine oocytes matured in vitro for 46 h: brownish (B), transparent (T) and large (L) fragments containing both B and T parts in a fragment. The production efficiencies of these types of fragments were 71.7, 91.0 and 17.8 fragments/100 oocytes, respectively. In experiments, L fragments were excluded because they contained both brownish and transparent components that were apparently intermediate between B and T fragments. Observations by confocal microscopy after staining with MitoTracker Red CMXRos (R) and transmission electron microscopy revealed highly condensed active mitochondria in B fragments in contrast to T fragments that contained only sparse organelles. We reconstructed oocytes by fusion of a karyoplast and two cytoplasts from B and T fragments (B and T oocytes, respectively). The B oocytes showed higher sperm penetration (95.8%) and male pronuclear formation rates (94.2%) by in vitro fertilization than T oocytes (66.7% and 50.0%, respectively). These results suggest that the active mitochondria in oocytes may be related to their ability for fertilization.

Research comparing the activation sensitivity of oocytes to chemical treatment among mammalian species remains limited. We compared the activation ability of oocytes from bovine and feline ovaries when treated with ethanol alone, with ethanol and cycloheximide, and without any chemical treatment; the oocytes were then cultured for 72 h. After in vitro maturation (IVM), 5% of feline oocytes were activated and 1% were cleaved, whereas there were no prematurely activated bovine oocytes. Activation rates with ethanol and ethanol/cycloheximide were significantly higher (P &lt; 0.01) in bovine oocytes than in feline oocytes (74.2% vs. 34.1% and 86.3% vs. 52.5%, respectively). Thus, our findings indicate that feline oocytes can be prematurely activated by the end of IVM, and that bovine oocytes may have a higher sensitivity of parthenogenetic activation to chemical treatment than do feline oocytes.

Primordial oocytes are a potential resource for medical and zoological application, but those of large animals have not yet been reported to show efficient embryonic development. In the present study, we established a pig model for production of blastocysts from primordial oocytes that had been grafted into nude mice and matured in vitro, in combination with fusion of cytoplasmic fragments. Neonatal porcine ovaries in which most follicles are at the primordial stage were minced and grafted into nude mice (Crlj:CD1-Foxn1(nu)). About 60 days after detection of vaginal opening, the mice were given 62.5 U/mL porcine FSH for 2 weeks by infusion to enhance follicular development. Developmentally competent oocytes collected from porcine ovaries (conventional oocytes) were matured in vitro and subjected to serial centrifugation to prepare cytoplasmic fragments without a metaphase plate (cytoplasts). Three cytoplasts were fused by electro-stimulation to an oocyte retrieved from a host mouse (xenogeneic oocyte) and matured in vitro. Then these fused oocytes were fertilized and subsequently cultured in vitro. No blastocysts were generated from xenogeneic oocytes without fusion of cytoplasm. When xenogeneic oocytes had been fused with three cytoplasts, the blastocyst rate increased significantly to 143%, comparable to that for untreated conventional oocytes (20.0%). The numbers of cells in blastocysts for these fused oocytes (37.2 cells/blastocyst) were not significantly different from those for conventional oocytes (25.4 cells/blastocyst). Our findings show that it is possible to use primordial oocytes of large mammals in combination with xenografting of ovarian tissue and also ooplasmic fusion. (C) 2013 Elsevier Inc. All rights reserved.

In porcine oocytes, the function of the zona pellucida (ZP) with regard to sperm penetration or prevention of polyspermy is not well understood. In the present study, we investigated the effects of the ZP on sperm penetration during in vitro fertilization (IVF). We collected in vitro-matured oocytes with a first polar body (ZP+ oocytes). Some of them were freed from the ZP (ZP- oocytes) by two treatments (pronase and mechanical pipetting), and the effects of these treatments on sperm penetration parameters (sperm penetration rate and numbers of penetrated sperm per oocyte) were evaluated. There was no evident difference in the parameters between the two groups. Secondly, we compared the sperm penetration parameters of ZP+ and ZP- oocytes using frozen-thawed epididymal spermatozoa from four boars. Sperm penetration into ZP+ oocytes was found to be accelerated relative to ZP- oocytes. Thirdly, we evaluated the sperm penetration of ZP+ and ZP- oocytes at 1-10 h after IVF (3 h gamete co-incubation). The proportions of oocytes penetrated by sperm increased significantly with time in both groups; however, the number of penetrated sperm per oocyte did not increase in ZP- oocytes. Finally, we performed IVF using ZP- oocytes divided into control (3 h) and prolonged gamete co-incubation (5 h) groups. Greater numbers of sperm penetrated in the 5 h group than in the control group. These results suggest that the ZP and oolemma are not competent factors for prevention of polyspermy in our present porcine IVF system. However, it appears that ZP removal is one of the possibilities for reducing polyspermic penetration in vitro in pigs.

Cryopreservation of immature testicular tissues is essential for increasing the possibilities of offspring generation by testicular xenografting for agricultural or medical purposes. However, successful production of offspring from the sperm involved has never been reported previously. In the present study, therefore, using intracytoplasmic sperm injection (ICSI), we examined whether xenogeneic sperm obtained from immature pig testicular tissue after cryopreservation would have the capacity to produce live piglets. Testicular fragments from 9- to 11-day-old piglets were vitrified after 10- or 20-min immersion in vitrification solution containing ethylene glycol (EG), polyvinyl pyrrolidone (PVP) and trehalose as cryoprotectants, and then stored in liquid nitrogen for more than 140 days. Thirty nude mice were assigned to each immersion-time group. Testicular fragments were transplanted under the back skin of castrated mice immediately after warming and removal of the cryoprotectants. Blood and testicular grafts were then recovered from the recipient mice on days 60, 120, 180 and 230-350 (day 0 = grafting). Histological assessment of the testicular grafts and analyses of inhibin and testosterone production revealed no significant differences between the two immersion-time groups, indicating equal growth activity of the cryopreserved tissues. A single sperm obtained from a mouse in each group on day 230-350 was injected into an in vitro-matured porcine oocyte, and then the ICSI oocytes were transferred to the oviducts of estrus-synchronized recipient gilts. One out of 4 gilts that had received oocytes fertilized using sperm from the 10-min immersion group delivered 2 live piglets, and one of another 4 gilts from the 20-min group delivered 4 live piglets. Thus, we have successfully generated porcine offspring utilizing sperm from immature testicular tissues after cryopreservation and transplantation into nude mice. The present model using pigs will be applicable to many large animals, since pigs are phylogenetically distant from the murine recipients.

Our aim was to optimize a cryoprotectant treatment for vitrification of immature porcine cumulus-oocyte complexes (COCs). Immature COCs were vitrified either in 35% ethylene glycol (EG), 35% propylene glycol (PG) or a combination of 17.5% EG and 17.5% PG. After warming, the COCs were in vitro matured (IVM), and surviving oocytes were in vitro fertilized (IVF) and cultured. The mean survival rate of vitrified oocytes in 35% PG (73.9%) was higher (P&lt 0.05) than that in 35% EG (27.8%). Oocyte maturation rates did not differ among vitrified and non-vitrified control groups. Blastocyst formation in the vitrified EG group (10.8%) was higher (P&lt 0.05) than that in the vitrified PG group (2.0%) but was lower than that in the control group (25.0%). Treatment of oocytes with 35% of each cryoprotectant without vitrification revealed a higher toxicity of PG on subsequent blastocyst development compared with EG. The combination of EG and PG resulted in 42.6% survival after vitrification. The maturation and fertilization rates of the surviving oocytes were similar in the vitrified, control and toxicity control (TC treated with EG+PG combination without cooling) groups. Blastocyst development in the vitrified group was lower (P&lt 0.05) than that in the control and TC groups, which in turn had similar development rates (10.7%, 18.1% and 23.3%, respectively). In conclusion, 35% PG enabled a higher oocyte survival rate after vitrification compared with 35% EG. However, PG was greatly toxic to oocytes. The combination of 17.5% EG and 17.5% PG yielded reasonable survival rates without toxic effects on embryo development. © 2013 by the Society for Reproduction and Development.

【目的】ブタ卵母細胞(以下，卵)の体外受精(IVF)時において，卵への精子侵入に関する透明帯(ZP)の機能については不明な点が多い。我々はこれまでに，体外成熟培養後，ZPを有する成熟卵(ZP+卵)と，ZP+卵からZPを除去したZP-卵との精子侵入状況の比較により，IVF時にZPが存在すると卵への精子侵入は促進され，ZPを通過した精子は全て先体を失い，効率よく卵細胞膜と融合することを示した（本学会第104，105回大会）。Izumoはマウス，ヒトおよびブタ精子において精子-卵子融合因子として報告されている。本研究ではブタのIVF時におけるZPの存在がIzumoの発現と卵への精子侵入に及ぼす影響を検討した。【方法】ZP-卵に対し抗Izumo抗体(0(対照区)，0.25ならびに0.5 μg/ml)を添加した体外受精培地と凍結融解精巣上体精子を用いてIVFを行い，開始後10時間での精子侵入状況を調べた。また，IVF開始後1，3および5時間での卵表面に付着した精子数を測定することでBinding Assayを行った。媒精時間は3時間，精子濃度は1×104 sperm/mlとした。次にZP+卵とZP-卵を用いてIVFを行い，開始後3時間でFITC-PNA染色，Hoechst染色および免疫蛍光染色を行い，ZP+卵においてはZP内部もしくは囲卵腔に存在しかつ先体を失った精子について，ZP-卵においては卵細胞膜へ付着しかつ先体を失った精子についてIzumoの発現状況を調べた。【結果】ZP-卵において抗体濃度を高くすると精子侵入率ならびに侵入精子数は減少し，0.5 μg/ml添加区では対照区と比較し有意に低かった(90.4％ならびに2.1個 vs 68.6％ならびに1.6個)(P<0.01，ANOVA/Tukey検定による)が，卵表面に付着した精子数に差は無かった。ZP内部もしくは囲卵腔に存在する精子はすべて先体を失い，ZP-卵に付着した先体を失った精子と比較してIzumoの発現率は有意に高かった（64.6％ vs 5.2％）（P<0.01，カイ二乗検定による）。【結論】精子がZPを通過することでIzumoの発現が促進されることが明らかとなった。それにより，精子と卵の膜融合が促され，卵への精子侵入が高まることが示唆された。

Epigallocatechin gallate (EGCG) is the major polyphenol in green tea (Camellia sinensis) and is known for its antioxidant effects. The objective of the present study was to examine the effects of EGCG during in vitro fertilization (IVF) on the sperm quality and penetrability into oocytes. In the first experiment, the effects of concentration and incubation period of EGCG on the motility and penetrability of spermatozoa were examined. When frozenthawed spermatozoa were incubated in IVF medium supplemented with 0 (control), 1, 50 and 100 mu M EGCG for 1, 3 and 5 h, supplementation with 50 and 100 mu M EGCG improved motility of the spermatozoa (p &lt; 0.05), but not viability, as compared with the control group. When frozenthawed spermatozoa were co-incubated with in vitro-matured (IVM) oocytes in IVF medium supplemented with 50 and 100 mu M EGCG for 5 h, supplementation of EGCG had positive effects on sperm penetration rates. In the second experiment, the effects of supplementation of EGCG in IVF medium on penetrability of sperm from different boars and development of fertilized oocytes were evaluated. When frozenthawed spermatozoa from six boars were co-incubated with IVM oocytes in IVF medium supplemented with 50 mu M EGCG, the effect of EGCG on sperm penetration and development of oocytes after fertilization was found to vary with individual boar. Our results indicate that motility and penetrability of boar spermatozoa are improved by co-incubation with 50 mu M EGCG, but the effects vary with individual boars.

For establishment of gonadal xenografting, it is essential to clarify whether offspring derived from gametes grown in host mice harboring xenografts have normal reproductive development. This study examined the secretory profiles of gonadal hormones in relation to sexual maturation or ovarian cyclicity in pigs generated by intracytoplasmic sperm injection using xenogeneic sperm (Xeno-ICSI pigs, four males and one female). We also assessed the developmental activity of gametes obtained from these pigs using in vitro culture systems, or by mating with conventionally produced (conventional) pigs. During the growth of male Xeno-ICSI pigs, serum inhibin and testosterone concentrations were generally within ranges for those hormones in conventional pigs. Histologically, there were no differences in the growth and differentiation of seminiferous tubules between Xeno-ICSI and conventional pigs. Parameters of semen quality, including volume, pH, sperm concentration, and the percentage of motile sperm were not different from those in conventional pigs. Among the Xeno-ICSI pigs, individual differences were noted in the ability of sperm to penetrate oocytes and to produce blastocysts. However, oocytes after in vitro fertilization using these sperm developed into blastocysts containing more than 31 cells. One conventional sow delivered 12 piglets after being mated with a male Xeno-ICSI pig. During growth of the female Xeno-ICSI pig, serum progesterone concentrations had a sudden increase at 41 wk of age, suggesting CL formation. After puberty, this animal showed cyclic changes in the serum concentrations of progesterone and inhibin, and delivered 10 piglets after AI using fresh sperm obtained from a conventional boar. In conclusion, these findings demonstrated that both male and female Xeno-ICSI pigs had normal reproductive abilities. (C) 2012 Elsevier Inc. All rights reserved.

This study reports about follicular development on the surface of canine ovarian tissue after autografting under the fascia of the thoracolumbar muscle and about meiotic resumption of follicle-derived oocyte after maturation culture. After ovarian excision from a bitch, each ovary of the pairs was cut approximately into half. The hemi-ovaries were transplanted into the bitch of origin at three different body sites (under the fascia of the quadriceps femoris muscle and the thoracolumbar muscle, and in the deltoid muscle in the scapular region). All grafted ovaries were recovered from the bitch at 35 days post-transplantation. A visible antral follicle was observed on the surface of the ovary grafted under the thoracolumbar fascia. Histological examination revealed viable follicles at different stages of development irrespective of graft site. Most granulosa cells in the follicles at different stages of development expressed proliferating cell nuclear antigen (PCNA). A total of three oocytes were collected from an ovary grafted under the fascia of the thoracolumbar muscle, wherein an oocyte reached metaphase I after maturation culture. This is the first report to demonstrate follicular development and meiotic resumption of oocytes recovered from autografted canine ovarian tissues.

The objectives of this study were to examine the characteristics and penetrability of Sumatran tiger spermatozoa cryopreserved with different type of cryoprotective sugars (lactose, glucose, and trehalose). Ejaculated semen were collected from two healthy male adult Sumatran tigers (SB#1099 and SB#1512) belonged to Taman Safari Indonesia, Bogor. The semen were cryopreserved in Niwa and Sasaki freezing (NSF) extender supplemented with 44 mg/ml of each sugar and 3% glycerol. Heterologous IVF with domestic cat oocytes was used to assess the penetrability of frozen-thawed Sumatran tiger spermatozoa. The value of sperm motility for semen cryopreserved with lactose was significantly higher than that for semen cryopreserved with trehalose (30.0 vs. 18.8, P < 0.05). There were no significant differences in the values of sperm viability and plasma membrane integrity among the groups. Frozen-thawed Sumatran tiger sperm were able to fertilize with in-vitro matured cat oocytes, irrespective of type of cryoprotective sugars. There were no differences in the rates of penetration and normal fertilization of sperm among the groups (28.8-45.5% and 65.3-72.4%, respectively). In conclusion, the type of cryoprotective sugar may affect the motility of frozen-thawed Sumatran tiger spermatozoa but not the in-vitro penetrability.

【背景】ブタ卵母細胞(以下，卵)において，体外受精(IVF)時の多精子侵入は他の哺乳類の卵に比較して頻発し，受精卵の発生率が低い原因の一つである。我々はこれまでに，体外成熟培養後，第一極体を放出し透明帯を有する成熟卵(ZP+卵)と，ZP+卵から透明帯を除去した卵(ZP-卵)においてIVF時の精子侵入状況を比較し，透明帯が存在すると精子の侵入を促進することを示した(本学会第104回大会)。その要因として透明帯による先体反応の誘起によるものと考えられた。本研究では卵表面の精子および囲卵腔内に存在する精子を観察し透明帯の機能を検討した。 【方法】ZP+卵と，ZP+卵を0.5%プロナーゼ処理後に機械的に透明帯を除去したZP-卵を用い，凍結融解精巣上体精子にてIVFを行った。媒精時間は3時間，精子濃度は1&times;10⁴/mlとした。Binding AssayによりIVF開始後1，3および5時間での卵表面に存在する精子数(ZP+卵では頭部が透明帯表面へ付着あるいは透明帯内部へ侵入した精子数，ZP-卵では頭部が卵細胞膜へ付着した精子数)を測定した。同時に0.1%プロナーゼ処理によりZP+卵の囲卵腔を拡張させ，囲卵腔に存在する精子数を調べた。また，各々FITC-PNA染色を行い先体の有無を観察した。加えて一部の卵をIVF開始後1，3，5および10時間で固定，染色し精子の侵入状況を調べた。 【結果】これまでの実験同様，ZP-卵と比較しZP+卵において精子侵入率および侵入精子数は有意に高く(P<0.05，ANOVA/Tukey検定による)，侵入精子数はZP+卵，ZP-卵共にIVF開始後5時間で最大となった(それぞれ3.23ならびに2.13個)。卵表面の精子数もZP+卵において有意に多かった(P<0.01)。囲卵腔内の精子数は各経過時間において平均0.45個/卵前後で推移し，すべて先体を失っていた。 【結論】透明帯を突破した精子は先体反応が誘起され，効率よく卵細胞膜と融合していることが示された。また，卵細胞膜による多精子拒否は起きていないことが示唆された。

Freeze-drying (FD) has been widely used for preserving viruses, bacteria, and fungi, because FD materials are easy to store and transport without cryopreservation in liquid nitrogen. In addition, sperm DNA is easy to be damaged by FD stress. EGTA (ethylene glycol tetraacetic acid) was reported to be a good chelator in FD buffer for sperm DNA protection. Recently, trehalose was also reported to have notable ability in stabilizing protein structure and bio-membrane in dry state. In this study, we examined effect of combination of EGTA and different concentrations of trehalose (0 M, 0.015 M, 0.03 M, 0.06 M and 0.09 M) in FD medium on sperm DNA integrity and in vitro development after ICSI using FD sperm.<br>Firstly, FD sperm in the groups were rehydrated and analyzed with Halomax kit to detect DNA lesion. DNA damage in 0.015 M trehalose group significantly decreased compared with the control group (P<0.001). However, higher concentrations of trehalose had no protective effects on DNA integrity. Secondary, we used FD sperm in trehalose 0 M and 0.015 M groups for ICSI. Sham injection and parthenogenetic activation were used as control groups. There were no significant differences in rates of normal fertilization and blastocyst formation (examined at 10 h and 6 days after insemination, respectively) using FD sperm between 0 M and .015M groups. The quality of blastocysts (measured by the total numbers) was not different between the groups. These parameters were also not different from those in the control groups. The results suggest that trehalose has protective effect against sperm DNA damage. Further experiments will be necessary to examine the DNA damage affect full-term development of ICSI-oocytes.

The influence of graft site on the survival of canine follicles and oocytes after autografting was investigated. Hemi-ovaries were autografted to three locations (quadriceps femoris muscle fascia, kidney capsule, and gastrosplenic ligament), and grafted ovaries were recovered (under anesthesia) 28 to 31 d after transplantation. The grafted hemi-ovaries were bisected: one-quarter ovary was used for histological assessment and another quarter for evaluation of oocyte viability. As controls, the remaining fresh hemi-ovaries were used to assess the viability of follicles and oocytes in non-transplanted ovaries. Most follicles in the histological sections of the grafts were classified as primordial or primary follicles. Antral follicles were not observed in the grafts, irrespective of the graft site. The percentages of viable follicles in the sections from control ovaries, and the ovaries grafted to the kidney capsule, the quadriceps femoris muscle fascia, and the gastrosplenic ligament were 17.4, 22.9, 18.3, and 32.4%, respectively. A total of 12 oocytes was recovered from the 15 hemi-ovaries grafted in five bitches, of which five (41.7%) oocytes from the ovaries grafted to the quadriceps femoris muscle fascia and the kidney capsule were cultured for assessment of meiotic competence. Three oocytes were viable but remained in the germinal vesicle stage after 72 h of maturation culture. The quadriceps femoris muscle fascia might be useful for grafting like the kidney capsule, but improvement of follicle survival and meiotic competence of oocytes in the grafts is necessary. (C) 2012 Elsevier Inc. All rights reserved.

The objectives of this study were to examine the effects of the presence or absence of serum during the in vitro culturing period of domestic cat embryos on their developmental potential into blastocysts as well as their tolerance to cryopreservation using a slow-freezing method. In vitro-fertilized cat oocytes were incubated in a modified synthetic oviduct fluid (mSOF) containing 4 mg/mL bovine serum albumin (BSA) throughout culturing (BSA group) or in mSOF containing 4 mg/mL BSA for the first 3 days followed by mSOF containing 5% fetal bovine serum (FBS group). The developmental potential of the embryos to the blastocyst and expanded blastocyst stages was evaluated 7 days after in vitro fertilization. The blastocysts were frozen-thawed by the slow-freezing method and cultured for 3 days to examine their viability in vitro. There were no differences in the formation rates of blastocysts or expanded blastocysts, or number of cells in the embryos between the two groups. After cryopreservation, the hatching rates of the expanded blastocysts in the BSA group were significantly higher (P &lt; 0.05) than those of the FBS group. The postthaw viability of blastocysts was lower than that of expanded blastocysts irrespective of culture medium. These results indicate that the developmental potential of cat embryos cultured in serum-free medium is comparable to those cultured in serum-containing medium. Furthermore, expanded blastocysts produced without serum exhibit better postthaw viability than those produced with serum. (C) 2011 Elsevier Inc. All rights reserved.

Chemical toxicity of cryoprotectants to in vitro developmental competence of porcine oocytes was examined. In vitro-matured oocytes were exposed to 40% ethylene glycol (EG), glycerol (GLY), or 1,2-propanediol (PD), fertilized with spermatozoa, and cultured for 8 d. Compared to treatment with other cryoprotectants, exposure to EG resulted in the development of significantly more blastocysts, but the rate was significantly lower than that of non-exposed control oocytes. In vitro-matured oocytes were also equilibrated in 40% EG by 3 multi-step methods, after which their developmental competence was evaluated. The rate of blastocyst development was higher in the 4-step method than in the 2- and 3-step methods of equilibrium. These results indicate that cryoprotectants and equilibration methods affect the developmental competence of porcine oocytes and that EG may be a superior cryoprotectant for vitrification of these cells.

Contents Culture techniques of antral follicle-like structure (AFLS) derived from cumulus-oocyte complexes (COCs) might provide important insights into follicular development and oocyte maturation. This study was undertaken to investigate the effects of embedding bovine COCs individually (one COC) or in groups (4-5 COCs) in collagen gels on the formation of AFLS and the meiotic status of oocytes. The observations of AFLS formation were performed every second day for 14 days. The AFLS was formed at Day 2 or 4 after the start of culture (Day = 0), irrespective of the culture methods. The mean diameters of AFLS during Days 4-14 using the individual culture method were significantly higher (p &lt; 0.05) than those using the group culture method. However, the AFLS formation rate in the individual culture method was significantly lower compared to that in the group culture method (26.1% vs 62.7%, p &lt; 0.01). Almost all oocytes had undergone the germinal vesicle breakdown stage, irrespective of the culture method or AFLS formation. In conclusion, comparison with the individual culture method revealed that the mean diameters of AFLS in the group culture method were smaller, but more COCs formed AFLS. The group culture method might be useful for evaluating the various hypotheses of follicular formation and interfollicular communication. However, improvement of the group culture system is necessary to prevent the meiotic resumption of oocytes, because the AFLS formation is dependent on the cumulus/granulosa cells surrounding oocytes.

We investigated the effects of a portable incubator with a CO2 chamber on the viability and development of porcine oocytes/embryos for their transportation and examined the operational suitability of a straw or dish as a container for culturing the oocytes or embryos in the portable incubator. In the first experiment, the cumulus-oocyte complexes (COCs) were placed either in a dish or straw; and they were then cultured for 44 h in a standard CO2 incubator, in the CO2 chamber in an incubator, or in the CO2 chamber in a portable incubator. The matured oocytes were fertilized with frozen-thawed spermatozoa and then cultured in a dish in the standard CO2 incubator for 8 days. There were no differences in the proportions of oocytes reaching metaphase II stage among the groups. However, the proportions of cleavage and development to blastocysts derived from oocytes matured in a straw were lower than those from oocytes matured in a dish, irrespective of the type of incubator used. In the second experiment, the COCs were matured in a dish in the standard CO2 incubator, and the matured oocytes were fertilized and then placed either in a dish or straw. These were then cultured for 8 days in the standard CO2 incubator or portable incubator. Some zygotes cultured in the portable incubator developed to the blastocyst stage. The proportions of cleavage and development to blastocysts were significantly lower for putative zygotes cultured in straw than for those cultured in dish, irrespective of the type of incubator used. Our results indicate that a portable incubator with a CO2 chamber can maintain the viability and development of oocytes/embryos, but the straw is not a suitable system for in vitro culture of the oocytes/embryos during transportation.

This study was conducted to examine the effects of (-)-epigallocatechin gallate (EGCG) supplementation on the developmental competence and quality of parthenogenetic porcine embryos during culture. Parthenogenetic embryos derived from in vitro matured oocytes were cultured for eight days in a modified North Carolina State University (NCSU)-37 solution supplemented with EGCG at different concentrations (0, 1, 5, 10 and 50 mu M). Supplementation of 1 and 5 mu M EGCG during in vitro culture of embryos showed no significant influence on the rate of cleavage or that of blastocyst formation or on the total cell number and DNA fragmentation indices of blastocysts when compared to those of a control group. However, when 10 and 50 mu M EGCG were supplemented into the culture medium, the cleavage rates were significantly lower than those of the other groups. No embryo developed to the blastocyst stage. Results suggest that treatment with low EGCG during in vitro culture has no influence on the developmental competence of porcine embryos but the presence of high concentrations of EGCG is apparently harmful for in vitro development of porcine parthenotes.

This study was conducted to investigate the influence of recipient cytoplasm on the development of somatic cell nuclear transfer (SCNT) embryos, using recipient oocytes and donor cells that were obtained from cats, cows, and pigs. Bovine and porcine oocytes were collected from ovaries obtained at a slaughterhouse, and cat oocytes were collected from ovaries obtained at local veterinary clinics following ovariohysterectomy. Cumulus cells from oocytes of each species were used as donors. When cat cumulus cells were transferred into cow, pig, and cat oocytes, the percentages of fusion and cleavage in the cow-cat and pig-cat interspecies groups were similar to those in the cat-cat intraspecies group. There were no significant differences in the percentages of fusion and cleavage between the interspecies (cow-cat and pig-cat groups) and intraspecies SCNT embryos (cow-cow and pig-pig groups) in each recipient oocyte species. However, none of the interspecies SCNT embryos developed to the morula and blastocyst stage. The percentages of fusion and cleavage were significantly higher (p&lt;0.05) in cow-cat SCNT embryos than in pig-cat SCNT embryos. In conclusion, bovine and porcine cytoplasm can be used to support the early embryonic development of interspecies SCNT with cat donor nucleus. However, the interspecies SCNT embryos could not develop to the late embryonic stage.

Roscovitine, a specific inhibitor of M-phase promoting factor kinase activity was used to inhibit the completion of meiotic maturation of bovine oocytes. The objectives of this study were to evaluate the nuclear maturation of bovine oocytes pre-cultured with various concentrations (0, 50, 100 and 200 mu M) of roscovitine before in vitro Maturation (IVM) and to examine the development of Somatic Cell Nuclear Transfer (SCNT) embryos derived from the oocytes pre-cultured with roscovitine. Before IVM, 72% of oocytes that were cultured without roscovitine (control) had reached the Metaphase II (MII) stage whereas culture with roscovitine decreased the rates of oocytes reaching MII (11-27%). After IVM, the maturation rate of oocytes pre-cultured with 200 mu M roscovitine was significantly higher than that of control oocytes (79 vs. 58%). Moreover, significantly more oocytes extruded the first polar body in the 50 mu M roscovitine group than in the control group (64 vs. 51%). The rate of blastocyst formation of reconstructed embryos derived from oocytes pre-cultured with 50 mu M roscovitine was significantly higher than that from the control oocytes (14 vs. 6%). In this study, the addition of roscovitine to culture medium delays the completion of meiotic maturation of bovine oocytes and the cytoplasm derived from oocytes pre-cultured under meiotic inhibition can support the development of SCNT embryos.

The present study was conducted to investigate the effects of the exposure length of ovaries to an elevated temperature (41 degrees C) on the meiotic competence and DNA damage of oocytes. Ovaries were stored in physiological saline at 41 degrees C for 0 h (control), 0.5, 1.0 and 1.5 h. After exposure of ovaries to the elevated temperature, oocytes were collected and then cultured for 44 h. The length of exposure of ovaries to 41 degrees C had no effect on the proportions of total oocytes with DNA-fragmented nuclei before maturation culture, but it did influence the proportions at the end of maturation culture. The proportion of oocytes reaching metaphase II (MII) significantly decreased with increasing exposure time. In addition, significantly more oocytes from ovaries exposed to 41 degrees C for 1.5 h had DNA-fragmented nuclei compared with control oocytes. These results indicate that the meiotic competence and DNA damage of porcine oocytes are dependent on the duration of exposure of ovaries to the elevated temperature. Moreover, the occurrence of DNA damage in oocytes becomes more apparent after maturation culture than before the culture.