Influence of freezing with liquid nitrogen on whole-knee joint grafts and protection of cartilage from cryoinjury in rabbits.

Improving survival rates for sarcoma patients are necessitating more functional and durable methods of reconstruction after tumor resection. Frozen osteoarticular grafts are utilized for joint reconstruction, but the joint may develop osteoarthritic change. We used a frozen autologous whole-rabbit knee joint graft model to investigate the influence of freezing on joint components. Thirty rabbit knee joints that had been directly immersed into liquid nitrogen (L) or saline (C) without use of cryoprotectants were re-implanted. Histological observations were made after 4, 8, and 12 weeks. Both groups had bone healing. In group L, despite restoration of cellularity to the menisci and ligaments, no live chondrocytes were observed and cartilage deterioration progressed over time. It was concluded that cryoinjury of chondrocytes caused osteoarthritic change. Then we tested whether a vitrification method could protect cartilage from cryoinjury. Full-thickness articular cartilage of rabbit knee was immersed into liquid nitrogen with and without vitrification. Histology, ultrastructure, and chondrocyte viability were examined before and after 24h of culture. Vitrified cartilage cell viability was >85% compared with that of fresh cartilage. Transmission electron microscopy revealed preservation of original chondrocyte structure. Our vitrification method was effective for protecting chondrocytes from cryoinjury. Since reconstructing joints with osteoarticular grafts containing living cartilage avert osteoarthritic changes, vitrification method may be useful for storage of living cartilage for allografts or, in Asian countries, for reconstruction with frozen autografts containing tumors.

Meiotic competence and DNA damage of porcine oocytes exposed to an elevated temperature.

The present study was conducted to investigate the effects of the length of exposure to an elevated temperature (41 degrees C) on the meiotic competence and DNA damage of porcine oocytes. Oocytes were recovered from ovaries, loaded into straws, and then exposed at 41.0 or 38.5 degrees C (sham control) for 0, 0.5, 1.0, or 1.5h, followed by culture for 44 h. The proportion of oocytes reaching the metaphase II (MII) stage gradually decreased with increasing exposure time, irrespective of the exposure temperature. A higher proportion of oocytes stored at 38.5 degrees C reached MII (57-63%) than those exposed to 41 degrees C (14-29%; P<0.01). The proportion of total oocytes with DNA fragmentation gradually increased with increasing exposure time, irrespective of the exposure temperature. The proportion of DNA fragmentation in total oocytes exposed to 41 degrees C (37-57%) was higher (P<0.01) than that in total oocytes stored at 38.5 degrees C (14-24%). When the oocytes were stored at 38.5 degrees C for up to 1.5 h, there were no differences in the proportions of MII-stage oocytes, with DNA-fragmented nuclei among all groups (P>0.05). However, a higher proportion of MII-stage oocytes exposed to 41 degrees C for more than 1h exhibited DNA-fragmented nuclei, compared with MII-stage oocytes stored at 38.5 degrees C (P<0.05). In conclusion, exposure of porcine oocytes to an elevated temperature had a detrimental effect on the meiotic competence and quality of oocytes; furthermore, the effect was dependent on the duration of exposure.

Effects of the reproductive status on morphological oocyte quality and developmental competence of oocytes after in vitro fertilization and somatic cell nuclear transfer in cat.

This study was conducted to examine the effects of the reproductive cycle of donor cat on the quality of oocytes at recovery and developmental competence of oocytes after in vitro fertilization (IVF) and somatic cell nuclear transfer (NT). Based on the presence or absence of follicles and corpora lutea, the ovarian pairs collected were classified into inactive, follicular or luteal stages. After collection of oocytes, the oocytes were classified into four grades according to the morphological condition of oocyte cytoplasm and cumulus cells. A total of 16 558 oocytes were obtained from 198 ovarian pairs. The total mean numbers of oocytes and the mean numbers of oocytes with high quality (grade I) were significantly higher in ovarian pairs at the inactive stage (111.1 and 19.0 oocytes, respectively) than in ovarian pairs at the follicular stage (67.1 and 11.4 oocytes, respectively). A significant difference in the proportions of oocytes with grade I out of the total examined oocytes was observed between the follicular and luteal stages of ovaries (14.9% vs 20.2%, p < 0.05). The proportions of IVF embryos cleaved and developed to blastocysts significantly decreased with decreased quality of oocytes at recovery, irrespective of the reproductive status of ovaries. Moreover, there were no significant differences in the proportions of cleavage and development to the blastocyst stage of IVF and NT embryos among three oestrous stages of ovaries. These results indicate that the reproductive cycle stage of donor cat ovaries has no apparent effects on the in vitro development of oocytes after IVF and NT, but the quality of oocytes at recovery influences the development of IVF embryos.

A rare giant scalp dermatofibrosarcoma protuberans.

BACKGROUND: Giant dermatofibrosarcoma protuberans (DFSP) of the scalp is a rare case, which is an intermediate grade soft tissue neoplasm originating from the dermal layer of the skin, which usually occurs in adults. CASE DESCRIPTION: We describe such a case in a 26-year-old male. A wide local excision of the tumor with a generous tissue margin was performed; microscopic and immunohistochemical findings established the diagnosis of recurrent DFSP. CONCLUSION: Our case is unique in that it is presented as a dermatofibrosarcoma protuberans of the scalp, which is an extremely rare clinical entity, and the patient remains well after 14 months with no further treatment, without any tumor recurrence.

Effects of oxygen tension and follicle cells on maturation and fertilization of porcine oocytes during in vitro culture in follicular fluid.

The objective was to investigate the effects of oxygen tension and follicle cells (FCs) during in vitro maturation of porcine oocytes in only porcine (Sus scrofa domesticus) follicular fluid (pFF), using static and non-static (rotating) culture systems, on the nuclear maturation and subsequent in vitro fertilization of the oocytes. In the first experiment, cumulus-oocyte complexes (COCs) were matured for 48 h in pFF supplemented with (+) or without (-) FCs (5.2x10(6) cells/mL), using the static (S) and rotating (R) culture systems (+FC/S, -FC/S, +FC/R, and -FC/R) under 5% or 20% O(2). Co-culture with FCs in the static culture system (+FC/S) had a detrimental effect on the meiotic competence of oocytes, whereas co-culture with FCs in the rotating culture system (+FC/R) increased maturation rates. In both culture systems, oxygen tension had no apparent effects on meiotic competence of oocytes, irrespective of culture system and FC addition. In the second experiment, COCs were matured under 5% or 20% O(2) using the -FC/S or +FC/R culture systems and then fertilized. Oxygen tension had no significant effects on fertilization parameters, irrespective of the culture system. The rotating culture system increased rates of sperm penetration and male pronuclear formation and decreased polyspermic fertilization compared with the static culture system (P < 0.05). In conclusion, both -FC/S and +FC/R culture systems supported meiotic competence, irrespective of oxygen tension. However, the +FC/R culture system may be superior to the -FC/S culture system for promoting fertilization.

Meiotic competence of canine oocytes embedded in collagen gel.

The present study was conducted to examine the meiotic competence of canine oocytes embedded in collagen gel, and to investigate the effects of timed exposure of the oocytes embedded in collagen gel to gonadotrophins during maturation culture, on their nuclear maturation. Cumulus-oocyte complexes (COCs) were collected from bitches at the anoestrous and dioestrous stages of the reproductive cycle. In the first experiment, half of the COCs were embedded in collagen gels. The COCs with or without collagen-gel embedding were cultured in a TCM-199 medium supplemented with 0.1 IU/ml human menopausal gonadotropin (hMG) and 10 IU/ml human chorionic gonadotropin (hCG) for 72 h. In the second experiment, the COCs embedded in collagen gels were cultured in TCM-199 medium with gonadotrophins (hMG and hCG) for various periods (0, 24, 48 and 72 h) and then cultured in the medium without gonadotrophins until reaching total culture period (72 h). The percentage of the oocytes reaching metaphase I and metaphase II (MI/MII) was significantly higher (p < 0.05) in COCs with collagen-gel embedding than in COCs without collagen-gel embedding. The percentage of oocytes that were arrested at the germinal vesicle stage was significantly lower (p < 0.05) in oocytes cultured with gonadotrophins than in oocytes cultured without gonadotrophins. However, there were no significant differences in the percentages of oocytes that reached each stage of meiosis among the groups, irrespective of the duration of exposure to gonadotrophins. These observations indicate that embedding of COCs by collagen gel enhances the meiotic competence of canine oocytes, but removal of hormone supplement from maturation medium does not improve the ability of the oocytes to reach MII stage.

Effect of the removal of cumulus cells on the nuclear maturation, fertilization and development of porcine oocytes.

The present study was conducted to investigate the effects of attachment of cumulus cells to porcine oocytes during the process of maturation and fertilization on the nuclear maturation, fertilization and subsequent development after in vitro fertilization (IVF). In the first experiment, the cumulus cells were removed from cumulus-oocyte complexes (COCs) at 0, 24 and 42 h after the onset of maturation culture and were then cultured until reaching 42 h of cultivation. In the second experiment, COCs were denuded as described in the first experiment, then fertilized and cultured for 7 days. As a control, cumulus cells were allowed to maintain attachment to the oocytes until the end of IVF. The proportion of oocytes reaching metaphase II significantly increased with the delay in the removal treatment of cumulus cells. The proportion of normal fertilization gradually increased with delay in the removal treatment of cumulus cells from COCs until the end of IVF. However, no significant difference in the proportion of normal fertilization was found between the 42-h and control groups. The removal treatment of cumulus cells in the 0- and 24-h group significantly (p < 0.05) decreased the proportion of cleaved embryos when compared with the control, and none of them developed to the blastocyst stage. The proportion of development to the blastocyst stage was significantly higher (p < 0.05) in the control group than in the 42-h group (18.1% vs 12.4%; p < 0.05). The present study indicates that the attachment of cumulus cells to the oocyte during maturation and fertilization is important to support oocyte nuclear maturation, fertilization and subsequent embryo development. Particularly, the attachment of cumulus cells to the oocyte during IVF promotes embryonic development.

Development of cat embryos produced by intracytoplasmic injection of spermatozoa stored in alcohol.

This study was conducted to examine whether cat spermatozoa stored in ethanol for 1 month was capable of developing into pronuclei and of supporting normal embryonic development. In vitro matured oocytes were injected with frozen-thawed spermatozoa and ethanol-stored spermatozoa. The status of oocytes and sperm nuclei was examined at 4 and 18 h after injection of spermatozoa, and the presumptive zygotes were cultured for 7 days to assess the development of oocytes injected with each storage sperm. The percentage of enlarged sperm head at 4 h after injection was higher in ethanol-stored spermatozoa than in frozen-thawed spermatozoa, but there was no significant difference in the development of oocytes and sperm nuclei at 18 h after injection between the two groups. The development of oocytes to the blastocyst stage after injection of spermatozoa was observed only in oocytes with frozen-thawed spermatozoa. Of oocytes injected with ethanol-stored spermatozoa, two (2.8%) oocytes developed to the 16-cell stage. These results indicate that cat spermatozoa stored in ethanol can decondense and form male pronuclei after intracytoplasmic injection. However, the oocytes injected with ethanol-stored spermatozoa did not have the ability to develop to the blastocyst stage.

Relationship between oxygen consumption and sex of bovine in vitro fertilized embryos.

The present study was conducted to examine the relationship between the oxygen consumption rate and sex ratio of bovine in vitro fertilized embryos on each day of blastocyst formation. The quality of blastocysts collected on day 7, 8, and 9 after in vitro fertilization (IVF) were categorized as ranks A and B (excellent and good, respectively) based on microscopic observation of the morphology. The oxygen consumption rate and sex of individual blastocysts were evaluated using two novel techniques: scanning electrochemical microscopy (SECM) and loop-mediated isothermal amplification (LAMP), respectively. The oxygen consumption rates of embryos of rank A were significantly higher (p < 0.05) than those of rank B, irrespective of the day of blastocyst appearance after IVF. Neither did the proportion of male embryos of ranks A and B differ significantly from each other at any of the days examined, nor from the average proportion (53%). The oxygen consumption rate of embryos of rank B collected on day 8 was significantly higher (p < 0.05) in female embryos than in male embryos collected on the same day. However, there were no apparent differences of oxygen consumption rates at each day of blastocyst appearance between male and female embryos of rank A. These results indicate that the oxygen consumption rate of individual embryos reflects their quality but does not correlate with the sex ratio of embryos of excellent quality.

Analysis of DNA fragmentation of porcine embryos exposed to cryoprotectants.

The chemical toxicity of cryoprotectants to porcine embryos was examined by the evaluation of survival and DNA damage after exposure to cryoprotectants. Porcine blastocysts were exposed to 10% of ethylene glycol (EG), 1,2-propanediol (PD) or glycerol (GLY) for 1 h at room temperature (23-25 degrees C) and then cultured in vitro for 24 h. The survival rates of blastocysts exposed to PD and GLY were significantly lower than those of control blastocysts in which the embryos were exposed to carrier solution without cryoprotectants. Significantly more DNA-fragmented nuclei occurred in the cryoprotectant-exposed blastocysts, compared with the control blastocysts. Moreover, the indices of DNA-fragmented nuclei in the blastocysts without blastocoele re-formation after culture were significantly higher than those with blastocoele re-formation, irrespective of the exposure treatment. These results indicate that the exposure of porcine blastocysts to cryoprotectant decreases the survival rates and increases the DNA-fragmented nuclei in embryos.

Blastocysts derived from in vitro-fertilized cat oocytes after vitrification and dilution with sucrose.

Experiments were conducted to find an optimal incubation period in a sucrose solution during dilution of cryoprotectants for obtaining a higher level of survival and development of cat oocytes cryopreserved by vitrification method. In the first experiment, in vitro-matured fresh oocytes were exposed to 0.5M sucrose solution for 1 or 5 min before in vitro fertilization (IVF). The percentage of development to the blastocyst stage significantly decreased in oocytes exposed for 5 min, compared with oocytes exposed for 1 min and control oocytes without exposure to sucrose (P<0.05). In the second experiment, oocytes that had been vitrified in 40% ethylene glycol and 0.3M sucrose were liquefied and then incubated in 0.5M sucrose for 0.5, 1 or 5 min to dilute the cryoprotectant. The percentage of cleavage (>or=2-cell stage) of vitrified-liquefied oocytes incubated for 0.5 min was significantly higher (P<0.05) than that of other groups. Development of vitrified-liquefied oocytes to the morula and blastocyst stages after IVF was observed only in oocytes incubated in sucrose for 0.5 min. The present study indicates that the oocytes have sensitivity to the toxic effect of sucrose and that the incubation period during dilution of the cryoprotectant is of critical importance for developmental competence of vitrified-liquefied cat oocytes.

Effects of cooling ovaries before oocyte aspiration on meiotic competence of porcine oocytes and of exposing in vitro matured oocytes to ambient temperature on in vitro fertilization and development of the oocytes.

Experiments were conducted to evaluate the effects of cooling porcine ovaries to low temperature (4 degrees C, 15 degrees C, 20 degrees C, 25 degrees C or 30 degrees C) for 1 h on the meiotic competence of their oocytes. Moreover, it was determined whether or not the exposure of in vitro matured oocytes to ambient temperature (20 degrees C, 25 degrees C or 30 degrees C) for 1 h affects the fertilization and developmental competence of the oocytes. There was no difference between the proportions of oocytes that underwent maturation to metaphase II when isolated from control ovaries held at 35 degrees C and ovaries exposed to 30 degrees C. However, the percentages of oocytes from ovaries exposed to 25 degrees C or less were significantly lower than those of oocytes from ovaries exposed to 30 degrees C and control ovaries. The proportions of total and normal fertilization of oocytes that had been exposed to 20 degrees C before in vitro fertilization (IVF) were significantly lower than those of control oocytes maintained at 38.5 degrees C. However, cooling in vitro matured oocytes had no effects on their cleavage and development to blastocysts after IVF. These data suggest that exposing porcine ovaries to a low temperature of 25 degrees C or less before aspiration of oocytes may adversely affect their subsequent in vitro maturation. It may be necessary to maintain the oocytes at a temperature of more than 25 degrees C during manipulation of oocytes for retaining the fertilizability of the oocytes.