Masayasu Taniguchi

Effect of sericin on preimplantation development of bovine embryos cultured individually

The silk protein sericin has been identified as a potent antioxidant in mammalian cells. This study was conducted to examine the effects of sericin on preimplantation development and quality of bovine embryos cultured individually. When two-cell-stage embryos were cultured individually for 7 days in CR1aa medium supplemented with 0, 0.1, 0.5, or 1% sericin, rates of total blastocyst formation and development to expanded blastocysts from embryos cultured with 0.5% sericin were higher (P < 0.05) than those from embryos cultured with 0 or 1% sericin. When embryos were cultured individually for 7 days in the CR1aa medium supplemented with 0 or 0.5% sericin under two oxidative stress conditions (50 or 100 μm H(2)O(2)), the addition of sericin significantly improved the blastocyst formation rate of embryos exposed to 100 μm H(2)O(2). However, the protective effect of sericin was not observed in development of embryos exposed to 50 μm H(2)O(2). When embryos were exposed to 100 μm H(2)O(2) during culture, the DNA fragmentation index of total blastocysts from embryos cultured with 0.5% sericin was lower than blastocysts derived from embryos cultured without sericin (4.4 vs. 6.8%; P < 0.01). In conclusion, the addition of 0.5% sericin to in vitro culture medium improved preimplantation development and quality of bovine embryos cultured individually by preventing oxidative stress.

Effects of (−)-Epigallocatechin Gallate on the Motility and Penetrability of Frozen–Thawed Boar Spermatozoa Incubated in the Fertilization Medium

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 frozen-thawed spermatozoa were incubated in IVF medium supplemented with 0 (control), 1, 50 and 100 μm EGCG for 1, 3 and 5 h, supplementation with 50 and 100 μm EGCG improved motility of the spermatozoa (p < 0.05), but not viability, as compared with the control group. When frozen-thawed spermatozoa were co-incubated with in vitro-matured (IVM) oocytes in IVF medium supplemented with 50 and 100 μm 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 frozen-thawed spermatozoa from six boars were co-incubated with IVM oocytes in IVF medium supplemented with 50 μm 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 μm EGCG, but the effects vary with individual boars.

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.

Melatonin Supplementation During In Vitro Maturation and Development Supports the Development of Porcine Embryos.

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 < 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 < 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 < 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.

Astaxanthin present in the maturation medium reduces negative effects of heat shock on the developmental competence of porcine oocytes

Astaxanthin, one of the most common carotenoids, elicits antioxidant effects on cellular viability and embryonic development. This study was conducted to investigate the effects of astaxanthin on maturation, fertilization and development of porcine oocytes matured in vitro under heat stress conditions, and then fertilized and cultured under standard conditions. Porcine oocytes were cultured in maturation medium supplemented with different concentrations of astaxanthin (0, 0.25, 0.5 or 1 ppm) for 46 h at either 38.5 or 41 °C. In comparison to oocytes cultured at 38.5 °C, the exposure of porcine oocytes to 41.0 °C during in vitro maturation (IVM) significantly inhibited maturation and development of fertilized oocytes to the blastocyst stage. Supplementation of maturation medium with astaxanthin (0.5 ppm) significantly improved oocyte maturation, fertilization and development to the blastocysts stage in both oocyte groups. However, the total cell number and apoptosis index of blastocysts did not differ among groups. Moreover, astaxanthin (0.5 ppm) significantly increased the rate of oocytes that reached metaphase II and decreased proportion of apoptotic oocytes exposed to H2O2 (1.0mM) during IVM. In summary, we demonstrated that supplementation of maturation medium with astaxanthin (0.5 ppm) exerted antioxidative effects and improved the ability of maturation, fertilization, and development of porcine oocytes exposed to heat stress.

Effects of green tea polyphenol on the quality of canine semen after long-term storage at 5°C.

The aim of the present study was to determine the effects of green tea polyphenol on the quality of canine semen after long-term storage at 5°C. The supplementation of a Tris-egg yolk extender with polyphenol (0.5, 0.75, or 1mg/mL) increased the motility and viability of sperm preserved for four weeks at 5°C.

Effect of Sericin Supplementation During In Vitro Maturation on the Maturation, Fertilization and Development of Porcine Oocytes

This study aimed to examine the effects of sericin supplementation during in vitro oocyte maturation on the nuclear maturation, fertilization and development of porcine oocytes. Cumulus-oocyte complexes (COCs) were cultured in maturation medium supplemented with 0 (control), 0.1, 0.5, 1.0, 2.5 or 5.0% sericin and were then subjected to in vitro fertilization and embryo culture. More COCs matured with 1.0% sericin underwent germinal vesicle breakdown and reached metaphase II compared with the control COCs matured without sericin (p < 0.01). The proportions of oocytes with DNA-fragmented nuclei did not differ between the groups, regardless of the sericin level. The total fertilization rate of oocytes matured with 1.0% sericin was higher (p < 0.05) than that of oocytes matured with 0.1%, 2.5% and 5.0% sericin. Supplementation with more than 1.0% sericin decreased the DNA fragmentation index of the blastocysts compared with the control group (p < 0.05). However, the supplementation of the maturation medium with sericin had no beneficial effects on the cleavage, development to the blastocyst stage and the total cell number of the embryos. Our findings indicate that supplementation with 1.0% sericin during maturation culture may improve the nuclear maturation and the quality of the embryos but does not affect blastocyst formation.

Motility and fertility of boar semen after liquid preservation at 5°C for more than 2 weeks.

This study investigated the effects of skim milk on the quality and fertility of boar spermatozoa under long-term chilled preservation. Semen samples were stored in Modena solution supplemented with 0 (control) to 50 mg/mL skim milk at 5°C for 4 weeks; spermatozoa stored with 7.5 and 15 mg/mL of skim milk (7.5-SM and 15-SM groups, respectively) exhibited significantly higher motility indices than those of the control group up to 3 weeks (P < 0.05), and the 7.5-SM group showed improved motility indices even after 4 weeks (P < 0.05). In vitro fertilization using spermatozoa in the 7.5-SM and 15-SM groups stored at 5°C for 2 weeks showed significantly higher fertilization rates of spermatozoa and the development rates to blastocyst than the control group (P < 0.05), and the 7.5-SM group showed similar rates of fertilization and blastocyst formation in the fresh non-stored spermatozoa group. After artificial insemination using spermatozoa stored for 2 weeks in the 7.5-SM group, healthy piglets were obtained. Boar spermatozoa can be stored at 5°C in a Modena solution containing skim milk. Supplementation of 7.5 mg/mL skim milk improves boar spermatozoa motility and fertility even after liquid preservation at 5°C for 2 weeks.

Development and subsequent cryotolerance of domestic cat embryos cultured in serum-free and serum-containing media.

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<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.

Histone Deacetylase Inhibitor Improves the Development and Acetylation Levels of Cat–Cow Interspecies Cloned Embryos

Abnormal epigenetic reprogramming, such as histone acetylation, might cause low efficiency of interspecies somatic cell nuclear transfer (iSCNT). This study was conducted to evaluate the effects of trichostatin A (TSA) on the developmental competence and histone acetylation of iSCNT embryos reconstructed from cat somatic cells and bovine cytoplasm. The iSCNT cat and parthenogenetic bovine embryos were treated with various concentrations of TSA (0, 25, 50, or 100 nM) for 24 h, respectively, following fusion and activation. Treatment with 50 nM TSA produced significantly higher rates of cleavage and blastocyst formation (84.3% and 4.6%, respectively) of iSCNT embryos than the rates of non-TSA-treated iSCNT embryos (63.8% and 0%, respectively). Similarly, the treatment of 50 nM TSA increased the blastocyst formation rate of parthenogenetic bovine embryos. The acetylation levels of histone H3 lysine 9 (H3K9) in the iSCNT embryos with the treatment of 50 nM TSA were similar to those of in vitro-fertilized embryos and significantly higher (p<0.05) than those of non-TSA-treated iSCNT embryos (control), irrespective of the embryonic development stage (two-cell, four-cell, and eight-cell stages). These results indicated that the treatment of 50 nM TSA postfusion was beneficial for development to the blastocyst stage of iSCNT cat embryos and correlated with the increasing levels of acetylation at H3K9.