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Sakagami N.,Kanagawa Prefectural Livestock Industry Technology Center | Nishida K.,Kanagawa Prefectural Livestock Industry Technology Center | Akiyama K.,Kanagawa Prefectural Livestock Industry Technology Center | Abe H.,Yamagata University | And 3 more authors.
Theriogenology | Year: 2015

Oxygen consumption rate of invivo-derived porcine embryos was measured, and its value as an objective method for the assessment of embryo quality was evaluated. Embryos were surgically collected 5 or 6days after artificial insemination (AI), and oxygen consumption rate of embryos was measured using an embryo respirometer. The average oxygen consumption rate (F×1014/mol s-1) of the embryos that developed to the compacted morula stage on Day 5 (Day 0=the day of artificial insemination) was 0.58±0.03 (mean±standard error of the mean). The Day-6 embryos had consumption rates of 0.56±0.13, 0.87±0.06, and 1.13±0.07 at the early blastocyst, blastocyst, and expanded blastocyst stages, respectively, showing a gradual increase as the embryos developed. Just after collection, the average oxygen consumption rates of embryos that hatched and of those that did not hatch after culture were 0.60±0.04 and 0.50±0.04 for Day 5 (P=0.08) and 1.05±0.09 and 0.77±0.05 for Day 6 (P<0.05), respectively. The value and probability of discrimination by measuring the oxygen consumption rates of embryos to predict their hatching ability after culture were 0.56 and 63.6% for Day-5 embryos and 0.91 and 68.4% for Day-6 blastocysts, respectively. When Day-5 embryos were classified based on the oxygen consumption rate and then transferred non-surgically to recipient sows, three of the seven sows, to which embryos having a high oxygen consumption rate (≥0.59) were transferred, became pregnant and farrowed a total of 20 piglets. However, none of the four sows, to which embryos having low oxygen consumption rate (<0.59) were transferred, became pregnant. These results suggest that the viability of invivo-derived porcine embryos and subsequent development can be estimated by measuring the oxygen consumption rate. © 2015 Elsevier Inc. Source


Watanabe A.,Yamagata University | Takayama-Watanabe E.,Research Institute for the Functional Peptides | Vines C.A.,University of California at Davis | Cherr G.N.,University of California at Davis
Development Growth and Differentiation | Year: 2011

Sperm motility-initiating substance (SMIS), a novel motility inducer from newt egg-jelly, is activated by the release from associated jelly substances at the beginning of internal fertilization and affects female-stored sperm. We examined motility initiation kinetics of newt sperm in response to SMIS by monitoring the changes of sperm intracellular calcium ([Ca2+]i). In quiescent non-motile sperm loaded with the Ca2+ indicator Fluo-4, intracellular free Ca2+ was observed around mitochondria using confocal scanning laser microscopy. A slight increase in [Ca2+]i occurred simultaneously and transiently at motility initiation in sperm treated with either heated jelly extract (hJE) containing activated SMIS, or a low osmotic solution, which naturally initiates motility in externally-fertilizing amphibians and can initiate motility in urodele sperm. When the increase of [Ca2+]i at motility-initiation was monitored using spectrofluorometry, large increases in [Ca2+]i occurred immediately in the low osmotic solution and within 1.5min in the hJE. In the intact jelly extract (no heating), small increases of [Ca2+]i irregularly occurred from around 1min and for about 4min, during which motility was differentially initiated among sperm. These results indicate that the SMIS induces differential initiation of sperm motility depending on the activational states of the SMIS and its overall activity. The motility initiation in the jelly extract was delayed in sperm whose intracellular Ca2+ had been chelated with BAPTA-AM. The relative levels of [Ca2+]i were variable with a mean of 414±256nmol/L among resting sperm, suggesting that the level of [Ca2+]i in the resting sperm modulates the responsiveness to the SMIS. © 2011 The Authors. Journal compilation © 2011 Japanese Society of Developmental Biologists. Source


Mito T.,Research Institute for the Functional Peptides | Yoshioka K.,Japanese National Institute of Animal Health | Noguchi M.,Japanese National Institute of Animal Health | Noguchi M.,Kagoshima University | And 2 more authors.
Molecular Reproduction and Development | Year: 2013

The biological functions of recombinant human follicle-stimulating hormone (FSH) and transforming growth factor-α (TGF-α) on in vitro maturation of porcine oocytes were investigated. Cumulus-oocyte complexes were matured in defined porcine oocyte medium containing 0-0.1IU/ml FSH in the presence or absence of 10ng/ml TGF-α. The percentage of oocytes reaching metaphase II was significantly higher with the addition of 0.01-0.1IU/ml FSH compared with no addition, and was further enhanced in the presence of TGF-α. The rates of sperm penetration and blastocyst formation were significantly higher with the addition of 0.05-0.1IU/ml FSH compared with no addition after in vitro fertilization and embryo culture. There was no beneficial effect of FSH and TGF-α on nuclear maturation of denuded oocytes. The specific EGF receptor inhibitor, AG1478, completely inhibited TGF-α-induced meiotic resumption, but did not completely prevent the stimulatory effect of FSH. Addition of both FSH and TGF-α significantly enhanced cumulus expansion compared with no addition. When cumulus expansion-related genes (HAS2, HAPLN1, and VCAN) mRNA expression in COCs was measured during in vitro maturaiton, addition of both of FSH and TGF-α upregulated the expression of HAS2 mRNA after 20hr culture and HAPLN1 mRNA after 44hr culture compared with no addition. Expression of VCAN mRNA was significantly higher in the presence of FSH compared with addition of TGF-α alone. These results suggest that FSH and TGF-α synergistically enhance porcine oocyte maturation via cumulus cells, and act through different signaling pathways. © 2013 Wiley Periodicals, Inc. Source


Mito T.,Research Institute for the Functional Peptides | Yoshioka K.,Japanese National Institute of Animal Health | Noguchi M.,Azabu University | Yamashita S.,Research Institute for the Functional Peptides | And 3 more authors.
Theriogenology | Year: 2015

We examined the effect of different embryo developmental stages, culture periods, and media on the cryotolerance of in vitro-produced porcine blastocysts. All media used for in vitro production, vitrification, and warming were chemically defined. When in vitro-produced embryos at the blastocyst and expanded blastocyst stages were vitrified using the Cryotop method on Day 5 or 6 (Day 0 = the day of IVF), the survival rate and hatching rate of expanded blastocysts after warming were higher than those of blastocysts. The viability after vitrification and warming of Day-6 embryos cultured in porcine blastocyst medium from Day 5 were higher than that of embryos cultured in porcine zygote medium 5. On the other hand, there were no significant differences on the cryotolerance between Day-5 embryos cultured in porcine zygote medium 5 and those replaced with porcine blastocyst medium on Day 4. There were no significant differences in viability between the embryonic ages of 5 and 6 days after vitrification and warming. When expanded blastocysts vitrified on Day 5 or 6 were surgically transferred to recipient gilts, all three recipients became pregnant in the Day-5 group, whereas only one out of three recipients became pregnant in the Day-6 group. These results indicate that the cryotolerance of porcine in vitro-produced blastocysts after vitrification appears to depend on the embryonic stage, culture period, and medium. © 2015 Elsevier Inc. Source


Sakagami N.,Kanagawa Prefectural Livestock Industry Technology Center | Nishida K.,Kanagawa Prefectural Livestock Industry Technology Center | Misumi K.,National Livestock Breeding Center | Hirayama Y.,National Livestock Breeding Center | And 6 more authors.
Animal Reproduction Science | Year: 2016

The aim of this study was to assess the viability of vitrified-warmed in vivo-derived pig embryos after measuring the oxygen consumption rate. Six days after artificial insemination, blastocysts were collected from gilts and vitrified by the micro volume air cooling method. The oxygen consumption rate was measured in 60 vitrified-warmed embryos, which were then cultured for 48. h to assess the viability. The survival (re-expansion) rate of embryos after warming was 85.0%. The average oxygen consumption rate of embryos immediately after warming was greater in embryos which could re-expand during subsequent culture (F=0.75 ± 0.04) than that in those which failed to re-expand (F=0.33 ± 0.05). Moreover, the oxygen consumption rate of vitrified-warmed embryos was greater in the hatched (F=0.88 ± 0.06) than that in the not-hatched group (F=0.53 ± 0.04). When the oxygen consumption rate of the vitrified-warmed embryos and the numbers of viable and dead cells in embryos were determined, there was a positive correlation between the oxygen consumption rate and the number of live cells (P< 0.01, r=0.538). A total of 29 vitrified embryos after warming and measuring the oxygen consumption rate were surgically transferred into uterine horns of two recipients. Both of the recipients become pregnant and farrowed 12 healthy piglets. These results demonstrate that the oxygen consumption rate of vitrified-warmed pig embryos can be related to the number of live cells and that the measurement of oxygen consumption of embryos after cryopreservation may be useful for estimating embryo survivability. © 2015 Elsevier B.V. Source

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