Masato Ooe

Effect of sucrose concentration used for one-step dilution upon in vitro and in vivo survival of bovine embryos refrigerated in glycerol and 1, 2-propanediol

Abstract Bovine embryos collected on Day 7 of pregnancy were suspended in 1.4 M glycerol or 1.6 M 1, 2-propanediol solution. Embryos were loaded into 0.25-ml plastic straws in phosphate buffered saline (PBS) containing 0, 0.2, 0.4 or 0.8 M sucrose. The straws were placed directly into a cooling chamber and cooled from 0°C to −6°C at 1°C/min and then seeded at −6°C and cooled at a rate of 0.3°C/min to −30°C. The straws were then plunged and stored in liquid nitrogen. Embryos frozen in each cryoprotectant were thawed by placing the straws into a 30°C water bath. The embryos were cultured in small dishes in Hams F-10 medium supplemented with 10% fetal calf serum (FCS). When the embryos were frozen in 0.0, 0.2, 0.4 or 0.8 M sucrose, respectively, in Hams F-10 the survival rates after culture were 0, 21, 82 or 88%, respectively, in 1.4 M glycerol and 88, 89, 84 or 72%, respectively, in 1.6 M 1, 2-propanediol. Embryos suspended in both cryoprotectants were cooled with 0.2 M sucrose in PBS, and the nonsurgical transfers were performed without removing the cryoprotectant. A pregnancy rate of 61% was obtained when embryos were suspended in 1.6 M 1, 2-propanediol and cooled with 0.2 M sucrose in PBS. However embryos suspended in 1.4 M glycerol and subjected to the same cooling regimen had a lower (P

Superovulation in the cow with a single intramuscular injection of FSH dissolved in polyvinylpyrrolidone

It is desirable to reduce the number of treatments required to induce superovulation in cows. In this study we examined whether dissolving FSH in polyvinylpyrrolidone(PVP) would reduce the rate of absorption of FSH and allow it to be administered in a single dose for superovulation. In Experiment 1, 10 cows each received a single dose of FSH which contains 0.6% luteinizing hormone (FSH-R; 30 mg i.m.) dissolved in 30% PVP (10 ml) or in saline. In Experiment 2, a single injection of 30 mg FSH-R dissolved in 30% PVP was given to 25 cows, and 32 cows were injected twice daily in declining doses to receive a total of 28 mg FSH-R dissolved in saline. Prostaglandin F2alpha was given to all the cows 48 h after the first FSH treatment. Embryos were collected on Day 7 or 8 post insemination. In Experiment 1, the effect of FSH dissolved in PVP was compared with that dissolved in saline (number of recovered ova and embryos; 9.4+/-4.1 vs. 0). In Experiment 2, the rate of transferable embryos by single injection of FSH-R in PVP were significantly higher (P<0.05) than that of treatment of multiple injection groups. Progesterone concentration measured in serum collected 4 times from estrus (Day-0) to the day of embryo collection, indicated similar patterns in the 2 treatment groups. These findings suggest that PVP is a suitable solvent for prolonging the absorption of FSH given in a single injection thus providing a more practical approach of FSH administration.

The effect of sperm-oocyte incubation time on in vitro embryo development using sperm from a tetraparental chimeric bull

The present study was designed as 5 x 4 factorial to investigate the effects of using sperm from 5 bulls, and varied sperm-oocyte incubation times (5, 10, 15 and 20 h) on the fertilization, cleavage rates and blastocyst formation on an in vitro bovine embryo production system. The bulls included a tetraparental Chimera, its sires (Japanese Black and Limousin), its maternal grand-sires (Japanese Brown and Holstein). The proportion of polyspermy, 2-pronuclei formation, fertilization, cleavage and development to blastocyst were affected (P < 0.001) by the duration of sperm-oocyte incubation, as well as by the interaction between bulls and their corresponding sperm-oocyte incubation time. Blastocyst rate observed after 5 h in oocytes inseminated with Chimera, Japanese Black and Limousin were higher (p < 0.05) than those observed at 20 h incubation. The proportion of blastocysts from oocytes inseminated with Japanese Black observed at 10 h of incubation did not differ from that of Chimera, but both were higher (p < 0.05) than those observed for the Limousin, Japanese Brown and Holstein sires. The present study showed that there was an effect by the duration of sperm-oocyte incubation on in vitro embryo development. The optimal time of sperm-oocyte incubation for the Chimera was similar to that of its sires (Japanese Black and Limousin) but differed from its maternal grand-sires (Japanese Brown and Holstein). The fertilization rates for the sperm from the Holstein bull increased up to 15 h suggesting that this might be the only bull that would benefit from a long incubation period for insemination.

Fertility of sperm from a tetraparental Chimeric bull

The purpose of the present study was to examine the ability of a tetraparental Chimera in producing IVF embryos. Cumulus oocytes complexes (COCs) were matured in vitro for 22 h. Frozen-thawed sperm of a Chimera (CH), as well as Japanese Black (JB), Limousin (L), Japanese Brown (JBr), Holstein (H) bulls were used for IVF. The chromosome preparations were made from peripheral lymphocytes. Based on chromosome analysis the Chimera had apparently normal chromosomes (29 acrocentric pairs, one large sub metacentric X chromosome and one small sub metacentric Y chromosome). The proportion of acrosome reacted spermatozoa after 1 h incubation was higher (P < 0.01) with the Chimera (CH) than with the Holstein and in Japanese Brown bulls, but did not differ from Japanese Black and Limousin bull sperm (79.0%, 71.2%, 72.5%, 57.8% and 57.0% for CH, JB, L, JBr and H sperm, respectively). Fertilization rates observed after 5 h of sperm-oocyte incubation with Chimera (O-CH) sperm were higher (P < 0.05) than with Japanese Brown (O-JBr) and (P < 0.01) than with Holstein (O-H) sperm, but did not differ from Japanese Black (O-JB) and Limousin (O-L) sperm (36/44, 81.8%; 28/35, 80.0%; 25/36, 69.4%; 19/43, 44.2% and 6/33, 18.2% for O-CH, O-JB, O-L, O-JBr and O-H, respectively). The cleavage rates of IVM oocytes inseminated with Chimera sperm were also higher (P < 0.001) than in Holstein, (P < 0.01) Japanese Brown and (P < 0.05) Limousin, but did not differ from Japanese Black sperm (181/239, 75.7%; 123/171, 71.9%; 108/186, 58.1%; 80/196, 40.8% and 30/186, 16.1% for O-CH, O-JB, O-L, O-JBr and O-H, respectively). The blastocyst rates of IVM oocytes inseminated with sperm were higher (P < 0.05) than in Limousin, Japanese Brown and Holstein, but did not differ from Japanese Black (69/181, 38.1%; 48/123, 39.0%; 27/108, 25.0%; 7/30, 23.3% and 16/80, 17.8% for O-CH, O-JB, O-L, O-JBr and O-H, respectively). Three findings suggested the sperm from this tetraparental Chimeric bull were able to be used for producing bovine IVF embryos.

Production of chimeric calves by aggregation of in vitro fertilized bovine embryos without zonae pellucidae

Abstract Bovine embryos produced by in vitro maturation (IVM), fertilization (IVF) and culture (IVC) were used to produce aggregation chimeras. An aggregated chimera was produced by combining bovine IVF embryos (Holstein × Japanese Black and Japanese Brown × Limousin breeds) which were cultured in vitro without the zonae pellucidae. Forty-eight hours after IVF, embryos at the 8 cell-stage were used to produce aggregation chimeras. In Experiment I, the zonae pellucidae was removed by a microsurgical method using a microblade or by treatment with 0.25% pronase. Holstein × Japanese Black embryos were aggregated with Japanese Brown × Limousin embryos after zonae removal by hand manipulation in culture medium. In Experiment II, the viability of the aggregated embryos developing into blastocysts was examined by measuring the extent of development. The number of aggregated embryos and embryos developed into blastocysts was 34 (91.9%) and 24 (70.6%), respectively, when the zonae pellucidae was removed by the microsurgical method; and 12 (92.3%) and 6 (50.0%), respectively, when the zonae pellucidae was removed using the 0.25% pronase treatment. The size of the aggregated embryos was significantly different from that of the normal embryos when cultured in vitro until Day 10, but not different thereafter. Five aggregated embryos were transferred nonsurgically to the recipients, resulting in 1 pregnancy and the birth of 2 chimeric calves. Skin color was used as evidence of chimerism.