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M. Kuwayama, S. Hamano, and T. Nagai

Summary. Two experiments were conducted to assess the viability of bovine blastocysts obtained by in vitro fertilization of oocytes matured in vitro (IVM–IVF) and cryopreserved by vitrification. In Expt 1, the optimal concentrations of glycerol and 1,2-propanediol in the basic medium (modified TCM199) for cooling and warming without formation of ice crystals were determined by plunging the solution into liquid nitrogen and then warming it in a water bath at 15°C; when both glycerol and 1,2-propanediol were present in the solution (>45% v/v), vitrification of the medium was observed. In Expt 2, IVM–IVF blastocysts were equilibrated to the mixture of glycerol and 1,2-propanediol (0% to 45%) at 15°C in a stepwise manner as follows: (i) in one step, for 18 min to the final vitrification solution; (ii) in two steps, for 8 min in the first step and 10 min in the second step; (iii) in four steps, for 4 min in the first three steps and 6 min in the last step; (iv) in eight steps, for 2 min in each step, but 4 min in the last step; and (v) in 16 steps, for 1 min in each step, but 3 min in the last step. After removal of cryoprotectants, the blastocysts were cultured for 24 h in vitro. The survival rates for the embryos equilibrated in 1, 2, 4, 8 and 16 step(s) were 56, 89, 100, 100 and 100%, respectively. The blastocysts equilibrated in 1, 2, 4, 8 and 16 steps were vitrified by plunging the straws containing them into liquid N2, thawed and cultured in vitro. Higher survival rates were obtained for blastocysts equilibrated in 4, 8 and 16 steps (79, 82 and 87%, respectively) than for those in one (0%) or two (10%) steps. Ten blastocysts that survived after vitrification were transferred to ten recipients and six of these became pregnant. These results indicate that vitrification can be used for cryopreservation of blastocysts obtained by in vitro culture of IVM–IVF bovine follicular oocytes.

Keywords: in vitro; vitrification; fertilization; embryo transfer; cow

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K. Kwamoto, K. Ikeda, N. Yonezawa, S. Noguchi, K. Kudo, S. Hamano, M. Kuwayama, and M. Nakano

The time for solubilization of the bovine zona pellucida in a hypotonic buffer containing 5% (v/v) β-mercaptoethanol and 7 mol urea l−1 increased by 10% after fertilization. Coupling with a specific fluorescent thiol probe, monobromobimane (mBBr), was markedly greater in the zona pellucida of ovarian eggs compared with fertilized eggs, indicating that the cysteine residues in the zona pellucida of unfertilized eggs are oxidized to cystines during fertilization. After endo-β-galactosidase digestion to remove N-acetyllactosamine repeats of the carbohydrate chains, three zona pellucida glycoproteins (ZPA, ZPB and ZPC) coupled with the fluorescent bimane groups were fractionated efficiently by reverse-phase HPLC. Estimation of bimane groups in the three components and SDS-PAGE revealed that intramolecular disulfide bonds in ZPA and intra- and intermolecular disulfide bonds in ZPB were formed during fertilization, but oxidation of cysteine residues in ZPC was low. Specific proteolysis of ZPA during fertilization was also observed. These results indicate that the formation of disulfide linkages together with specific proteolysis result in the construction of a rigid zona pellucida structure, which is responsible for hardening of the zona pellucida.

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T. Nagai, T. Takahashi, H. Masuda, Y. Shioya, M. Kuwayama, M. Fukushima, S. Iwasaki, and A. Hanada

Summary. In Exp. 1 pig oocytes matured in vitro were used to evaluate the fertilizability in vitro of frozen epididymal (4 boars) and ejaculated (3 boars) spermatozoa that were preincubated in modified TCM-199 for 4 h at 37°C. The percentages of penetrated oocytes with the frozen epididymal spermatozoa were 0–40%. In contrast, none of the occytes were penetrated with the frozen ejaculated spermatozoa. In Exp. 2, oocytes matured in vivo were inseminated in vitro with the frozen epididymal spermatozoa that were known to penetrate oocytes matured in vitro. The penetration rate was 79% and the percentage of polyspermic oocytes was 57%. Culture for 30 h of oocytes matured in vivo and fertilized in vitro resulted in 51% (34/67) developing to the 2-cell stage. These embryos were transferred to 2 recipient gilts. One gilt became pregnant and a litter of 3 (1 live and 2 dead) was born. These results indicate that frozen epididymal spermatozoa can be used for in-vitro fertilization in the pig.

Keywords: in vitro; oocyte; fertilization; pig; embryo transfer

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M Endo, R Kawahara-Miki, F Cao, K Kimura, T Kuwayama, Y Monji, and H Iwata

Antrum formation and estradiol (E2) secretion are specific features of oocyte and granulosa cell complexes (OGCs). This study investigates the effect of E2 on the in vitro development of bovine OGCs derived from early antral follicles as well as on the expression of genes in granulosa cells (GCs). The supplementation of culture medium with either E2 or androstenedione (A4) improved the in vitro development of OGCs and the nuclear maturation of enclosed oocytes. When OGCs were cultured in medium containing A4, developmentally competent OGCs secreted more E2 than OGCs that were not competent. In addition, fulvestrant inhibited the effect of both E2 and A4 on OGCs development. Comprehensive gene expression analysis using next-generation sequence technology was conducted for the following three types of GCs: i) GCs of OGCs cultured for 4 days with E2 (1 μg/ml; E2(+)), ii) GCs of OGCs cultured for 4 days without E2 (E2(−)) or iii) OGCs that formed clear antrum after 8 days of in vitro culture in medium containing E2 (1 μg/ml; AF group). GCs of the E2(+) group had a similar gene expression profile to the profile reported previously for the in vivo development of large follicles. This genetic profile included factors implicated in the up-regulation of E2 biosynthesis and down-regulation of cytoskeleton and extracellular matrices. In addition, a novel gene expression profile was found in the AF group. In conclusion, E2 impacts the gene expression profile of GCs to support the in vitro development of OGCs.

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T. Kuwayama, K. Shimada, N. Saito, T. Ohkubo, K. Sato, M. Wada, and K. Ichinoe

Summary. Gifujidori hens were allowed to repeat a breeding cycle in one season. In the first breeding cycle the duration of the brooding (raising chicks) stage was limited to 3 weeks, whereas in the second breeding cycle it was limited to 1 week by removing all chicks from mother hens. In the first breeding cycle, plasma prolactin (PRL) was high during the incubation period, but rapidly decreased on the day of hatching and reached minimum values about 1 week after hatching. In contrast, plasma luteinizing hormone (LH) concentrations were low during the incubation period, but after hatching they gradually increased and reached peak values immediately after removal of chicks. Concentrations of oestradiol in plasma were low in the incubation and brooding stages but increased significantly immediately after removal of chicks. In the second breeding cycle, changes in PRL and LH concentrations were similar to those observed in the first breeding cycle except that even greater increases in plasma LH and oestradiol concentrations were observed one week after hatching when the chicks were removed. These results suggest that coexistence of newly hatched chicks may suppress LH secretion from the pituitary of the hen in the natural breeding cycle.

Keywords: prolactin; LH; oestradiol; chicks; hen

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Y Du, C S Pribenszky, M Molnár, X Zhang, H Yang, M Kuwayama, A M Pedersen, K Villemoes, L Bolund, and G Vajta

The purpose of the present study was to improve cryotolerance using high hydrostatic pressure (HHP) pretreatment of porcine in vitro matured (IVM) oocytes, to facilitate their further developmental competence after parthenogenetic activation. A total of 1668 porcine IVM oocytes were used in our present study. The pressure tolerance and optimal duration of recovery after HHP treatment were determined. Oocytes were treated with either 20 or 40 MPa (200 and 400 times greater than atmospheric pressure) for 60 min, with an interval of 10, 70, and 130 min between pressure treatment and subsequent vitrification under each pressure parameter. Oocytes from all vitrification groups had much lower developmental competence than fresh oocytes (P<0.01) measured as cleavage and blastocyst rates. However, significantly higher blastocyst rates (P<0.01) were obtained in the groups of 20 MPa pressure, with either 70 (11.4±2.4%) or 130 (13.1±3.2%) min recovery, when compared with the vitrification control group without HHP treatment where no blastocysts were obtained. The influence of temperature at HHP treatment on further embryo development was also investigated. Treatments of 20 MPa with 70 min recovery were performed at 37 °C or 25 °C. Oocytes pressurized at 37 °C had a significantly higher blastocyst (14.1±1.4%) rate than those treated at 25 °C (5.3±1.1%; P<0.01). Our results demonstrate that HHP pretreatment could considerably improve the developmental competence of vitrified pig in vitro matured (IVM) oocytes. The HHP pretreatment will be tested as a means to improve survival and developmental competence at different developmental stages in different species including humans.