The Journal of Reproduction and Development Supplement

The current study aimed to evaluate the diagnostic value of plasma anti-Müllerian hormone (AMH) as a biomarker for sex cord-stromal tumors (SCST) in cattle in comparison to immunoreactive inhibin (ir-INH), estradiol-17β (E₂) and testosterone (T). Therefore, we conducted two experiments. In experiment 1, four clinically healthy Japanese Black cows had blood samples taken daily over one estrous cycle to characterize their AMH profiles throughout the normal estrous cycle. Additionally, single blood samples were collected from 21 cyclic cows to clarify the physiological range of AMH. In experiment 2, animals with histologically confirmed SCST (SCST group, n = 10) and cows affected with cystic ovarian disease (COD group, n = 8) had one blood sample taken before extraction of SCST or treatment of COD. Blood samples (n = 105) from cyclic cows (n = 25) in experiment 1 were assigned as a physiologically cyclic group (PC group). Plasma AMH, ir-INH, E₂ and T concentrations were assayed in all samples. Plasma AMH concentrations did not show substantial fluctuation throughout the estrous cycle. Only plasma AMH, ir-INH and E₂ were significantly elevated in SCST group in comparison to PC group. The area under the receiver operating characteristic curve (AUC_ROC) of AMH for diagnosis of SCST was 0.99 and was significantly (P < 0.01) higher than that of ir-INH and E₂. Plasma AMH at a cutoff point of ≥ 0.36 ng/mL had the highest diagnostic accuracy (99.2%), sensitivity (100%), and specificity (99.1%) compared with ir-INH and E₂. In conclusion, unilateral ovarian enlargement accompanied with plasma AMH concentrations ≥ 0.36 ng/mL is considered diagnostic for bovine SCST.

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Identification of polyspermic embryos by non-invasive selection markers could be a possible way to select only diploid embryos for embryo transfer. Although previous studies suggested altered cleavage patterns in polyspermic porcine embryos, convincing evidence has not been provided to support this theory. Therefore, we compared the early developmental kinetics of monospermic and polyspermic porcine embryos by time lapse cinematography. Follicular oocytes were in vitro matured, fertilized (IVF) and cultured according to Kikuchi et al. (2002 Biol Reprod). Ten h after IVF, zygotes were centrifuged at 10000 × g and those with 2 pronuclei (monospermy, 2PN zygotes ; n=32) or more than 2 pronuclei (polyspermy, PPN zygotes; n=32) were selected under an inverted microscope. PPN, 2PN and non-centrifuged zygotes (n=64) were cultured separately in the well of well dishes. The timing and pattern of the first embryonic cleavage of each embryo were recorded by a Primo Vision Timelapse System for 48 h. Four replications were performed. Data were analyzed by paired t-test and ANOVA. There was no difference in the pattern of first cleavage between 2PN and PPN zygotes regarding the frequencies of fragmentation, protrusions or direct cleavage resulted in the first cleaved embryos with 3 or 4 blastomeres. However, PPN zygotes cleaved significantly earlier (P<0.05) compared to 2PN zygotes (29.1±1.2 and 31.0±1.1 h post IVF, respectively). Centrifugation treatment had no effect on the timing and pattern of first cleavage. In conclusion, the timing of the first cleavage but not its pattern can be considered as a potent marker for polyspermy in pigs. This project was supported by JSPS-HAS/JHRCP and 8526-5/2014/TUDPOL.

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Oxygen consumption is a useful parameter for evaluating mammalian embryo quality, since individual bovine embryos was noninvasively quantified by scanning electrochemical microscopy (SECM). Recently, several approaches have been used to measure the oxygen consumption rates of individual embryos, but relationship between oxygen consumption and pregnancy rates of hanwoo following embryo transfer has not yet been reported. In this study, we measured to investigate the correlation between oxygen consumption rate and pregnancy rates of hanwoo embryo using a SECM. In addition to, the expression of apoptosis-related genes was determined using real-time PCR by extracting RNA according to the oxygen consumption of in vivo embryo. First, we found that the oxygen consumption significantly increased in blastocyst-stage embryos(blastocyst) compared to early blastocyst stage embryos, indicating that oxygen consumption reflects the embryo quality (Grade I). The oxygen consumption or G I blastocysts were significantly higher than those of G II blastocysts(10.2×10¹⁴/mol s^-1 versus 6.4×10¹⁴/mol s^-1, p<0.05). Pregnant rate in recipient cow was 0, 60 and 80% in the transplantation of embryo with the oxygen consumption of below 10.0, 10.0~12.0 and over 12.0 × 10¹⁴/mol s^-1, respectively. Apoptosis regulatory genes, Hsp-70.1 were significantly increased in over-10.0 group than below 10.0 groups but in Caspase-3, Bax and P53 gene, there was no significant difference. In conclusion, these results suggest that measurement of oxygen consumption maybe help increase the pregnant rate of hanwoo embryos.

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Cryopreservation is able to store the surplus pre-embryos for freezing and furthermore thawing and transfer in a subsequent cycle. This study was carried out to evaluate the effects of embryonic stage, cryoprotectant, and freezing-thawing method on the rates of survival and development of the cryopreserved mouse early embryo and finally to establish the cryopreservation method of surplus embryos obtained during assisted reproductive technology (ART). Female ICR mice (6–8 weeks old) were induced to superovulate by sequential intraperitoneal injection of 5 IU PMSG and 5 IU hCG 48h apart. Mouse embryos were collected according to its developmental stage after the injection of hCG. Embryos were cryopreserved not only by cryoprotectant step (1 step–4 step) but also in a variety of media (HT F, IVF medium, D-PBS) and cell stage. The results were as follows : There is no clear advantage in these freezing media of rapid method, but 4 cell an d 8 cell of slow method (2, 3, 4 step) have advantage in D-PBS. The development of embryos according to cell stage become greater in 8 cell stage. In the treatment steps of cryopreservation, the development of embryo to blastocyst was similar among rapid method, but the development of 4 cell and 8 cell embryos to blastocyst according to slow method was better than rapid method.

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