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Approximately 20% of the cumulus-oocyte complexes (COC) collected from living animals during repeated ovum pick-up (OPU) sessions develop to the blastocyst stage. An increase in the blood supply to individual follicles appears to be associated with follicular growth rates, while a reduction seems to be closely related to follicular atresia (Acosta TJ et al. 2003; Reproduction, 125, 759-767). Recently, it has been shown that differences in perifollicular blood flow during repeated OPU sessions once weekly were predictive of oocyte competence. The purpose of this study was to determine whether qualitative perifollicular blood flow changes affect the quality of oocytes collected during repeated OPU sessions once or twice weekly as well as the quality of the resulting blastocysts. Lactating Holstein cows (n = 20) were used as oocyte donors. After dominant follicle removal, OPU was performed twice (group 1, for 3 weeks) or once (group 2, for six weeks) weekly employing a 7.5 MHz transducer (GE 8C-RS) of an ultrasound scanner (GE Logiq Book). Doppler characteristics were recorded by transvaginal ultrasonography just before COC collection using the color flow imaging. Because of technical limitations for measurement of blood flow in small individual follicles, only the presence or absence of blood flow was assessed for each follicle. When a clearly visible blue or red spot (blood flow) was detected in the follicle wall, it was considered a follicle with detectable blood flow. Follicles with or without detectable blood flow from each individual cow were aspirated separately. After morphological classification of COC, standard protocols for IVP were used for blastocyst production. For mRNA analysis, denuded COC and blastocysts were frozen at -80°C to analyze the relative transcript abundance using RT-qPCR. The transcripts studiedplay important roles during oocyte and embryo development [DNA methyltransferase 1a, 1b, 3a (DNMT1a, DNMT1b, DNMT3a); histone deacetylase 2 (HDAC2); growth differentiation factor 9 (GDF9); bone morphogenetic protein 15 (BMP15); maternal effect gene zygotic arrest (ZAR); heat shock protein 70.1 (HSP); glucose transporter1, 3 (GLUT1, GLUT3); glucose-6-phosphate dehydrogenase (G6PD); and desmocollin II (DCII)]. Data were tested using analysis of variance (ANOVA) followed by multiple pairwise comparisons using Tukey’s test. The relative abundances of ZAR, BMP15, GDF9, DNMT1a, DNMT3a, and HDAC2 transcripts were significantly upregulated in oocytes stemming from OPU sessions twice weekly, whereas qualitative blood flow changes did not influence the mRNA abundance. At the blastocyst stage, G6PD mRNA was upregulated in blastocysts generated from oocytes collected in OPU sessions twice weekly. These results show that the time interval between the individual OPU sessions had an effect on the quality of oocyte and embryos at the molecular level, whereas differences in the perifollicular blood flow did not. Ruthe Research Farm, Germany for providing the animals; Masterrind GmbH, Germany for donation of the semen, and the HW Schaumann Stiftung for financial support.

In vitro production (IVP) of bovine embryos has been greatly improved over the last couple of years. However, only one-third of the total number of embryos transferred worldwide are of in vitro origin. The IVP embryos still show remarkable differences compared with their in vivo-derived counterparts (i.e. bovine embryos produced in vitro are more sensitive to cryopreservation). So far, vitrification seems to be the most promising method to cryopreserve in vitro-produced bovine embryos. The aim of this study was to determine the effect of 2 different cryopreservation methods on the quality of in vitro-produced bovine embryos at the molecular level using a sensitive RT-qPCR assay. Bovine blastocysts were produced using abattoir ovaries and a standard protocol for IVP (Wrenzycki et al. 2001). They were randomly vitrified employing PBS plus ethylene glycol and DMSO or cryopreserved using a programmable freezer and 1.5 M ethylene glycol. After thawing, embryos from both groups were cultured for 48 h. After 24 h of culture re-expansion rates were documented, and after 48 h hatching rates were documented. After hatching, blastocysts were stored at -80°C for subsequent RT-qPCR analysis. The following gene transcripts known to play important roles during preimplantation development were analyzed: HSP70, GLUT-1, GLUT-3, E-CAD, ZO-1, DNMT3a, IFNτ, DCII. Re-expansion rates were 74.7% (68/91) and 75.0% (87/116) for vitrified and conventionally cryopreserved blastocysts, and 57.1% (52/91) and 55.2% (64/116) of re-expanded embryos hatched. The relative abundances of HSP70, GLUT-1, and ZO-1 transcripts were significantly affected in both groups of cryopreservation compared with the control group (hatched blastocysts without cryopreservation). Conventional cryopreservation had a significant effect on the amount of GLUT-3, DNMT3a, and IFNτ mRNA, whereas vitrification significantly affected DCII transcripts. E-CAD mRNA expression was similar in all groups of embryos. These results suggest that not only the cryopreservation process itself but also the method used to freeze the embryos had a significant influence on the mRNA expression of developmentally important genes in hatched bovine blastocysts. The support of the H.W. Schaumann Stiftung (Germany) and Gynemed Medizinprodukte GmbH & Co. KG (Germany) is gratefully acknowledged.

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