Non-invasive metabolomic profiling of culture media of ICSI- and IVF-derived early developmental cattle embryos via Raman spectroscopy.

The aim of the present study was to compare differences in composition between in vitro cultured early developmental embryos resulting from either in vitro fertilization (IVF) or intracytoplasmic sperm injection (ICSI). Non-invasive metabolomic profiling of culture media was conducted with laser tweezer Raman spectroscopy (LTRS), providing molecular information that was used to aid the diagnosis or treatment of embryos that were adversely affected by ICSI treatment, ultimately improving the ICSI embryonic developmental potential. Cattle embryos were generated via ICSI and IVF with development to the 2-, 4-, 8-, 16-,32-cell, and blastocyst stages with individual in vitro culturing occurring for 4 h. The culture media for embryos in different developmental stages were separately analyzed using LTRS. The resulting composition of culture media used for culturing IVF- and ICSI-derived embryos was mainly altered in contents of carbohydrates, lipids, DNA, and proteins. Bands at 1004 cm-1 (phenylalanine) and 1529 cm-1 (-C = C-carotenoid) had specific patterns related to the metabolicactivity of embryos; using LTRS, and these may be considered as biomarkers for embryonic development. Furthermore, the vibrations of lipids at different stages increased more with assessment of ICSI culture media than in IVF media. Discriminant function analysis can be utilized for the classification of culture media used for culture of ICSI- and IVF-derived embryos. In conclusion, LTRS can be used for development of an independent assay to assess embryo status during both ICSI and IVF procedures, which provides novel insights into differences in structure and components of single cells.

Successful vitrification of bovine immature oocyte using liquid helium instead of liquid nitrogen as cryogenic liquid.

The objectives of this study were to compare the effectiveness of liquid helium (LHe) and liquid nitrogen (LN2) as cryogenic liquid for vitrification of bovine immature oocytes with open-pulled straw (OPS) system and determine the optimal cryoprotectant concentration of LHe vitrification. Cumulus oocyte complexes were divided into three groups, namely, untreated group (control), LN2 vitrified with OPS group, and LHe vitrified with OPS group. Oocyte survival was assessed by morphology, nuclear maturation, and developmental capability. Results indicated that the rates of normal morphology, maturation, cleavage, and blastocyst (89.3%, 52.8%, 42.7%, and 10.1%, respectively) in the LHe-vitrified group were all higher than those (79.3%, 43.4%, 34.1%, and 4.7%) in the LN2-vitrified group (P < 0.05) although the corresponding rates in both treated groups decreased compared with the control group (100%, 75.0%, 64.9%, and 40.8%; P < 0.05). Normal calves were obtained after the transfer of blastocysts derived from LHe- and LN2-vitrified oocytes. The effects of the different vitrification solutions (EDS30, EDS35, EDS40, EDS45, and EDS50) in LHe vitrification for bovine immature oocytes vitrification were examined. No difference was found in the rates of morphologically normal oocytes among the EDS30 (87.9%), EDS35 (90.1%), EDS40 (89.4%), and EDS45 (87.2%) groups (P > 0.05). The maturation rate of the EDS35 group (65.0%) was higher than those of the EDS30 (51.3%), EDS40 (50.1%), EDS45 (52.1%), and EDS50 groups (36.9%; P < 0.05). No significant differences were observed in the cleavage and blastocyst rates between the EDS35 (49.0% and 12.1%) and EDS40 (41.7% and 10.2%) groups. However, the cleavage and blastocyst rates in the EDS35 group were higher (P < 0.05) than those of the EDS30 (36.2% and 6.8%), EDS45 (35.9% and 5.8%), and EDS50 (16.6% and 2.2%) groups. In conclusion, LHe can be used as a cryogenic liquid for vitrification of bovine immature oocytes, and it is more efficient than LN2-vitrified oocytes in terms of blastocyst production. EDS35 was the optimal cryoprotectant agent combination for LHe vitrification in this study.

Developmental competence and gene expression of immature oocytes following liquid helium vitrification in bovine.

The objective of this study was to develop an effective ultra-rapid vitrification method and evaluate its effect on maturation, developmental competence and development-related gene expression in bovine immature oocytes. Bovine cumulus oocyte complexes were randomly allocated into three groups: (1) controls, (2) liquid nitrogen vitrification, and (3) liquid helium vitrification. Oocytes were vitrified and then warmed, the percentage of morphologically normal oocytes in liquid helium group (89.0%) was significantly higher (P<0.05) than that of the liquid nitrogen group (81.1%). When the vitrified-thawed oocytes were matured in vitro for 24h, the maturation rate in liquid helium group (50.6%) was higher (P<0.05) than liquid nitrogen group (42.6%). Oocytes of liquid helium vitrification had higher cleavage and blastocyst rates (41.1% and 10.0%) than that of liquid nitrogen vitrification (33.0% and 4.5%; P<0.05) after in vitro fertilization. Moreover, the expression of GDF9 (growth/differentiation factor-9), BAX (apoptosis factor) and ZAR1 (zygote arrest 1) was analyzed by quantitative real-time polymerase chain reaction (qRT-PCR) when the vitrified-thawed oocytes were matured 24h. The expression of these genes was altered after vitrification. Expression of GDF9 and BAX in the liquid helium vitrification group was not significantly different from that of the control, however there were significant differences between the liquid nitrogen vitrification group and control. In conclusion, it was feasible to use liquid helium for vitrifying bovine immature oocytes. There existed an association between the compromised developmental competence and the altered expression levels of these genes for the vitrified oocytes.