31 Effect of vitrification of porcine oocytes on production of ATP and mitochondrial DNA copy number

Abstract

This experiment evaluated the effects of vitrification at different time points of in vitro maturation (IVM) on ATP production and mitochondrial DNA (mtDNA) copy number of porcine oocytes. Treatments included vitrification at 24 h of IVM (V24), vitrification at 44 h of IVM (V44), and a control group consisting of fresh oocytes after 48 h of IVM. Porcine cumulus–oocyte complexes (COCs) were obtained from a commercial vendor and underwent the first 24 h of IVM during shipment in a portable incubator. Upon arrival, COCs were randomly allocated into treatments. The oocytes in the V44 and control groups were incubated at 38.8°C and 5.5% CO₂ to continue IVM. Before vitrification, COCs were denuded in hyaluronidase by vortexing, followed by 3 washes in holding medium (Hanks’ balanced salt solution–HEPES + 4% BSA). Denuded oocytes were vitrified using a 3-step, dimethyl sulfoxide (DMSO)- and ethylene glycol-based protocol (VitriCool kit, IVF Bioscience), Cryolocks as carriers, and liquid nitrogen as cryogenic agent. All steps were carried out at room temperature. Warming was achieved using the VitriWarm kit (IVF Bioscience) consisting of 4 dilution steps. After warming, the oocytes were washed in holding medium and incubated in IVM medium to complete 48 h of maturation (24 h for V24 and 4 h for V44). All warming steps were performed at 38.5°C. Oocytes destined for ATP production assessment (Control n = 26, V44 n = 27, V24 n = 28) were frozen in 50 µL of ultra-pure water, whereas oocytes destined for mtDNA copy number quantification (Control n = 32, V44 n = 30, V24 n = 32) were snap-frozen in ∼1 µL of holding medium. Samples were kept at −80°C until further processing. The ATP content of single oocytes was determined using an ATP bioluminescent somatic cell kit (FLASC, Sigma-Aldrich). The assessment of mtDNA copy number in single oocytes was performed by amplifying the porcine Mt-ND4 gene (F atccaagcactatccatcacca, R ccgatgattacgtgcaaccc; NC_000845.1) and quantification was carried out using a Droplet Digital PCR system (Bio-Rad Laboratories). Results for ATP production and mtDNA copy number were analysed through ANOVA with Tukey’s adjustment (SAS 9.4; Sas Institute Inc.). No differences were found in mtDNA copy number among groups (Control 178 004.69 ± 19 207.23, V44 170 483.67 ± 18 127.18, V24 176 767.50 ± 27 211.09; P = 0.36). In contrast, all groups differed in ATP content (pg/µL) among each other (Control 26.36 ± 4.99, V44 20.26 ± 6.61, V24 16.54 ± 8.07; P < 0.0001). These data indicate that although there was no effect on mitochondrial number, ATP production/storage ability is significantly reduced as a result of vitrification-warming. Vitrification at 44 h of IVM followed by a 4-h post-warming incubation showed the highest ATP content among the vitrification treatments.