98 Effects of maternal age on oxygen consumption of oocytes and in vitro-produced equine embryos

Abstract

Mitochondrial replication is arrested during early cleavage stages, leaving the embryo dependent on maternally derived mitochondria for oxidative phosphorylation. Numbers of mitochondrial DNA (mtDNA) are used as indicators of functional mitochondria; however, direct comparisons for mtDNA and oxygen consumption rate (OCR) have not been performed for horses. The objectives of this study were to compare equine oocyte mtDNA copy numbers with a measure of mitochondrial function (OCR) and to determine whether maternal age of the oocyte donor impacts OCR of early-stage embryos. We hypothesised that (1) OCR in oocytes is not directly associated with mitochondrial numbers and (2) aerobic metabolism (OCR) is lower in early embryos from old than from young mares. Mares ages 6-13 years (Young, n = 7) and = 20 years (Old, n = 8) were used as oocyte donors. Oocytes were collected from dominant follicles (=35 mm) during oestrus and at 16 ± 2 h after induction of follicular maturation. Recovered oocytes were incubated in tissue culture medium 199 with 10% fetal calf serum, 25 mg mL⁻¹ of gentamicin, and 0.2 mM pyruvate for 26 ± 2 h. Metaphase II oocytes (Young, n = 14; Old, n = 15) were fertilized by intracytoplasmic sperm injection (ICSI) using frozen-thawed sperm from one stallion. Presumptive zygotes were cultured in global medium (LifeGlobal Group). Other oocytes and early embryos were used for OCR. A microchamber containing an electrochemical sensor was used to measure OCR from individual oocytes (Young, n = 9; Old, n = 14) and early embryos (Young, n = 8; Old, n = 10). After analyses, oocytes were snap frozen, and mtDNA was later quantified by qPCR. Metabolic assays of embryos that cleaved were performed at Day 2 after ICSI. After the assay, embryos were placed back to culture until blastocyst formation at Day 7 or 8. Two-tailed Student's t-tests were used for OCR and mtDNA comparisons, and Fisher's exact tests were used to compare development rates. We found that OCR was higher (P = 0.007) for oocytes from Young (mean ± s.e.m.: 1.8 ± 0.2) than from Old (1.3 ± 0.1 fmol s⁻¹). However, mtDNA numbers were not different (P = 0.3) for Young (5.6 ± 0.4 × 10⁵) and Old (6.2 ± 0.4 × 10⁵). Cleavage rates were similar (P = 0.6) between Young (11 out of 14, 79%) and Old (13 of 15, 87%). Day 2 embryos from Young had higher basal OCR compared with Old (3.8 ± 0.1 and 3.2 ± 0.2 fmol s⁻¹, respectively; P = 0.05). Blastocyst rates per cleaved oocytes were similar for Young (5 of 11, 45%) and Old (4 of 13, 31%; P = 0.7). Lower OCR was observed in oocytes and early embryos from Old, which indicates that mitochondrial metabolic function is reduced for mitochondria originating in the oocytes of Old compared with Young. Use of mtDNA was not indicative of mitochondrial metabolic function. Although sample numbers were limited, cleavage and blastocyst development were not significantly different between Young and Old. Further developmental competence was not determined, although the compromised metabolic capacity of oocytes and embryos from old mares could ultimately contribute to lower fertility outcomes.