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A simple, non-invasive, rapid and sensitive oxygen microsensor system was developed to investigate correlations between oxygen respiration rates of individual bovine embryos and their morphology, sex, diameter and developmental stage. Bovine IVP-embryos (n = 78; Holm et al. Theriogenology 52, 683?700) were analysed around the 8-cell stage (Day 3; n = 18) and at various blastocyst stages (Day 7; n = 60). Each embryo was morphologically evaluated, its outer diameter measured and was then loaded into a glass tube (i.d. 0.68 mm, length 3 mm). After 1 h, oxygen concentration gradients generated by the embryo?s respiration were measured over app. 8 min with an oxygen microelectrode (www.unisense.com). Five embryos were measured in one round together with an empty tube as control. The procedure was repeated twice for each embryo with app. 1 h interval. Individual respiration rates in nL O2/embryo/h (nL/h) were calculated from these gradients. The measurements were performed at 38.5C under constant flow of humidified 5% CO2 in air (app. 19% O2). After this, 64 embryos (14 Day 3; 50 Day 7) were lysed for sex diagnosis by PCR. Values are given as mean SD. The sensitivity of the oxygen measurement system was high (controls: 0.034 0.035 nL/h, n = 15) and its repeatability from 1st to 2nd measurement was 99.7 9.8% (n = 71). The average embryo respiration rate was 0.39 0.05 nLl/h on Day 3 (n = 18) and 1.31 0.52 nLl/h on Day 7 (n = 60). For Day 7 embryos, the respiration rates varied according to their morphological quality, being 1.87 0.46a (n = 18), 1.17 0.32b (n = 23), 0.95 0.27b,c (n = 14) and 0.72 0.24c (n = 4) nL/h for quality 1, 2, 3, and 4 embryos, respectively (Proc Mixed,a,b,c: P < 0.05; values with different superscripts differ significantly). The sex ratio (male:female) was 9:5 (Day 3) and 32:18 (Day 7), and on Day 7 this ratio varied between qualities: 11:2, 12:8, 8:4, and 1:3 for quality 1, 2, 3, and 4, respectively. The average respiration rate on day 3 was the same for males and females, as it was on day 7 (1.22 0.43 nL/h (females) and 1.31 0.58 nL/h (males), P > 0.05). There was a correlation between embryo diameter and respiration rate (r2 = 0.65, n = 74), which was even stronger for Day 7 male embryos (r2 = 0.72, n = 32). In conclusion, a highly reliable, repeatable and sensitive system was established for measuring respiration rates in single bovine embryos, even at early developmental stages. The respiration rate was lower on day 3 compared to Day 7 embryos, and it was correlated with the morphological embryo quality on Day 7. Oxygen consumption could be a valuable supplementary indicator of embryo viability, especially in difficult evaluations (e.g. quality 2 and 3 after IVP). It remains to be demonstrated if such measurements can also reveal quality differences already at Day 3, which would be of interest in, e.g. the human field.  ASL is supported by FCT, Portugal.

We have studied the effect of suppressed IVM on the developmental competence of bovine oocytes, aiming at elucidating the importance of cytoplasmic maturation in fertilization and embryo development. Six replicates of abattoir-derived oocytes were randomly divided into three IVM groups. Control (n ? 950): TCM-199 with glutamax-I (Gibco, Grand Island, NY, USA), 0.25 mM Na-pyruvate, 100 IU mL-1 penicillin and 100 ?g mL-1 streptomycin, 50 ng mL-1 FSH, and 10? fetal bovine serum (FBS) (Gibco); Serum?FSH-free (n ? 944): same as control but without FSH and FBS; ?-amanitin (n ? 977): same as control but with 10 ?g mL-1 ?-amanitin. Nuclear maturation of oocytes was studied 24 h after the onset of IVM, the formation of sperm aster structure 10 hours post-insemination (hpi) and the formation of pronuclei 20 hpi. Sperm aster was visualized with ?-tubulin antibody (modified from Navara et al. 1999 Dev. Biol. 162, 29?40). Presumptive zygotes were cultured until Day 7 in modified SOFaaci ? 4 mg mL-1 fatty acid-free BSA in 5? O2. Cumulus cell expansion was seen only in the control group. The results of nuclear maturation, fertilization, and embryo development are summarized in Table 1. Serum and FSH deprivation did not have a statistically significant effect on the parameters studied (vs. control). ?-amanitin exposure during IVM reduced nuclear maturation, fertilization, and Day 3 embryo cleavage vs. control, and resulted in total blockage of Day 7 blastocyst development. The treatment groups had significantly smaller mean diameters of male pronuclei (control: 14 0.6 ?­m; serum?FSH-free: 12 0.5 ?­m, P < 0.05; ?-amanitin: 10 0.6 ?­m, P < 0.001) and sperm asters (control: 86 4 ?­m; serum?FSH-free: 82 4 ?­m, P < 0.01; ?-amanitin: 49 7 ?m, P < 0.001) (nonparametric Kruskall Wallis and Mann-Whitney U tests) vs. control group. Despite reduction in pronucleus and sperm aster diameter, serum and FSH deprivation during IVM did not affect in vitro developmental competence of bovine oocytes, suggesting a need for re-evaluation of the components of IVM. ?-Amanitin exposure in IVM disturbed nuclear maturation, fertilization, and embryo development, indicating the essence of early transcription.