Effect of Conventional Controlled-Rate Freezing and Vitrification on Morphology and Metabolism of Bovine Blastocysts Produced In Vitro1

Oxygen consumption is a useful parameter for evaluating embryo quality, since it provides a valuable indication of overall metabolic activity. Over the years, several approaches have been used to measure the respiration rates of individual embryos, but a convincing method has not yet been reported. In this study, we introduce and have validated a novel high resolution microsensor technology to determine the respiration rates of individual embryos at different developmental stages. We have employed this technology to investigate the correlation between respiration rate and embryo morphology, diameter and sex. Following morphological evaluation, individual respiration rates of day 3 (n 5 18) and day 7 (n 5 60) bovine in vitro-produced embryos were determined. Of the measured embryos, 64 were lysed for sex diagnosis by PCR. Average respiration rates of day 7 embryos (1.30 6 0.064 nl/h) were 3.4-fold higher than day 3 embryos (0.38 6 0.011 nl/h). On day 7, the average respiration rate of quality 1 blastocysts was significantly higher than the respiration rates of the lower qualities. For both day 3 and day 7 embryos, respiration rates were directly influenced by embryo diameter but did not differ between sexes. These results have demonstrated that the novel microsensor technology can be used to accurately and rapidly (8 min) measure the respiration rates of individual embryos at different developmental stages. Respiration rates were only in partial agreement with embryo morphology, suggesting a slight discrepancy between these two methods in assessing embryo quality. It is likely that a combined assessment of embryo respiration and morphology would improve embryo classification and subsequent selection.

Section: Discussioncontrasting

confidence: 92%

Respiration rates of individual bovine in vitro-produced embryos measured with a novel, non-invasive and highly sensitive microsensor system

Lopes¹,

Larsen²,

Ramsing³

et al. 2005

“…This value is closer to the value of 0.14 £ 10 213 moles/s for bovine morula reported by Shiku et al (2001) than for values reported by Thompson et al (1996) of , 9 £ 10 211 moles/s for bovine embryos from day 0 -4 of culture. Reported values for bovine blastocysts range from 0.4 -1.5 £ 10 29 moles/s , Kaidi et al 2001, Donnay et al 2002. Reasons for the differences in absolute amounts are not known but could include methodology since the lower values in the present study and by Shiku et al (2001) are based on electrochemical measurements of oxygen, while other papers cited above rely on quenching of pyrene fluorescence by oxygen.…”

Section: Discussionmentioning

confidence: 99%

Actions of thermal stress in two-cell bovine embryos: oxygen metabolism, glutathione and ATP content, and the time-course of development

Rivera¹,

Dahlgren²,

Paula³

et al. 2004

The mechanism by which heat shock disrupts development of the two-cell bovine embryo was examined. The reduction in the proportion of embryos that became blastocysts caused by heat shock was not exacerbated when embryos were cultured in air (20.95% O 2 ) as compared with 5% O 2 . In addition, heat shock did not reduce embryonic content of glutathione, cause a significant alteration in oxygen consumption, or change embryonic ATP content. When embryos were heat-shocked at the two-cell stage and allowed to continue development until 72 h post insemination, heat-shocked embryos had fewer total nuclei and a higher percentage of them were condensed. Moreover, embryos became blocked in development at the eight-cell stage. The lack of effect of the oxygen environment on the survival of embryos exposed to heat shock, as well as the unchanged content of glutathione, suggest that free radical production is not a major cause for the inhibition in development caused by heat shock at the two-cell stage. In addition, heat shock appears to have no immediate effect on oxidative phosphorylation since no differences in ATP content were observed. Finally, the finding that heat shock causes a block to development at the eight-cell stage implies that previously reported mitochondrial damage caused by heat shock or other heat shock-induced alterations in cellular physiology render the embryo unable to proceed past the eight-cell stage.

“…Embryos transferred fresh included unexpanded and expanded blastocysts, while embryos transferred after vitrification and warming were predominantly expanded blastocysts. For vitrification, embryos were loaded into straws containing PBS (Invitrogen) with glycerol, ethylene glycol and FCS, adjacent to two fractions of galactose solution (Kaidi et al 2001). Embryos were warmed for 5 s in air and 10 s in a water bath at 30 8C.…”

Section: Embryo Transfer (Et)mentioning

confidence: 99%

Oocytes recovered from cows treated with retinol become unviable as blastocysts produced in vitro

Hidalgo¹,

Díez²,

Duque³

et al. 2005

Retinoids have been shown to enhance developmental competence of the oocyte in cattle, sheep and pigs. In this study we investigated whether exogenous retinol stimulates the bovine oocyte during its intrafollicular growth and the time limits of exposure to exogenous retinol. In addition, we also determined the efficiency of ovum pick-up techniques in combination with retinol treatment and the viability of embryos after transfer to recipients. In Experiment 1, heifers were injected with retinol or vehicle, and concentrations of retinol in the blood were analysed on Day 0 (prior to injection), Day 1 and, together with follicular fluid, Day 4. Blood retinol increased by Day 1 and cleared on Day 4, but retinol remained higher within the follicle. In Experiment 2, oocyte donors were injected weekly with retinol or vehicle four times during a twice-per-week cycle of eight recovery sessions (starting 4 days before the first session), followed by a second eight-session cycle without treatment. Oocytes recovered were fertilized and culturedin vitro.Retinol treatment yielded higher numbers of low-quality oocytes throughout, although retinol measured during cycles did not change. Total oocytes, and morulae and blastocyst rates, increased during the first five sessions following treatment with retinol. As previously shown with oocytes from slaughterhouse ovaries, retinoic acid stimulated blastocyst development. Following transfer to recipients, blastocysts from oocytes exposed to retinol were unable to establish pregnancy. Our study confirms the existence of an effect of retinol on the intrafollicular oocyte in the cow and provides evidence regarding the teratogenic effect of retinol.