P–213 Comparative optical analysis of spindle reformation after oocyte vitrification with media containing differing basal formulations

Study question Do the base ingredients and total composition of vitrification media have an effect on meiotic spindle reformation in warmed donor oocytes? Summary answer Meiotic spindle reformation occurs more readily in donor oocytes vitrified and warmed with a contemporary culture media based vitrification formulation than in traditional vitrification media. What is known already Embryo culture trends continue towards more freeze-all cycles and oocyte preservation is becoming more prevalent across all age groups, thus vitrification continues to serve a pivotal role in today’s laboratory; however, the formulation of vitrification media remains largely unchanged from the m199 or mHTF base composition of antiquated slow freeze media. Literature identifies temperature as a key determiner in spindle reformation. Our preliminary data utilizing a robust vitrification medium with a basal formulation specifically designed for human embryo growth demonstrates a pronounced positive effect on oocytes post-warm in regard to spindle recovery time. Study design, size, duration In a prospective study, 30 oocytes obtained from a diverse population of 10 oocyte donors donating to an egg bank were imaged prior to vitrification to identify the meiotic spindle using the Oosight Imaging System (Hamilton Thorne). Donor oocytes were then split between two vitrification media groups, Vitrification Kit NX (FujiFilm Irvine Scientific) and Vitrification Media (Kitazato USA). After warming, oocytes were again imaged at various time points and meiotic spindle retardance was noted. Participants/materials, setting, methods Oocytes from approved egg bank donors were imaged, vitrified and warmed using protocols recommended by the manufacturers. The oocyte donors were all cycled and retrieved in the same private IVF Clinic and Laboratory between October 2019 and February 2020. Temperature of media, culture dishes, incubators and work stations were all monitored and maintained. Retrieved oocytes were imaged prior to vitrification. Oocytes were imaged immediately upon warm, and at 1 and 3 hours post warm. Main results and the role of chance Oocyte survival was similar across the two media groups, with Vitrification Kit NX at 86.7% and Vitrification Media at 80.0% survival. 100% of the degenerated oocytes in Vitrification Kit NX did not display an initial meiotic spindle when imaged prior to vitrification as opposed to 67% of the degenerate oocytes in Vitrification Media. Upon warming, immediate imaging showed oocytes from Vitrification Kit NX displayed a spindle at a rate of 41.7%, while Vitrification Media allowed this in 0% of the oocytes. At 1-hour post warm, Vitrification Kit NX and Vitrification Media displayed meiotic spindles in 83.3% and 22.2% of oocytes, respectively. The final time point was imaged at 3-hours post warm and showed meiotic spindle reformation in 91.7% of Vitrification Kit NX oocytes and 66.7% of Vitrification Media oocytes. While none of the time points imaged show significance, there is a defined trend towards a faster rate of meiotic spindle recovery in vitrification media formulated with a more modern culture media base. Limitations, reasons for caution The preliminary findings of this study offer that the composition and formulation of the vitrification medium perhaps do have an effect on the reformation of the meiotic spindle post-warm; however, more information is needed and a larger population size must be examined prior to discerning any significance. Wider implications of the findings: If, through further study and imaging, it is ascertained that meiotic spindle reformation is determined in part by the vitrification media composition, it could lead to potentially healthier warmed oocytes for the patient and less workload and scheduling stress on the embryologists due to shorter wait times prior to ICSI. Trial registration number Not applicable

Assessing the Extinction Risk of Heterocypris incongruens (Crustacea: Ostracoda) in Climate Change with Sensitivity and Uncertainty Analysis

Organisms respond to climate change in many different ways and their local extinction risk may vary widely among taxa. Crustaceans from freshwater temporary ponds produce resting eggs to cope with environmental uncertainty and, as a consequence, egg banks have a fundamental role for population persistence. The egg bank dynamics of six clonal lineages of Heterocypris incongruens (Ostracoda) from Northern Italy were simulated. Clonal lineages W1 and W2 are the most common “winter ecotypes”, clonal lineages S1 and S2 are allochthonous “summer ecotypes” and clonal lineages I1 and I2 are relatively rare and generalist in terms of seasonality. Fecundity and proportion of resting eggs vary by clonal lineage, temperature and photoperiod. The clonal extinction risk was estimated in present climate conditions and under climate change. For comparison, and to assess the potential colonization of northern ponds, clonal lineages from Lampedusa Island (Southern Italy), L, were considered. Cohen’s general model was used for simulating egg bank dynamics and the extinction rate of each clonal lineage was estimated with uncertainty analysis. A 30 year simulation in present and climate change conditions was carried out. Extinction rates were lower in climate change conditions than in present conditions. Hydroperiod, hatching rate and egg deterioration rate were the critical factors that affected extinction rates. Extinction rates varied among clonal lineages. This suggests that H. incongruens might be able to have multiple responses to climate change due to its genetic diversity. In climate change conditions, W clonal lineages underwent a niche expansion, while a mismatch between photoperiod and hydroperiod might generate a detrimental effect on the phenology of summer S clonal lineages that might cause their extinction. Southern clonal lineages L, showing an intermediate extinction rate, might colonize northern temporary ponds.

Reversed evolution of grazer resistance to cyanobacteria

Exploring the capability of organisms to cope with human-caused environmental change is crucial for assessing the risk of extinction and biodiversity loss. We study the consequences of changing nutrient pollution for the freshwater keystone grazer, Daphnia, in a large lake with a well-documented history of eutrophication and oligotrophication. Experiments using decades-old genotypes resurrected from the sediment egg bank revealed that nutrient enrichment in the middle of the 20th century, resulting in the proliferation of harmful cyanobacteria, led to the rapid evolution of grazer resistance to cyanobacteria. We show here that the subsequent reduction in nutrient input, accompanied by a decrease in cyanobacteria, resulted in the re-emergence of highly susceptible Daphnia genotypes. Expression and subsequent loss of grazer resistance occurred at high evolutionary rates, suggesting opposing selection and that maintaining resistance was costly. We provide a rare example of reversed evolution of a fitness-relevant trait in response to relaxed selection.

Influence of restoration age on egg bank richness and composition: an ex situ experiment

Zooplankton community assembly after flooding in temporary ponds depends mostly on abundance and diversity of the dormant propagule bank. However, our understanding of the hatching patterns of zooplankton is imperfect. We performed an ex situ experiment to study the species composition and temporal sequence of zooplankton emergence from sediments in three ponds that were restored at different times: 1998, 2003 and 2007. The patterns of species hatching observed in our microcosms were compared with the emergent zooplankton community of their equivalent, synchronically inundated ponds. An escalation in species richness and a delay in hatching time were detected with increasing restoration age. In addition, some discrepancies between the egg bank and active populations were observed, especially in the oldest pond. This study illustrates the use of hatching experiments in comparing ponds with different characteristics. Nevertheless, egg bank hatching and water column sampling should complement each other in order to afford a full reflection of past diversity and contribute to a better understanding of the efficiency of restoration practices.