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Over the last decade, the African turquoise killifish, Nothobranchius furzeri, has emerged as an important model system for the study of vertebrate biology and ageing. Propagation of laboratory inbred strains of Nothobranchius furzeri, such as GRZ, however, can pose challenges due to the short window of fertility, the efforts and space requirements involved in continuous strain maintenance, and the risks of further inbreeding. The current method for long term strain preservation relies on arrest of embryos in diapause. To create an alternative for long term maintenance, we developed a robust protocol to cryopreserve and revive sperm for in vitro fertilization (IVF). We tested a variety of extender and activator buffers for sperm IVF, as well as cryoprotectants to achieve practical long-term storage and fertilization conditions tailored to this species. Our protocol enabled sperm to be preserved in a cryogenic condition for months and to be revived with an average of 40% viability upon thawing. Thawed sperm were able to fertilize nearly the same number of eggs as natural fertilization, with an average of ~ 25% and peaks of ~ 55% fertilization. This technical advance will greatly facilitate the use of N. furzeri as a model organism.

15Over the last decade, the African turquoise killifish, Nothobranchius furzeri, has 16 emerged as an important model system for the study of vertebrate biology and 17 ageing. However, rearing this fish in captivity can pose challenges, due to the 18 short window of fertility, inbreeding problems, and the continuous maintenance of 19 different strains and transgenic lines. To date, the main means of long term 20 strain maintenance is to arrest embryos in diapause, a poorly understood and 21 unreliable method. To solve these problems, we developed a robust protocol to 22 cryopreserve sperm and to revive them for in vitro fertilization (IVF), as a better 23 option for long term storage of N. furzeri lines. We tested a variety of extender 24 and activator buffers for sperm in vitro fertilization, as well as cryoprotectants to 25 achieve maximal long term storage and fertilization conditions tailored to this 26 species. Our optimized protocol was able to preserve sperm in a cryogenic 27 condition for months and to revive an average of 40% upon thawing. Thawed 28 sperm were able to fertilize nearly the same number of eggs as natural 29 fertilization, with an average of ~25% and peaks of ~55% fertilization. This 30 technical advance will greatly facilitate the use of N. furzeri as a model organism. 31 32 Abbreviations used: 33 HBSS: Hank's Balanced Salt Solution 34 BSMIS: Buffered sperm motility-inhibiting solution 35 FBS: Fetal bovine serum 3 36 DMSO: Dimethyl sulfoxide 37 DMF: Dimethylformamide 38 MetOH: Methanol 39 DMA: Dimethylacetamide 40 IVF: In vitro fertilization 41 42 Introduction 43 Over the last few years the African killifish, Nothobranchius furzeri, has emerged 44 as important model system for the study of vertebrate ageing. The life cycle of 45 these fish is characterized by a fast growth rate, reaching sexual maturity by 4-5 46 weeks, and a maximum lifespan of 6,5-7 months (as mean lifespan of the most 47 long-lived 10% of a given cohort)[1], [2], making them among the shortest-lived 48 vertebrate species bred in captivity and a unique platform for the rapid 49 exploration of aging and age-associated diseases[3]. 50 Unfortunately fast growth and aging features also carry some drawbacks that 51 make the maintenance in captivity of this species quite challenging. Rapid 52 growth and maturation lead to a rapid passing of successive generations, which 53 can often result in inbreeding [4], [5]. Indeed, it is virtually impossible to establish 54 an ancient founder to maintain an original genotype because of the marginal 55 overlap of generations. Fertility in this species is limited to the 5 th -20 th week of life 56 and is maximal between the 6 th and the 11 th [6]-[8], marking an extremely narrow 57 breeding window, especially compared to other fish species where this can 4 58 correspond to years. Moreover the success of transposon-mediated 59 transgenesis [9] and CRISPR-mediated mutagenesis [10] in this fish means that 60 more genetically engineered lines require continuous maintenance and space 61 u...

The measles, mumps and rubella (MMR) vaccine protects against all-cause mortality in children, but the immunological mechanisms mediating these effects are poorly known. We systematically investigated whether MMR can induce long-term functional changes in innate immune cells, a process termed trained immunity, that could at least partially mediate this heterologous protection. In a randomized placebo-controlled trial, 39 healthy adults received either the MMR vaccine or a placebo. By using single-cell RNA-sequencing, we found that MMR caused transcriptomic changes in CD14-positive monocytes and NK cells, but most profoundly in γδ T cells. Surprisingly, monocyte function was not altered by MMR vaccination. In contrast, the function of γδ T cells was significantly enhanced by MMR vaccination, with higher production of TNF and IFNγ, as well as upregulation of cellular metabolic pathways. In conclusion, we describe a new trained immunity program characterized by modulation of γδ T cell function induced by MMR vaccination.

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