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We developed a multi-period chance-constrained optimization model to rationalize collections of endangered livestock breeds at risk of in situ extinction in Spain. The model configures collections by determining the least-cost optimal collection locations, timing and material quantities (semen doses). A decision variable defining an “acceptable level of risk” allows decision makers to specify tolerable levels of in situ breed endangerment when taking ex situ collection and storage decisions. Using data from 18 gene banks we demonstrate how collections can be rationalized, and derive cost curves relating marginal ex situ collection cost and accepted probability of in situ extinction covering the period 2018 to 2060. The modelling framework can be replicated in countries seeking to rationalize ex situ collections under limited conservation budgets and uncertain in situ extinction risks.