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• Serum 25(OH)D concentrations from ewes on a farm in NZ were measured at mating. • The scanning result was significantly associated serum total 25(OH)D concentrations. • Triplet bearing ewes had lower 25(OH)D and 25(OH)D 3 compared with single bearing ewes. • Weight was significantly associated with scanning result. Research on Soay Island sheep showed a positive association of serum 25(OH)D concentration and fecundity, while similar research on Scottish sheep didn't show an association with fecundity but did show an association with weaned lamb weight. Therefore the aim of this study was to determine if serum 25-hydroxyvitamin D (25(OH)D) concentrations of ewes at mating, on a commercial sheep farm, was associated with subsequent prolificacy. On a sheep and beef farm in the North Island of New Zealand 200 mixed-age Romney ewes (Ovis aries) had weights recorded and blood was collected by jugular venepuncture in April (autumn) at mating. Pregnancy testing was conducted 12 weeks after mating, using real time ultrasound to confirm pregnancy and determine whether the dams were carrying singletons, twins or triplets. Serum 25(OH)D 2 and D 3 concentrations, total calcium, and phosphorus concentrations were measured in the 200 pregnant ewes. A multinomial logistic regression model was fitted to the data in order to determine significant predictors of the ultrasound scanning result. The mean serum total 25(OH)D concentrations of single, twin and triplet bearing ewes were 102.0 ± 3.4, 101.0 ± 2.4 and 83.0 ± 4.5, respectively. In a multinomial logistic regression model serum total 25(OH)D and 25(OH)D 3 concentrations were also negatively correlated with ewes pregnant with triplets (P = 0.015), while weight was significantly associated with both twin and triplet bearing ewes (P < 0.001). The reason for the lower serum 25(OH)D concentration in sheep bearing triplets is unclear, and further research is required in order to determine the mechanisms behind this. The results suggest that an effect on ewe fecundity may only be seen when serum 25(OH)D concentrations are marginal. [ABSTRACT FROM AUTHOR]