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Objectives  Currently, there is no consensus protocols regarding the combination of glycerol (GLY), gelatin or foetal bovine serum (FBS) with dimethyl sulphoxide (DMSO) as cryoprotectants for  Mycoplasma bovis  in bovine milk samples. This study aimed to compare different cryopreservation compounds and storage temperatures for  M. bovis .    Results  There were significant differences in the survival of  M. bovis  on different media. Differences were also observed between different storage conditions. All additives improved the survival of  M. bovis  in comparison to control (CON). The combination of GLY and DMSO was shown to be significantly different to CON with 57.1% (95% CI = 21.43–133.34) and 19.1% (95% CI = 11.73–60.27), respectively at week 16, and its use should be encouraged as a cryoprotectant for  M. bovis  at − 20 and − 80 °C. GEL/DMSO showed the highest survival rate for  M. bovis  with 57.14% (95% CI = 21.43–133.34) at 4 °C in comparison with CON 14.29% (95% CI = 9.60–50.39). FBS/DMSO showed the highest survival rate for the short-term preservation similarly to other additives. The evaluated cryopreservative compounds would improve survivability of  M. bovis  in milk for both transport and long-term storage. Hence, it is recommended to use the mentioned methods for routine transportation or storage purposes for suspicious  M. bovis  milk samples.

Evaluation of three cryoprotectants used with bovine milk affected with Mycoplasma bovis in different freezing conditions