Biocompatibility and heat stress stabilisation of bovine sperm cryopreserved with ectoine

Extremolytes are compatible solutes that protect macromolecules and cell structures under extreme stress. The objectives of this study were to assess the biocompatibility of the extremolyte ectoine with bull sperm during cryopreservation, and its potential to protect sperm under osmotic and heat stress. Semen from mature Limousin and Charolais bulls (n = 8) was diluted in OptiXcell containing 0 (control), 0.5, 5 or 50 mM ectoine, packaged into 0.25 mL straws, cooled overnight at 4 °C, and cryopreserved as per routine commercial procedures. Post-thaw assessments included motility (CASA), viability, membrane fluidity, acrosomal integrity, MMP, superoxide, intracellular calcium and DNA fragmentation (flow cytometry). Thawed sperm were exposed to heat stress (42 °C for 4 h) or osmotic stress (150 mOsm for 15 min). Fertilisation and embryo development were evaluated via in vitro fertilisation. Cleavage was assessed on Day 2 and blastocyst development on Days 7-8. Ectoine (0.5-5 mM) had no adverse effect on sperm function post-thaw (P > 0.05). At 50 mM ectoine, however, kinematic parameters (VCL, VSL, VAP and BCF) were reduced (P < 0.05). Osmotic stress reduced viability by 24% and was not affected by ectoine. Under heat stress, motility declined by 36% in the control after 2 h (P < 0.001), while ectoine-treated groups maintained motility (P < 0.01). Ectoine did not affect cleavage or blastocyst rates (P > 0.05). Overall, ectoine did not enhance standard post‑thaw sperm quality but was biocompatible across concentrations tested. Its modest effect during heat exposure highlights potential stabilising effects under defined post‑thaw challenges.