Optimization of sperm cryopreservation in common marmoset (Callithrix iacchus)

摘要

The common marmoset (callithrix jacchus) is a New World primate species with many advantages compared to other laboratory primates for studying reproductive biology; they are small, readily adaptable to the laboratory and have high fecundity. Many reproductive technologies, however, are less well developed in marmoset. Cryopreservation of marmoset sperm has also yet to be optimized. Therefore, the objective of the present study was to determine the optimal conditions for sperm cryopreservation. Ejaculated semen was collected in TYH medium using vibrator. After washing TYH, sperm were resuspended in TCG (Tris-Citrate acid-Glucose) solution containing 1.5 % BSA and subjected to following experiments. [Experiment 1] Sperm suspensions were diluted 1:1 v/v with each glycerolated TCG solutions to give a final concentration of 0 %, 3.75 % and 7.5 % glycerol, followed by freezing in liquid nitrogen. [Experiment 2] Sperm suspensions were diluted 1:1 v/v with each TCG solutions containing egg yolk to give a final concentration of 0 %, 10 % and 20 % egg yolk, followed by freezing in liquid nitrogen. [Experiment 3] Sperm suspensions were diluted 1:1 v/v with each TCG solutions containing raffinose to give a final concentration of 0 M, 0.1 M and 0.2 M raffinose, followed by freezing in liquid nitrogen. Sperm motility was assessed using microscopy and its viability was evaluated by LIVE/DEAD Sperm Viability Kit (Molecular Probes) throughout the all experiments. Sperm cryopreserved with TCG solution containing 3.75 % and 7.5 % glycerol, or 10 % and 20 % egg yolk exhibited a higher motility and viability compared to that cryopreserved with TCG solution. Addition of raffinose had no effect on sperm cryopreservation. In conclusion, cryopreservation with TCG solution containing 3.75 % glycerol and 10 % egg yolk enhances the freezability of ejaculated sperm in marmoset.