An Open-Hardware Insemination Device for Small-Bodied Live-Bearing Fishes to Support Development and Use of Germplasm Repositories.

Small-bodied live-bearing fishes attract broad attention because of their importance in biomedical research and critical conservation status in natural habitats. Artificial insemination is an essential process to establish hybrid lines and for the operation of sperm repositories. The existing mouth-pipetting technique for artificial insemination of live-bearing fishes has not been substantially upgraded since the first implementation in the 1950s. The goal of this work was to develop a standardized artificial inseminator device (SAID) to address issues routinely encountered in insemination by mouth-pipetting, including lack of reproducibility among different users, difficulty in training, and large unreportable variation in sample volume and pressure during insemination. Prototypes of the SAID were designed as relatively inexpensive (<USD 80) open hardware based on commercially available and 3-D printed components to enable broad community access. A linear actuator was used to accurately control the position of a piston for fluid transfer with a standard deviation of <0.1 mm over a 4 mm range of travel. The volume of sample transfer was precisely controlled with a linear relationship ( r 2 > 0.99) between the piston position and volume. Pressure generation from eight mouth-pipetting operators and SAID prototypes were assessed by pressure sensors. The pressure control by SAID was superior to that produced by mouth-pipetting, yielding lower pressures (31-483 Pa) and smaller variations (standard deviation <11 Pa). These pressures were sufficient to deliver 1-5 μL of fluid into female reproductive tracts yet low enough to avoid physical injury to fish. Community-level enhancements of the SAID prototype could enable standardized insemination with minimal training and facilitate the participation of research communities in the use of cryopreserved genetic resources.