Zebrafish is an invaluable animal model that has been extensively used to study toxicology, developmental biology, and human-related diseases. This animal model possesses a higher order of genetic similarity with humans, and its genomic structure can be custom edited to incorporate new genes making it an excellent animal model to study genetics and epigenetics of human diseases. This process results in a thousand of mutant & transgenic zebrafish lines, making it difficult to nurture them in laboratories due to limited availability of space and resources. To avail the zebrafish lines for futuristic research purposes, zebrafish sperms are collected and cryopreserved in various centers. To regenerate a specific zebrafish line, the cryopreserved sperms are collected, manually activated, and subsequently introduced with freshly collected eggs in a well-regulated environment. These manual processes are not only labor-intensive but also harmful towards sperm health, overall affecting the fertilization success. To address these issues, this thesis has devised microfluidic platforms that can facilitate cryopreserved zebrafish sperm manipulation towards On-Chip Fertilization (OCF) success. To initiate the motility, zebrafish sperms need to be diluted in an aqueous environment through the process of mixing towards altering their osmolality. As the zebrafish sperm exhibit burst motility (< 15 s), a microfluidic platform is in demand where specific tasks such as uniform mixing can be achieved within a minimal timeframe. In this aspect, by mimicking nature, an artificial cilia embedded microfluidic device was developed in Chapter 2, where micromixing efficiency of 84% was achieved within a timeframe of 5 s. Considering the delicate nature of zebrafish sperms, hydrodynamic forces imposed on them due to artificial cilia beating hypothesized to be crucial towards their activation. Hence, artificial cilia beating parameters were optimized in Chapter 3 towards cryopreserved sperm activation. With the optimized artificial cilia beating parameters, a serpentine microfluidic device was further designed, with which 74.44 ± 6.07% of the total number of sperms were activated. Considering that the cryopreservation protocols significantly alter the genetic integrity of sperm cells, a baffle based microfluidic device was designed in Chapter 4 towards progressively motile zebrafish sperm collection. All these proposed microfluidic devices were designed in such a way that they can be easily integrated as a single Lab-on-a-Chip device towards zebrafish OCF.

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