Simple SummaryThe Bengal tiger (Panthera tigris tigris) is critically endangered, so artificial insemination (AI) is an important conservation tool. Electroejaculation (EE) protocols have been optimized to obtain sufficient amounts of viable sperm for artificial insemination in many felid species; however, less attention has been paid to animal wellbeing during the process. This study examined the effects of three EE protocols (Low, 2–5 volts; Medium, 3–6 volts; and High, 4–7 volts) on semen quality, testicular size, serum creatine kinase (CK, as a proxy for muscle damage), serum testosterone, and cortisol (as a proxy for stress) concentrations, and seminal plasma and sperm pellet malondialdehyde (MDA, as a measure of oxidative stress) measured after each EE series. Primary sperm abnormalities and seminal plasma MDA were higher in the Low compared to Medium and High voltage groups (p< 0.05). Serum CK in the High voltage group increased during the EE series suggesting the potential for muscle damage (p< 0.05). However, no significant differences were observed for serum cortisol, testosterone, or MDA concentrations across voltage groups. Results suggest the Medium voltage protocol produced good quality samples at lower voltages and with no increase in CK compared to the High protocol, which might be better for animal welfare.AbstractThe Bengal tiger (Panthera tigris tigris) is critically endangered, so assisted reproductive technologies, including artificial insemination, are important conservation tools. For wild and domestic felids, electroejaculation (EE) is the most common semen collection method, with protocols optimized to obtain sufficient amounts of viable sperm for artificial insemination. However, less attention has been paid to ensuring animal wellbeing during the process. This study examined the effects of three EE protocols (Low, 2–5 volts; Medium, 3–6 volts; High, 4–7 volts) on semen quality, testicular size, serum testosterone, creatine kinase (CK), and malondialdehyde (MDA) concentrations, and serum cortisol as a proxy for stress. Blood samples were collected before, during, and after each EE series. Seminal plasma pH, and sperm motility, viability, and morphology were evaluated after each procedure. Seminal plasma and sperm pellet MDA concentrations were also determined. Primary sperm abnormalities and seminal plasma MDA were higher in the Low compared to Medium and High voltage groups (p< 0.05). Serum CK in the High voltage group increased during the EE series (p< 0.05), suggesting the potential for muscle damage. However, no significant changes were observed for serum cortisol, testosterone, or MDA concentrations. Results suggest the Medium voltage protocol produced good quality samples at lower voltages than the High protocol with no negative effect on muscle function, which might be better for animal welfare.