Swim-up is a sperm purification method that is being used daily in andrology labs around the world as a simple step for in vitro sperm selection. This method accumulates the most motile sperm in the upper fraction and leaves sperm with low or no motility in the lower fraction. However, the underlying reasons are not fully understood. In this article, we compare metabolic rate, motility and sperm tail length of bovine sperm cells of the upper and lower fraction. The metabolic assay platform reveals oxygen consumption rates and extracellular acidification rates simultaneously and thereby delivers the metabolic rates in real time. Our study confirms that the upper fraction of bull sperm has not only improved motility compared to the cells in the lower fraction but also shows higher metabolic rates and longer flagella. This pattern was consistent across media of two different levels of viscosity. We conclude that the motility-based separation of the swim-up technique is also reflected in underlying metabolic differences. Metabolic assays could serve as additional or alternative, label-free method to evaluate sperm quality.

Sperm-driven micromotors are promising devices for developing autonomous, noninvasive diagnostic and therapeutic tools, specifically in reproductive biology and medicine. In this article, we present template-based microstructures fabricated from gelatin for sperm capture, activation, and antioxidant protection. The cartridge-like structures with one closed end-capture single bovine sperm cells and are propelled by the sperm's flagellum. We demonstrate the pH-dependent release of heparin from the gelatin microcartridges to induce capacitation, a crucial maturation process prior to fertilization. Further, the gelatin cartridges exhibit a protection against oxidative stress, one of the main causes for sperm damage. Finally, we investigate the stability and degradability of the microstructures in various physiological environments, such as endometrial cell culture, different pH, and in the presence of proteases. Overall, we prove that these gelatin spermbots display great potential for the development of noninvasive theranostic tools in reproductive biology and medicine for the protection and activation of sperm, especially useful for studying sperm migration. We present their beneficial features which comprise not only biocompatibility and biodegradability but also pH response, loading stability, and antioxidant protection.

18Swim-up is a sperm purification method that is being used daily in andrology labs around the 19 world as a simple step for in vitro sperm selection. This method accumulates the most motile 20 sperm in the upper fraction and leaves sperm with low or no motility in the lower fraction but 21 the underlying reasons are not fully understood. In this article, we compare metabolic rate, 22 motility and sperm tail length of bovine sperm cells of the upper and lower fraction. The 23 metabolic assay platform reveals oxygen consumption rates and extracellular acidification rates 24 simultaneously and thereby delivers the metabolic rates in real time. Our study confirms the 25 upper fraction of bull sperm has improved motility compared to the cells in the lower fraction 26 and shows higher metabolic rates. This pattern was consistent across media of two different 27 levels of viscosity. Sperm with longer flagella are selected in the upper fraction. We conclude 28 that the motility-based separation of the swim-up technique is based on metabolic differences. 29Metabolic assays could serve as additional or alternative, label-free method to evaluate sperm 30 quality, which is likely particularly useful in cases of asthenozoospermia and teratospermia. 31Furthermore, metabolic measurements of sperm cells can reveal differences in metabolic 32 pathways in different environments. 33 2 1 Keywords 2 swim-up, bovine sperm, metabolism, sperm selection, sperm quality 3