# A Descriptive Study of Brown Bear (Ursus arctos) Sperm Quality and Proteomic Profiles Considering Sperm Origin

## TL;DR

This study compares methods for collecting brown bear sperm and finds that direct collection from reproductive organs yields the highest quality sperm for conservation efforts.

## Contribution

The first comparative analysis of sperm quality and proteomic profiles from different sperm origins in brown bears.

## Key findings

- Epididymal sperm showed the highest viability, motility, and lowest apoptosis.
- Sixty-three proteins related to metabolism and stress response were differentially expressed based on sperm origin.
- Ejaculated sperm had higher oxidative stress and reduced metabolic protein expression.

## Abstract

The brown bear population in the Cantabrian mountains of Spain is small and genetically vulnerable, which makes its conservation a priority. One way to help preserve this species is by developing reproductive techniques that enable scientists to collect, store, and utilize bear sperm to support breeding programs. However, there is still limited information about which methods are most effective for collecting and preserving brown bear sperm. In this study, we compared the sperm obtained using three different methods: directly from the reproductive organs, collected just before ejaculation via urethral catheterization, and collected via ejaculation using electrical stimulation. We examined the activity of the sperm and analyzed the types of proteins present, which provides clues about the sperm’s condition. We found that the sperm collected from the reproductive organs were in the best condition, with higher survival rates, increased movement, and greater stress resistance. This information will help scientists determine the most effective methods for collecting and storing brown bear sperm, thereby enhancing global efforts in wildlife conservation.

The conservation of small and genetically vulnerable brown bear populations, such as the Cantabrian subpopulation in Spain, depends on developing species-specific assisted reproductive technologies and genetic resource banks. However, the lack of standardized sperm collection and cryopreservation protocols hinders their application. This study provides the first comparative analysis of sperm quality and proteomic profiles from three different origins: epididymal, pre-ejaculated, and ejaculated. Sperm quality parameters —motility and kinetic, viability, apoptosis, and oxidative stress— and protein expression were assessed. Although yields were similar, marked differences were observed in sperm quality and protein profiles. Sixty-three proteins involved in metabolism, stress response, and oxidative balance were differentially expressed depending on sperm origin. Epididymal sperm showed the highest viability and motility, lowest apoptosis, and a proteomic profile indicative of active spermatogenesis and enhanced oxidative stress defense. In contrast, ejaculated sperm had increased oxidative stress and reduced expression of metabolic proteins, while pre-ejaculated sperm exhibited lower motility, likely due to urine contamination and mitochondrial protein alterations, despite comparable viability and apoptosis. These findings offer novel insights into brown bear sperm biology and highlight the importance of sperm origin in developing optimized assisted reproduction strategies, ultimately supporting ex situ conservation efforts for this species.