# Agarose Gel-Supported Culture of Cryopreserved Calf Testicular Tissues

## TL;DR

This study shows that agarose gel-supported culture better preserves and supports the development of cryopreserved calf testicular tissues compared to direct adherent culture.

## Contribution

The study introduces agarose gel as a superior method for in vitro culture of cryopreserved bovine testicular tissues.

## Key findings

- Agarose gel culture preserved seminiferous cord architecture and supported seminiferous epithelium development better than direct adherent culture.
- Agarose culture reduced apoptosis and enhanced key gene expression related to spermatogenesis and testosterone secretion.
- The agarose system improved spermatogenic differentiation and Leydig cell function in cultured testicular tissues.

## Abstract

The ability to culture testicular tissues in vitro provides an important tool for studying male fertility. In this study, we compared two methods for culturing cryopreserved calf testicular tissues: Agarose-Supported culture and Direct Adherent culture. Our findings showed that agarose gel offered a better microenvironment, maintained the tissue structure, reduced apoptosis, and supported germ cell proliferation and differentiation. It also promoted the expression of spermatogenesis-related genes and regulated testosterone secretion. These results suggest that Agarose-Supported culture is a promising approach for preserving and studying testicular tissue development in large livestock.

Optimizing the cultivation system is crucial for tissue culture. The culture of cryopreserved testicular tissues is of great importance for the germplasm preservation of endangered animals and especially to ensure high-quality and high-output livestock. In this study, we compared two cultivation systems (Agarose-Supported system and Direct Adherent system) by evaluating their effects on tissue morphology, cell proliferation, apoptosis, gene expression, and endocrine function in cryopreserved testicular tissues from 30-day-old calves. The testicular tissues were cultured for 18 and 27 days with three biological replicates per group, aiming to identify which system better supports tissue preservation, cellular viability, and spermatogenic differentiation. This allowed us to clarify how different cultivation systems influence the structural maintenance and developmental potential of immature bovine testicular tissues. Histological and gene expression analyses revealed that the Agarose-Supported system better preserved the seminiferous cord architecture and supported the development of the seminiferous epithelium compared to the Direct Adherent system. The Agarose system significantly reduced the apoptosis and enhanced the expression of some key genes, including spermatogonial stem cell (SSC) markers (GFRα-1, UCHL1), meiotic marker (SYCP3), mature sperm marker (CRISP1), and testicular somatic cell markers (STAR, SOX9, ACTA2). The Agarose-Supported system also benefited spermatogenic differentiation and testosterone secretion. These findings demonstrate that the Agarose-Supported system facilitates the in vitro development of spermatogenic cells and Leydig cells in post-cryopreserved immature bovine testicular tissues.