# Using Cholesterol-Loaded Cyclodextrin to Improve Cryo-Survivability and Reduce Capacitation-Like Changes in Gender-Ablated Jersey Semen

## TL;DR

Adding cholesterol to sex-selected bull semen improves sperm quality and fertility after freezing, without harming motility or other key traits.

## Contribution

This is the first study to use cholesterol-loaded cyclodextrin with gender-ablated semen to improve cryo-survivability and fertility.

## Key findings

- Cholesterol treatment increased sperm with intact acrosomes and membranes from 28.9% to 34.1%.
- Fertilization rates improved to 82% in cholesterol-treated groups after 12 hours, compared to 74% in controls.
- CLC preserved mitochondrial activity, capacitation progress, and oviduct cell binding ability in post-thaw sperm.

## Abstract

Sex-selected semen is now an integral feature of dairy cattle production and is essential to profitability and sustaining productivity. The sexing procedures and subsequent cryopreservation can damage sperm membranes, lower sperm lifespan, and ultimately reduce the fertility of sex-selected semen when compared to conventional semen. This study aimed to improve the quality of gender-ablated semen. Our approach was to increase the cholesterol content of sperm plasma membranes to reduce membrane damage and keep them intact until insemination. Adding cholesterol to sperm cells did not increase sperm motility. However, the portion of sperm cells with intact acrosomes and membranes significantly increased from 28.9 ± 1.2% to 34.1 ± 1.2% after adding cholesterol. Mitochondrial activity, maturation progress, and binding ability to oviduct cells (required for sperm lifespan inside the cow reproductive system) were maintained. Cholesterol treatment did not delay fertilization time but significantly increased fertilizing ability. After 12 h of co-incubation with oocytes, the fertilization rate rose to 82 ± 3% in the cholesterol-treated group, compared to 74 ± 3% in the control group (p < 0.05). Overall, the addition of cholesterol added minimal improvement to the quality of post-thaw gender-ablated semen, and further studies are still needed to maximize the benefits of cholesterol.

Sexing procedures and subsequent freezing still impact sperm cells, leading to decreased fertility of gender-ablated semen. This study aimed to enhance cryo-survivability and reduce the capacitation-like change rate of gender-ablated semen by adding 2 mg of cholesterol-loaded cyclodextrin (CLC) per mL of extended semen containing 67 × 106 sperm cells. This marks the first use of CLC with gender-ablated semen. Semen from four Jersey bulls was used for this study. Viability, motility, and mitochondrial activity were evaluated and adjusted to account for the inactivation of undesired sex sperm cells during processing. Binding ability to oviduct cells, fertilizing ability, and acrosome status were also evaluated. Adding CLC did not increase sperm motility. The population with intact membranes and acrosomes was significantly increased (p < 0.05) from 28.9 ± 1.2% to 34.1 ± 1.2% in the CLC-treated group. Mitochondrial potential, capacitation status at the membrane, calcium levels, and binding ability to oviduct cells were maintained. CLC treatment did not delay capacitation while significantly improving fertilization rates after 8 and 12 h of co-incubation (77 ± 3% vs. 67 ± 3% and 82 ± 3% vs. 74 ± 3%, respectively; p < 0.05). In conclusion, CLC addition significantly improved gender-ablated post-thaw sperm viability, acrosome integrity, and fertilizing ability while preserving motility, capacitation progress, and binding ability to oviduct cells.