# Field-Compatible Cytometric Assessment of Epididymal Alpaca Sperm Viability and Acrosomal Integrity Using Fluorochrome

## TL;DR

This study introduces a practical method to assess alpaca sperm quality in the field using dyes and simple tools, enabling storage and analysis up to one week later.

## Contribution

A field-compatible cytometric method for evaluating alpaca sperm viability and acrosomal integrity using fluorochrome staining and formaldehyde fixation.

## Key findings

- Fixed samples showed higher viability and acrosomal integrity values after 24 hours and one week of storage.
- Strong correlations and Bland–Altman analysis confirmed consistent results across time points.
- The method allows accurate sperm quality evaluation for up to one week without immediate laboratory analysis.

## Abstract

Alpacas are important animals in the Andes, especially for rural families who rely on them for income. Ensuring good reproductive performance in males is essential, but evaluating sperm quality often requires advanced laboratory tools that are not available in remote areas. This study offers a simple and practical method to assess the quality of alpaca sperm collected after death, using two special dyes that identify whether the sperm cells are alive and whether they are structurally intact. After staining, the samples were preserved in a fixative and stored at 5 °C for up to one week before being analyzed. The results showed that the method is reliable, with consistent findings even after storage. This approach is helpful for veterinarians and researchers who work in the field and need a way to collect and preserve sperm samples until they can be analyzed in a laboratory. While this study used sperm from the epididymis, future research should focus on ejaculated semen. Overall, this method can support better reproductive management and genetic conservation programs in alpacas, especially in rural regions where access to equipment is limited but fertility monitoring remains important.

In remote alpaca breeding regions, access to advanced sperm analysis laboratories is limited. This study validates a practical cytometric method for evaluating sperm viability and acrosomal integrity in epididymal alpaca sperm using early fluorochrome staining, formaldehyde fixation, and intermediate storage. Thirty-two testes were transported at 5 °C, and spermatozoa were collected from the cauda epididymis. After morphometric screening, 26 samples were included. Aliquots were stained with Zombie Green (viability) and FITC–PSA (acrosomal integrity), at time zero. Each aliquot was divided for cytometric analysis at T0 (immediately), T24 (24 h after formaldehyde fixation) and T1w (1 week post-fixation). Fixed samples showed higher viability and acrosomal integrity values (T24: 70.75%, 97.24%; T1w: 71.80%, 97.21%) than T0 (67.63%, 95.89%). This may reflect fluorescence alterations associated with fixation. Strong correlations and Bland–Altman analysis confirmed consistency across time points. This method enables accurate sperm quality evaluation up to one week after collection, offering a useful tool for reproductive monitoring in field conditions without immediate analysis. Further research on ejaculated semen and field protocols is recommended.