Utilize point formationLUNA-FX7 ™ Optimization of sperm cell evaluation using an automatic cell counter

keywordsAutomatic cell counter, image-based sperm cell evaluation, cell counting, survival rate evaluation, double staining

1 Introduction

Sperm cell assessment (also known as semen analysis) plays an important role in determining male fertility potential. Although it is not a direct indicator of fertility, male fertility has a significant impact on both human fertility and animal husbandry breeding, making sperm cell evaluation highly concerned.

At present, sperm cell evaluation can be performed through flow cytometry and image-based analysis tools. Click on LUNA-FX7 ™ Automated devices such as automatic cell counters can use images to quickly and conveniently analyze sperm samples. However, due to the unique morphology and characteristics of sperm cells compared to mammalian cells, it is necessary to optimize analysis parameters and select appropriate dyes.

This study compared and analyzed fluorescent dyes such as SYBR14, acridine orange (AO), and propidium iodide (PI) to optimize the spot forming LUNA-FX7 ™ Ability to evaluate sperm cells. This study aims to evaluate the effectiveness of different dye combinations and optimize the parameter settings for sperm cell evaluation.

2 Methods and Materials

2.1 sample preparation

Thaw frozen semen samples from cows and replace the buffer with PBS. According to the sample situation, dilute the semen sample in PBS at a ratio of approximately 1:20 to 1:40.

2.2 Fluorescent staining

1. Mixing:

Sperm cells in 18 μ L PBS

2 μ L mixed green and red fluorescent dyes

Incubate at 37 ℃ for 10 minutes

3. Load the sample onto a suitable glass slide

4. Use Diancheng LUNA-FX7 ™ analyze

Table 1. Click on LUNA-FX7 ™ Optimization parameter settings for sperm cell evaluation in advanced mode

3 Optimize the cell counting scheme and dye combination for sperm cell evaluation

summaryBoth SYBR14/PI and AO/PI double staining are suitable for evaluating sperm motility, but SYBR14/PI performs better.

Click on LUNA-FX7 ™ The default protocol has been optimized for common mammalian cells, but sperm cell evaluation requires adjusting parameters such as exposure level, cell size, and de aggregation sensitivity. For example, the minimum search size was adjusted from 3 µ m to 7 µ m, and the de aggregation sensitivity was adjusted from 5 to 7 to improve detection performance (Table 1). In addition, the exposure levels for SYBR14/PI and AO/PI staining were set to GF 6 and RF 9, respectively.

On this basis, we compared and analyzed the SYBR14/PI and AO/PI staining methods. Given the known effectiveness of SYBR14/PI staining method in sperm cell evaluation, we focus on the performance of AO/PI staining method. The research results indicate that both staining methods can be used for sperm cell evaluation (Figure 1). However, the applicability of SYBR14 in sperm cell staining is superior to AO, indicating that the chemical properties of SYBR14 may be more suitable for this application.

Figure 1. Fluorescence images of sperm cells stained with two different dyes (SYBR14/PI and AO/PI). This combination image displays the analyzed bright field, green light, red light, and marker images. The labeled image is a composite fluorescence channel image, where red circles represent dead cells and green circles represent surviving cells.

4 Conclusion

This study found that both SYBR14/PI and AO/PI staining methods can be used to evaluate sperm cell survival rate, but SYBR14/PI performed better. In addition, adjusting exposure levels and other parameters is crucial for overall detection performance. Although labeled as LUNA-FX7 ™ It is not specifically optimized for sperm samples, but through recommended sample dilution ratios and imaging conditions optimization, it can still provide a fast and convenient sperm cell evaluation scheme.