Comparison of microscopy techniques for the assessment of boar sperm morphometry

Bc. Bhavika Sanjaykumar Upadhyay

Master's thesis

Abstract:

The study was conducted to examine the suitable technique for assessing sperm morphometry in boar. Sperm morphology and morphometry are very important factors to predict fertility. Sperm are considered fertile and to be normal if the size of head, midpiece and tail fall under the classification of given species. Several studies had been directed to evaluate the sperm head and flagellum morphometry in boars. One of the main issues for the evaluation of sperm morphology and morphometry is that the different staining methods affect the accuracy of the analysis. The method of staining, as well as the evaluating specimens can remarkably affect the results of morphometric measurements, thus it is important to select the appropriate technique for staining the sperm of a given species. Moreover, most of the staining methods do not allow to properly discern the different structures of the sperm flagellum such as the midpiece, which contains the mitochondria. For this reason, alternative methods for the assessment of sperm morphometry without the use of staining, like phase contrast microscopy, could provide accurate measurements of the sperm flagellum structures. In this study, two different microscopy techniques were used: bright field and phase contrast. For bright field microscopy, Farelly and Hemacolor staining were used, while unstained samples were analyzed by phase contrast microscopy. Semen samples from 5 boars were evaluated for each technique. Measurements had been taken under imageJ software. The following sperm morphometric parameters were determined: head length, head width, head area, head perimeter, flagellum length, midpiece length, and principal plus terminal piece length. From these measurements, several head shape factors and the total sperm length were also determined. There were significant differences (p<0.05) between Hemacolor and Farelly staining in several sperm head parameters. By using the phase contrast technique, the structures of the flagellum were clearly defined allowing a faster analysis of the morphometric parameters. Even though the phase contrast technique did not show significant differences (p>0.05) in any sperm measures in comparison with the other techniques, it shows the lowest coefficient of variation (CV) for several sperm parameters, especially the midpiece (p<0.05). In conclusion, the phase contrast was the most suitable technique for assessing boar sperm morphometry because of the reduced CV obtained and a faster analysis of the samples.

Keywords

Boar, Bright field microscopy, Coefficient of variation, Phase contrast microscopy, spermatozoa, sperm morphometry, Staining

Language used: English

Date on which the thesis was submitted / produced: 25. 4. 2021

Thesis defence

• Supervisor: Jose Luis Ros-Santaella
• Reader: Nicole Sjahrirová, externi

Czech University of Life Sciences Prague

Faculty of Agrobiology, Food and Natural Resources

Master programme:
Sustainable Agriculture and Food Security