Flow cytometric assessment of the quality of equine spermatozoa after different storage lengths

Abstract

Introduction - Artificial insemination is a common breeding technique in the equine industry. Insemination can be either performed, using fresh, frozen/thawed or cooled-stored semen. Freezing and thawing as well as cooling of semen in thought to induce capacitation-like changes and reduces the ability of spermatozoa to perform physiological capacitation. Semen quality is, until now, assessed in a rather classical way, considering sperm count, motility and morphology. Nowadays, more modern techniques, such as flow cytometry are available, making it possible to assess characteristics such as the capacitation state of spermatozoa.

Aim - The aims of the study were I) to assess sperm quality, by flow cytometric determination of sperm viability, acrosome integrity, intracellular [Ca2+] and the capacitation state of spermatozoa, during cooled-storage over time and II) to determine the ability of cooled- stored spermatozoa to perform capacitation over time.

Material and methods - Semen of six breeding stallions, attending the Faculty of Veterinary Medicine, Utrecht University, was collected. Insemination doses, containing 50 x 106 sperm/mL were cooled-stored (°5C) in Styrofoam boxes for up to 96 hours. Computer Assisted Sperm Analysis and flow cytometric assessment were performed at 1 hour after collection but before cooling and 8, 24, 48, 72 and 96 hours after cooled storage. In addition, temperature curves were made, during the first 8 hours of storage.

Results - Motility significantly declined earlier than viability (at 24 hours and 48 hours of storage, respectively). The decline in the percentage of non-capacitated sperm cells was accompanied by an increase in intracellular [Ca2+], indicating that capacitation-like changes in stallion spermatozoa result in a rise in intracellular [Ca2+], instead of in an increase of membrane fluidity as is the case in physiological capacitation. Storage seemed to induce these capacitation-like changes, rather than the cooling process. A high correlation could be found between the progressive motility and the size of the population of “optimal” spermatozoa. The percentage of spermatozoa, able to capacitate, showed a declining trend from 24 hours on.

Conclusion - No significant changes in parameters were found, during the first 24 hours of storage, except for the trends in the size of the “optimal” sperm population and the percentage of spermatozoa, able to capacitate. AI with semen stored for more than 24 hours might theoretically result in lower pregnancy rates, because the number of spermatozoa, able to capacitate declines.