Seasonal changes in ROS concentrations and sperm quality in unfrozen and frozen-thawed stallion semen

Beatrice Mislei; Diego Bucci; Eleni Malama; Heirich Bollwein; Gaetano Mari

doi:10.1016/j.theriogenology.2019.12.016

Seasonal changes in ROS concentrations and sperm quality in unfrozen and frozen-thawed stallion semen

Theriogenology. 2020 Mar 1:144:89-97. doi: 10.1016/j.theriogenology.2019.12.016. Epub 2019 Dec 24.

Authors

Beatrice Mislei¹, Diego Bucci², Eleni Malama³, Heirich Bollwein³, Gaetano Mari⁴

Affiliations

¹ AUB-INFA National Institute of Artificial Insemination, University of Bologna, Italy. Electronic address: beatrice.mislei@unibo.it.
² Department of Veterinary Medical Sciences, University of Bologna, Italy.
³ Clinic of Reproductive Medicine, Vetsuisse-Faculty University of Zurich, Switzerland.
⁴ AUB-INFA National Institute of Artificial Insemination, University of Bologna, Italy; Department of Veterinary Medical Sciences, University of Bologna, Italy.

PMID: 31927419
DOI: 10.1016/j.theriogenology.2019.12.016

Abstract

Oxidative stress is regarded as an important cause of sperm damage during cryopreservation. However, seasonal changes in oxidative status in unfrozen and frozen-thawed stallion sperm have not been well established. We tested the hypothesis that sperm ROS concentrations and lipid peroxidation change between breeding and non-breeding seasons and influence quality of unfrozen and frozen-thawed sperm. Eighteen ejaculates from six Warmblood stallions (8-21 y) known to be fertile, were collected in winter and summer and processed for freezing. After 90 min at +4 °C, some straws from each ejaculate were not frozen (unfrozen), whereas the remainder were frozen by N2 vapors, plunged in N2 and thawed (frozen-thawed). Rapid cells (RAP; determined by CASA), plasma membraneacrosome integrity (PMAI), high mitochondrial membrane potential (Mpos), low intracellular Ca2+ concentration (Fneg), membrane lipid peroxidation (BODIPY), intracellular ROS concentrations (DCFH, MitoSOX) and chromatin fragmentation (DFI%) were evaluated by flow cytometry in both groups and at intervals during incubation at +37 °C for 24 h. Overall, ROS concentrations and lipid peroxidation were higher and faster (P < 0.0001) in winter versus summer, DFI% was lower in winter versus summer (P < 0.0001), but similar between the two groups within season. There were moderate positive correlations in both seasons between DFI% and MitoSOX, DCFH, BODIPY in both groups, whereas a negative correlation, stronger in winter, was evident between sperm quality (RAP, PMAI, Mpos, Fneg) and BODIPY, DCFH, MitoSOX. There were no differences between seasons for RAP, PMAI, Mpos and Fneg. In conclusion, ROS-related parameters were higher in winter than in summer, without a negative effect on sperm quality. We concluded that increased ROS concentrations were less deleterious to sperm than freezing-thawing. Furthermore, incubation at +37 °C and sequential analysis were useful to assess sperm resistance.

Keywords: Freezing-thawing; Horse; Oxidative status; Seasonality; Sperm.

MeSH terms

Animals
Cryopreservation / veterinary*
Freezing*
Horses / physiology*
Male
Photoperiod
Reactive Oxygen Species
Seasons*
Semen Analysis / veterinary*
Sperm Motility

Substances

Reactive Oxygen Species