Effects of the cryopreservation process on dog sperm integrity.

Carmen Cecilia Sicherle,Frederico Ozanam Papa,Fabiana Ferreira De Souza,Carlos Roberto Padovani,Maria Denise Lopes,Gabriele Barros Mothé,Camila De Paula Freitas-Dell Aqua

doi:10.21451/1984-3143-ar2019-0081

Carmen Cecilia Sicherle, Frederico Ozanam Papa + Show 5 more

Open Access

https://doi.org/10.21451/1984-3143-ar2019-0081

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Journal: Animal Reproduction	Publication Date: Jan 1, 2020
Citations: 12	License type: cc-by

Affiliation: Universidade Estadual Paulista (Unesp)

Abstract

Sperm cryopreservation has become an indispensable tool in reproductive biology. However, frozen/thawed semen has a short lifespan due to loss of sperm cell integrity. To better understand which sperm cell structures are compromised by the cryopreservation process and apoptosis markers, the sperm of five healthy mature dogs was analyzed in this study. Analysis was performed after collection, cooling, and thawing via computer assisted sperm analyzer (CASA) and evaluation of membrane fluidity and permeability, phosphatidylserine translocation (Annexin V), membrane integrity, mitochondrial membrane potential, membrane lipid peroxidation (LPO) and activity of the apoptotic markers caspases 3 and 7 by flow cytometry. Cryopreservation decreased total and progressive motility and the percentage of rapid sperm (P < 0.01). Damage to sperm cells was confirmed by Annexin V (P < 0.01), indicating that capacitation-like changes were induced by the cryopreservation procedures. An increase in sperm membrane fluidity was also noted in frozen/thawed samples (P < 0.01). Plasma and acrosomal cell membranes were affected (P < 0.01), with decreases in the subpopulation displaying high membrane potential (P < 0.01). Membrane LPO was increased in thawed sperm compared to cooled sperm (P < 0.05) but was not different from that in fresh sperm. No differences were observed in caspase 3 and 7 activity after cooling, freezing, or thawing. In conclusion, total and progressive motility, plasma membrane integrity and mitochondrial membrane potential suffered from the deleterious effects caused by cryopreservation, unlike the activity of caspases that remained stable during the freezing process.

Highlights

Cryopreservation of semen offers practical advantages because there is no dependence on time or distance, and it can indefinitely preserve the genetics of higher animals
To investigate which sperm cell structures are compromised by the cryopreservation process and apoptosis markers, the objective of the present study was to evaluate the effects of cryopreservation on sperm cell membrane integrity, mitochondrial membrane potential, membrane lipid peroxidation (LPO) and caspase activity
Compared to fresh and cooled semen, cryopreservation treatment of sperm led to a significant decrease in total and progressive motility and in the percentage of cells displaying rapid movements (P < 0.01) (Table 1)

Summary

Introduction

Cryopreservation of semen offers practical advantages because there is no dependence on time or distance, and it can indefinitely preserve the genetics of higher animals. Frozen/thawed dog semen has a short lifespan due to loss of sperm cell integrity during the processes of cooling, freezing and thawing (Alhaider and Watson, 2009). Regardless, the viability of sperm when analyzed by motility is usually greater than the actual fertilization capacity due to changes that occur in cell membranes during the cryopreservation process (Maxwell and Watson, 1996).

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