Effect of plasma separation techniques and sperm selection on sperm recovery and viability of cooled pony stallion semen for 48h at 5°C

G.A Pessoa,J.M Trentin,A.P Martini,T Minela,M.I.B Rubin

doi:10.1590/1678-4162-11986

Abstract

ABSTRACT Separation techniques of seminal plasma [centrifugation (SC) and Sperm Filter® (SF)] and sperm selection [Androcoll-E (SCA) and filtration glass wool (GW)] were used in 24 ejaculates from 6 stallions. In experiment 1, the ejaculates were allocated into control (no spin), centrifugation at 600 g x 10min, SF and GW. In experiment 2, semen was submitted to SC, SGA and filtered through GW. Following the treatments in both experiments, samples were kept chilled at 5°C to 50 x 106 sperm/ml for 48h. The variables measured on fresh and cooling semen were pH, motility, membrane viability function by 6-carboxyfluorescein diacetate and propidium iodide (CFDA / PI), viability or vitality (eosin / nigrosine) and mitochondrial activity. In experiment 1, centrifugation to remove seminal plasma resulted in greater damage to sperm than separation by sperm filter, and selection by glass wool was more efficient in separating viable cells and maintaining viability during cooling. In experiment 2 Androcoll-E and glass wool treatments resulted in higher (P <0.0001) motility, membrane function, mitochondrial activity, and viability than centrifuged semen. Both selection by Androcoll- E and glass wool improved the quality of semen pony stallions for preservation for up to 48h to 5ºC.

Highlights

Cooled or frozen semen in horses has become an important biotechnology applied to the reproduction management of this species (Ball et al, 2001)
The pH was similar between the fresh groups for the control (7.52), centrifuged (7.55), glass wool (7.54) and SpermFilter (7.53) (P = 0.96) groups
After 24h cooling, the pH did not differ between the control groups (7.25), centrifuged (7.26), glass wool (7.22) and SpermFilter (7.14) (P = 0.08)

Summary

Introduction

Cooled or frozen semen in horses has become an important biotechnology applied to the reproduction management of this species (Ball et al, 2001). Studies about reproduction of some breeds demonstrate reduced daily sperm production due to their size (Neves et al, 2006). The specific reasons for declining viability of the sperm cell during cooling and freezing processes involve several factors, among them osmotic and oxidative stress, pH changes and osmolarity that may be a consequence of toxic products eliminated by dead cells pre-processing (Ball, 2008). The production of oxygen-reactive species is high in the presence of cells damaged by the cryopreservation process, non-viable spermatozoa, or with morphological defects, among which we highlight intermediate part changes (Ball & Vo, 2001). Damage to the sperm membrane and mitochondria is greater when equine semen is subjected to cooling or cryopreservation

Methods

Results

Conclusion