Sperm Susceptibility to Oxidative Stress in the Retuertas Endangered Horse

Abstract

In order to determine whether the high level of inbreeding of the Retuertas endangered Horses may increase the vulnerability to oxidative stress of the sperm, the susceptibility to lipid peroxidation (LPO) of sperm membranes was evaluated in cooled extended ejaculates. Extended ejaculates (INRA 96) from four Retuertas horses and nine ejaculates from pure Spanish horses were received within 24 hours of collection at our laboratory. An aliquot was analyzed and served as time 0 control. The rest of the ejaculates were split into two additional aliquots: the first aliquot served as an incubation control and the second aliquot received an oxidative insult (Fe₂SO₄). Membrane LPO, motility parameters, membrane integrity, and mitochondrial membrane potential were evaluated. The oxidative insult induced a significant increase in the percentage of sperm with LPO in the Retuertas horses (P < .05). Total and progressive motility were not affected by the oxidative insult in both groups. However, curvilinear velocity (VCL), average velocity (VAP), straightness index (STR), and amplitude of lateral head displacement (ALH) were significantly affected by the treatment (P < .05) in the Retuertas horse population. The membrane integrity of sperm in this group was also affected: the percentage of sperm with intact membranes decreased (57.31% ± 7.76% vs. 47.52% ± 8.28%); and the percentage of necrotic sperm increased (33.27% ± 7.83% vs. 44.85% ± 9.16%) (P < .05). Finally, the oxidative insult increased the population with low mitochondrial membrane potential in these wild horses. We concluded that the high level of inbreeding of this particular breed apparently leads to a higher susceptibility to lipid peroxidation.

Introduction

The Retuerta horse is a population of endangered feral horses that has lived in an isolated area covering approximately 300 km² in the Doñana National Park in southern Spain. This endangered breed relies on 127 animals (50 males and 77 females) to preserve the genetics of the population [1]. Because of the reduced number of animals, in the 1980s, a program of conservation of these equines was started by scientists at the Doñana Biological Reserve. One of the main problems with endangered wild species is the inbreeding depression caused by mating between closely related animals; in this case, the increase in inbreeding rate year round was 0.1% [2]. This inbreeding may depress the immune defenses due to decline in genetic variability, leading to a high vulnerability to diseases and pathogens [3], [4]. Moreover, a negative effect on fertility and/or semen quality has been demonstrated in several species [5], [6]. The development of assisted reproductive techniques to ensure the preservation of genetic diversity may reduce and prevent the inbreeding depression in these threatened species. The advances in reproductive biotechnologies and the used of captive breeding programs have been able to recover species in danger of extinction like Prezwalski's horse. The development of a genetic bank of frozen semen that preserves semen indefinitely could facilitate the genetic management of this species, providing mating strategies. Currently, cryopreservation has become an area of increased interest for the equine industry, especially in superior sires [7], although this technology is still in a suboptimal level of development [8]. Recently, new advances in cryopreservation protocols [9], [10], [11], [12] and new extenders [13], [14] have been designed to reduce the variability and to improve the obtained results. One of the major insults that spermatozoa of domestic horse suffer during cryopreservation or refrigeration is oxidative stress [15], [16], [17]. Previous findings from our laboratory demonstrated that lipid peroxidation (LPO) of sperm membranes triggers an apoptosis-like phenomenon during stallion sperm cryopreservation [18]. Moreover, differences in the susceptibility to LPO can explain stallion-to-stallion variability in the ability of their ejaculates to survive the process of cryopreservation. However, there are no studies that evaluate whether inbreeding in stallions could affect susceptibility to LPO. Recent studies in other endangered species demonstrated that parameters such as motility, morphology, and proportion of spermatozoa with intact acrosomes were affected by a high inbreeding coefficient [19], [20]. Therefore, to support the conservation program for Retuertas horses, we determined whether the high inbreeding of this particular breed might instigate a higher susceptibility to oxidative stress of the sperm membranes and whether motility might be affected. The aim was to use susceptibility to oxidative stress to mimic the stress after cryopreservation and compare it with a control domestic population.