Impact of Seminal Plasma Antioxidants on Donkey Sperm Cryotolerance

Abstract

This study investigated whether the activities of the antioxidant components of donkey seminal plasma (SP)—both enzymatic (superoxide dismutase (SOD), catalase-like (CAT), glutathione peroxidase-like (GPX), and paraoxonase type 1 (PON1)) and non-enzymatic (measured in terms of total thiol, copper-reducing antioxidant capacity (CUPRAC), ferric-reducing ability of plasma (FRAP), and Trolox equivalent antioxidant capacity (TEAC))—and oxidative stress index (OSI) are related to sperm cryotolerance. For this purpose, 15 ejaculates from jackasses (one per individual) were collected and split into two aliquots. The first one was used for measuring the activities levels of enzymatic and non-enzymatic antioxidants and OSI in SP, whereas the other aliquot was cryopreserved. Before cryopreservation, sperm quality parameters (concentration, motility, and viability) were evaluated. After thawing, sperm motility, plasma membrane integrity, lipid disorder, mitochondrial membrane potential, reactive oxygen species (ROS), and calcium intracellular levels were also determined. Based on the percentages of total motility (TM) and of sperm with an intact plasma membrane (SYBR14+/PI−) after thawing, samples were classified as good-freezability (GFE) or poor-freezability (PFE) ejaculates through cluster analyses. The SP activity levels of enzymatic (SOD and PON1) and non-enzymatic antioxidants (CUPRAC, FRAP, and TEAC) were higher (p < 0.05) in GFE than in PFE, whereas SP-OSI was higher (p < 0.05) in PFE than in GFE. In addition, the activity levels of SOD, PON1, GPX, CUPRAC, FRAP, and TEAC were positively (p < 0.05) related to post-thaw sperm motility and plasma membrane integrity and negatively to intracellular ROS levels. The SP-OSI was negatively correlated (p < 0.05) to post-thaw sperm quality parameters and positively to intracellular ROS levels. It can thus be concluded that donkey SP antioxidants are related to sperm cryotolerance and that measurements of antioxidants PON1, SOD, CUPRAC, FRAP, and TEAC, as well as SP-OSI, could be used as markers of sperm cryotolerance. Further research addressing the relationship of these antioxidants and SP-OSI with sperm cryotolerance and their potential use as freezing markers is warranted.

Seminal Plasma Antioxidants Are Related to Sperm Cryotolerance in the Horse

“…Consequently, non-enzymatic antioxidants of SP (assessed by TEAC) could be key to maintaining the functional integrity of cryopreserved horse sperm, as the positive relationship between TEAC in SP and post-thaw sperm motility parameters demonstrated. This would be also in line with the results of the aforementioned studies in frozen-thawed pig [42] and donkey [28] sperm. It is also worth noting that the fact that the CUPRAC of SP did not differ between GFE and PFE was in agreement with previous results obtained in frozen-thawed pig sperm [42].…”

“…Specifically, SOD neutralizes O 2 -, which, in excess, triggers a dangerous oxidative chain reaction leading to lipoperoxidation of plasma membrane and thereby leads to the loss of motility and sperm death [15]. This would explain the positive relationship of SOD activity with sperm motility and acrosome integrity, as well as the differences between GFE and PFE observed herein and in previous studies with frozen-thawed horse [14] and donkey [27,28] sperm. In addition, it is important to mention that the activity levels of SOD in horse SP observed in this work were lower than those reported in a recent study in donkey SP [28], but higher than those observed in pigs [42], which would indicate a great inter-species variability in SP composition.…”

“…In addition, a ROS/antioxidants imbalance may cause mitochondrial dysfunction, leading to adenosine triphosphate depletion; this decrease in the energy available to sperm results in a reduction of their motility [88]. Interestingly, OSI values in horse SP were higher than those reported in donkey [28] and in pig SP [45]. One may, therefore, posit that the control of oxidative stress is highly complex and species-specific [14].…”

“…Horse sperm are particularly susceptible to oxidative stress due to the large amount of polyunsaturated fatty acids (PUFAs) in plasma membrane phospholipids [ 13 , 19 , 20 ], and their low intracellular antioxidant activity [ 21 , 22 , 23 , 24 ]. Under this scenario, seminal plasma (SP) represents the main antioxidant defense source, as it is endowed with enzymatic and non-enzymatic antioxidants that scavenge excessive ROS [ 25 , 26 , 27 , 28 ]. Yet, current equine semen cryopreservation protocols remove SP by centrifugation [ 14 , 29 ].…”

“…In the horse, as in other mammalian species, differences in sperm cryotolerance between breeds [ 32 ], individuals [ 33 ] and even between ejaculates from the same animal [ 6 ] have been reported. Because these differences, which allow ejaculates to be classified as with “good” (GFE) or “poor” freezability (PFE) [ 14 , 28 , 34 ], are related to the composition of SP [ 35 , 36 ], elucidating the function of antioxidants is very relevant. In horses, some SP antioxidant enzymes (such as superoxide dismutase (SOD), catalase (CAT) and the glutathione peroxidase/glutathione reductase system (GPX/GSR)) are responsible for reducing oxidative stress [ 14 , 26 , 37 ].…”