Effect of Antioxidants on Pig Semen Cryopreservation to Preserve Sperm Fertility after Thawing

Special Article - Veterinary & Animal Sciences

Ann Agric Crop Sci. 2021; 6(5): 1090.

Effect of Antioxidants on Pig Semen Cryopreservation to Preserve Sperm Fertility after Thawing

da Costa Silva RJ, da Silva MHM, Valadão L and da Silva FM*

Department of Animal Reproduction, Faculty of Agrarian Sciences and Environment Animal, University of the Azores, IITAA, Portugal

*Corresponding author: Fernando Moreira da Silva, Department of Animal Reproduction, Faculty of Agrarian Sciences and Environment Animal, University of the Azores, IITAA, 9701-851 Angra do Heroísmo, Portugal

Received: July 02, 2021; Accepted: July 28, 2021; Published: August 04, 2021

Abstract

Boar semen cryopreservation has a high potential in the swine industry, allowing the large-scale use of genetically superior animals, improving efficiency, product quality, helping to reduce the risk of disease spread and gathering needs from the market. From a genetic point of view, semen freezing is desirable for genetic diversification, favouring a more efficient reproduction as well as the constitution of germplasm banks, including for repopulation in case of disease outbreak. However, freezing this semen for long periods for practical use is limited by the reduced viability and fertilization potential caused to sperm during the cryopreservation process and consequently low conception rates and smaller litters after artificial insemination. In part, the decrease in the fertilizing power of frozen spermatozoa may be associated with oxidative damage due to excessive formation of Reactive Oxygen Species (ROS), osmotic stress and cell damage due to ice formation during cryopreservation.

To suppress the damage caused by ROS, the present study was conducted to determine the impact of supplementation with three antioxidants, these being ascorbic acid, a-tocopherol and reduced glutathione, evaluating the parameters of semen quality, viability, total and progressive motility, vigour and agglutination rate after thawing. For this purpose, semen was collected from five boars, each being collected three times, at weekly intervals, always at the same time. Immediately after harvesting, the macroscopic (colour, appearance, and volume) and microscopic evaluation of the semen (mass motility, concentration, progressive individual motility, spermatic vigour and spermatic morphology) were evaluated. Subsequently, the semen was placed at 15°C for two hours and centrifuged at 800 x g for 10 minutes also at 15°C, removing the supernatant. For the freezing medium, a base medium consisting of a commercial MR-A extender, supplemented with 3% v/v glycerol, 10% v/v egg yolk and 0.20% w/v Sodium Dodecyl Sulfate (SDS) was used. The nine treatments used in the study were, respectively, ascorbic acid at concentrations of 100, 200 and 400μL, a-Tocopherol at concentrations of 200, 400 and 800μM and reduced Glutathione at concentrations of 2.5, 5 and 10 mg/l and numbered as T1 to T9, respectively. In the control group, semen was frozen in a medium without adding any antioxidant. The semen belonging to the different treatments and to the control was placed in 0.25ml insemination French straws and incubated at 6°C for two hours. The subsequent freezing was carried out in nitrogen vapours (-120°C) for ten minutes and immersed in liquid nitrogen after this period. After 7 days, the semen was thawed in a water bath at 37°C for 20 seconds, the straws dried on paper, placed on a microscope slide heated to 37°C and evaluated according to the parameters described above.

Regarding the comparison between the different treatments, it was observed that the sperm viability obtained in the treatments with ascorbic acid as well as glutathione reduced, was not statistically different from the control group. Higher values of ascorbic acid and reduced glutathione reduced sperm viability after thawing. As for the use of a-tocopherol at a concentration of 400μM, the best results of the entire study were obtained, with sperm viability of 31.52% (±1.50). Regarding sperm motility and agglutination rate, a-tocopherol also showed the best results at the concentration of 200μM, in which the mean sperm motility was 2.57 ± 0.15 and 2.07 ± 0.15, respectively.

The results of the present study allow us to infer that the addition of 200μM or 400μM of a-tocopherol to the swine semen-freezing medium has a positive effect on sperm viability parameters after thawing.

Keywords: Reactive oxygen species; Antioxidants; Freezing; a-tocopherol; Glutathione; Ascorbic acid; Semen

Introduction

The principle of cryopreservation is to conserve biological material by reversibly reducing cell metabolism, allowing the conservation of cells and tissues for indeterminate periods. In animal production, the cryopreservation of embryos and especially of gametes, has allowed, in addition to the constitution of germplasm banks, a great advance, since mainly for semen, after thawing and its use in artificial insemination has allowed a genetic evolution continuous improvement of phenotypic and genotypic characteristics of animals.

Regarding pigs, the conservation of semen through cryopreservation remains limited, given the reduction in the viability and fertility of the semen after its thawing, taking into account the percentage of returns and the number of piglets produced per litter, when compared with the results after using fresh or refrigerated semen. The low viability of spermatozoa observed after their thawing is associated with several factors, including oxidative damage, intracellular and extracellular ice formation during the semen cryopreservation/thawing processes, excess formation of Reactive Oxygen Species (ROS), osmotic stress, among others [1]. Free radicals produced by cells, and particularly sperm, are highly reactive groups of molecules, with one or more unpaired electrons. Its structure makes free radicals very susceptible to react with other molecules, oxidizing them, leading to a decrease in sperm motility, increased damage to sperm DNA, decreased efficiency in sperm fusion in oocytes, lipid peroxidation of the cell membrane, among others [2].

Given that the damage caused to swine sperm during their cryopreservation/thawing is more severe when compared to other species, there is also the need to understand the processes that can enable the maintenance of metabolism throughout the freezing and thawing procedure that allows keep its metabolic characteristics unchanged, making this semen capable of being used for artificial insemination. Antioxidants that, according to Halliwell and Gutteridge [3], can be defined as substances that, when present in low concentrations when compared to the oxidizable substrate, are capable of significantly inhibiting or delaying the oxidation of a substrate, have been widely studied with the aim to reduce damage caused by oxidative stress of sperm during cryopreservation [4]. Work carried out in this area has shown that the addition of antioxidants to semen has been beneficial in its protective action against oxidative damage. Estrada et al. demonstrated that the addition of ascorbic acid to semen significantly reduces (p >0.05) sow returns to heat, increasing the total number of live born piglets per litter [5]. Giaretta and collaborators have also shown that reduced glutathione and ascorbic acid, when combined, have a beneficial effect on sperm freezing and thawing [6]. Particularly in swine, despite the known existence of enzymatic and non-enzymatic antioxidants, in seminal plasma, an imbalance between the total of antioxidants and ROS may occur, causing a state of oxidative stress. In a bibliographical research work, Moreira da Silva et al. [7] postulated that ROS might be of endogenous origin, through reductions of oxygen for energy production and as a byproduct, and of exogenous origin, for example, through chemical products and radiation forming free radicals from oxygen. Among these, the most common anion superoxide (O2-), hydrogen peroxide (H2O2), peroxyl radical (ROO-) and hydroxyl radical (OH-). ROS, when in controlled amounts, are necessary for sperm capacitation, activation and fusion of sperm in oocytes, however, when they are present in high amounts, there is a disruption of the balance in cell metabolism, which can lead to cell death [8]. In order to counteract the imbalance caused by excess oxidants and associated cellular damage, antioxidants have the function of suppressing excess ROS and allowing stability in the levels of free radicals, which can act at three different levels: prevention, interception and repair.

Thus, the aim of this study was to evaluate the survival capacity of sperm during the freezing/thawing process of swine semen, changing the base medium with different concentrations of antioxidants, namely a-Tocopherol, Ascorbic Acid and Reduced Glutathione. Several studies have demonstrated the success of antioxidant supplementation in freezing media, improving semen quality in response to increased fertility after sperm thawing [9]. The use of these antioxidant agents may represent a new approach to the preservation of frozen swine semen, allowing the development of Artificial Insemination (AI) strategies and protocols in the swine industry using frozen semen, avoiding the harmful imbalance of free radicals in the semen and obtaining results more consistent enabling its application on a large scale.

Materials and Methods

Semen collection was carried out in boars belonging to the artificial insemination center for pigs of the Agrarian Development Services of Ilha Terceira. In total, five males were used, each being collected 3 times, at weekly intervals, always at the same time.

The method used in all collections was the gloved hand technique with the use of a thermal cup with a filter on top, and the set was kept at a temperature of 37°C to avoid thermal shocks. The first fraction of the ejaculate was rejected because it contained little sperm concentration, and only the sperm-rich fraction of the ejaculate was used. After collection, the filter was removed and immediately macroscopic and microscopic evaluation of the semen was carried out using the protocol described by Westendorf et al. [10] with some modifications.

For the macroscopic evaluation, the semen was analysed taking into account the colour, appearance, and volume of the ejaculate, only samples whose appearance was opaque and creamy were used. Samples with different appearance were discarded. Regarding the microscopic evaluation, mass motility, concentration, progressive individual motility, spermatic vigor and spermatic morphology were evaluated using a Leica DFC 320 phase contrast microscope coupled to a digital image recording system.

Sperm motility was determined by evaluating at least 400 sperm in each semen sample. Each sperm was categorized as belonging to one of the four motility categories (fast progressive, slow progressive, non-progressive and immobile), using evaluation and quality control techniques. The values of motile sperm are, therefore, the sum of the two categories of motile sperm: fast progressive, slow progressive, based on Table 1 and 3, and collections whose semen presented motility below 70% were rejected.