Cooled preservation of semen is usually associated with artificial insemination and genetic improvement programs in livestock species. Several studies have reported an increase in reactive oxidative species and a decrease in antioxidant substances and sperm quality parameters during long-term semen storage at refrigerated temperatures. The supplementation of antioxidants in extenders before refrigeration could reduce this detrimental effect. Various antioxidants have been tested, both enzymatic, such as superoxide dismutase and catalase, and non-enzymatic, such as reduced glutathione, vitamins E and C and melatonin. However, the problem of oxidative stress in semen storage has not been fully resolved. The effects of antioxidants for semen-cooled storage have not been reviewed in depth. Therefore, the objective of the present study was to review the efficiency of the supplementation of antioxidants in the extender during cooled storage of semen in livestock species.
1. Introduction
Semen preservation, either by freezing or refrigeration allows the separation of the moment of extraction from that of use in artificial insemination (AI) or in vitro fertilization, providing multiple applications in livestock and human species
[1,2][1][2]. In the case of livestock, semen preservation is usually associated with the AI technique and genetic improvement programs, allowing its use in places far from AI centers. AI is used for progeny testing of young males and for disseminating genetic improvement
[3]. Cryopreservation or cooled liquid storage have different pros and cons
[3], and the choice of the preservation method will depend on the AI efficiency in the specific species and the objective of the AI program. For example, frozen semen is usually used in bovine, while in porcine, AI is mainly performed with semen doses refrigerated at 15–18 °C and stored for several days. In general, fertility after AI is higher when using cooled rather than frozen/thawed semen.
In humans, spermatozoa are frozen to preserve fertility for the future (for example, prior to chemotherapy treatment
[2]) or for depositing in donor banks. Sometimes cooled preservation can be useful for transporting raw semen samples from one laboratory or collection place to another for additional tests or uses
[4]. Cooled semen is commonly used in domestic animals; therefore, the majority of the research studies concerning liquid cooled storage of semen referred to in this review were carried out with livestock species. Although freezing/thawing and refrigeration of semen are routine procedures in laboratories of livestock AI centers or human assisted reproduction clinics
[1], these procedures are not always optimized, and a worsening of several important sperm quality parameters has frequently been observed
[5,6][5][6].
Oxidative and nitrosative stress occurs when there is an excess of oxidants (reactive oxygen species (ROS) and reactive nitrogen species (RNS)), a deficiency of antioxidants, or both
[7,8][7][8]. When sperm samples were stored cooled for a certain time, an increase in ROS
[9,10,11][9][10][11] and a decrease in antioxidants
[11] were observed. Treatments with antioxidants to avoid damage due to oxidative stress (OS) in gametes can be approached from different perspectives
[12]. The first would be oral antioxidant supplementation, an approach widely discussed in different reviews on both male and female gametes in humans
[6,13,14][6][13][14]. The second would be the supplementation of antioxidants in media used during assisted reproductive technologies, mainly in semen extenders used to preserve samples. In this context, the use of antioxidants in the frozen/thawing process has been extensively discussed in several works
[15,16,17][15][16][17]. However, the effect of the inclusion of antioxidants in extenders used for semen cooled storage has not been reviewed in depth. Conclusions obtained in cryopreservation studies may not be applicable to refrigeration. There are substantial differences between the freezing and the refrigeration process, such as osmotic shock, cryoprotectant toxicity and/or the presence of ice crystals. In addition, cellular metabolism is practically stopped in frozen samples while in cooled storage sperm metabolism does not stop completely and the number of dead sperm progressively increases over time.
2. Treatments with Antioxidants in the Preservation Process
2.1. Enzymatic Antioxidants
Antioxidant enzymes present in spermatozoa and/or seminal plasma include SOD, CAT and GPx. As
Table 1 shows, SOD and CAT are the most extensively studied enzymatic antioxidants. SOD is the main antioxidant enzyme in seminal plasma
[15,99][15][18] and protects the cell against O
2•—, as it catalyzes the dismutation of this anion to H
2O
2. Additionally, this reaction prevents the formation of the highly reactive ·OH which happens when O
2•— and H
2O
2 react with ferric ion by the Haber-Weiss reaction
[100][19]. However, SOD activity promotes the formation of H
2O
2, a more stable and long-lived ROS, which can be removed by the cell using other enzymatic antioxidants such as CAT and GPx. In general, the addition of SOD to extenders, both alone or in combination with other antioxidants, has been found to increase sperm motility and viability in comparison with control groups in several species, although in canine and ovine these effects were not always evident (
Table 1;
[9,77,87,101,102,103,104][9][20][21][22][23][24][25]). In dogs, supplementation with SOD or SOD plus GPx did not improve the majority of sperm quality parameters in comparison with the control group
[101][22].
Table 1. Effects of enzymatic antioxidants in liquid cooled storage on sperm parameters.
Antioxidant |
Concentration |
Opt |
A/C |
Temp |
Time |
Species |
In Vitro Effects |
Ref. |
CAT |
50–150 U/mL |
100 |
A |
4 °C |
30 h |
bovine |
Increased sperm motility and decreased dead or abnormal spermatozoa, and acrosomal abnormalities compared with control group. |
[79] | [26] |
CAT |
100 U/mL |
|
A |
4 °C |
72 h |
canine |
Reduced total ROS, increased sperm motility. |
[105] | [27] |
CAT |
BHT90–3600 U/mL |
|
A |
5 °C |
72 h |
equine |
No effect or detrimental effect at high concentrations. |
[95] | [ |
SOD + GPx |
100 and 5 U/mL respectively |
|
C |
4 °C |
96 h |
canine |
No effect in sperm motility, DNA or acrosome status with compared with control group. Increased viability. |
[ | 101 | ] | [ | 22 | ] |