**3. Oxidative stress and antioxidants**

#### **3.1. Oxidative stress (OS)**

**2.1. Semen cryopreservation media**

have [2], include:

storage unit (right).

106 Cryopreservation in Eukaryotes

metabolism.

**4.** Provide nutrition for the sperm metabolism.

**5.** Control microbial contaminants.

to 5°C.

A cryopreservation media, which is also known as semen diluent or extender, plays a cru‐ cial role in the quality of sperm after thawing and consequently, it affects significantly the success of AI in the livestock industry. This is because, the survival of the sperm after going through all the physical and biochemical challenges during cooling, freezing, and thawing is dependent mainly on the protective roles provided by the different components of the semen extender used. There are a number of ready‐to‐use commercially available semen extenders for the production of animals, which include Bioxcel®. Semen extend‐ ers such as tris‐egg‐yolk can also be prepared freshly in the laboratory provided that the components to be mixed are available. In spite of some variations among extenders in their composition, the ultimate objectives intended to be achieved are usually common. These objectives, which have been described as properties that a good semen extender should

**Figure 2.** Liquid nitrogen tank used to freeze and store semen in straws (left) and a cross section of a typical semen

**2.** To have a buffering capacity to maintain pH by neutralizing acid produced by sperm

**3.** To protect the sperm from cold shock injury during cooling from body temperature down

**6.** Provide sufficient protection to the sperm from damage during freezing and thawing.

**1.** To have the same free ion concentration or to be isotonic with semen.

**7.** Preserve the life of the sperm with minimum drop in fertility.

Before discussing about antioxidants and their role as supplements to protect sperm from oxidative damage, it is also important to review on what oxidative stress mean and evidence that shows sperm quality is indeed affected by oxidative stress. Oxidative stress (OS) refers to a disturbance in the balance between the production of reactive oxygen species (ROS) (free radicals) and the antioxidant defense that helps to counteract or detoxify their harmful effects. As discussed earlier, cryopreservation of sperm is a routine practice especially in cattle breed‐ ing industries for the purpose of artificial insemination. The freezing‐thawing procedures of cryopreservation are known to produce ROS in sperm samples. Exposure of semen to cold shock and atmospheric oxygen during cryopreservation increases the susceptibility to lipid peroxidation (LPO) due to higher production of ROS [5, 6].

Oxidants, such as reactive oxygen species (ROS) which are produced physiologically in liv‐ ing cells during respiration as well as by abnormal or dead sperm and phagocytic cells of both the ejaculate and female reproductive tract, affect the quality of postthawed sperm in animals [7, 8]. These ROS can inhibit sperm motility, capacitation, and acrosome reaction mediated by lipid peroxidation (LPO) of sperm membrane. Lipid peroxidation has been correlated with exposure of spermatozoa to ROS and it has been demonstrated that sper‐ matozoa undergoing freeze‐thaw cycles produces ROS [9]. A lipid peroxidation rate can be assessed by measuring malondialdehyde (MDA) level in the semen [10] which is one of the final products of polyunsaturated fatty acids (PUFAS) peroxidation in the cell and considered to be an oxidative stress biomarker.

Oxidative stress, which occurs when oxidants outnumber antioxidants in tissues or cells caus‐ ing pathological effects, is known to play a significant role in the pathophysiology of infertility in human [11]. Factors causing oxidative stress such as ROS are known to be involved in mul‐ tiple physiological processes from oocyte maturation to fertilization, embryo development, and pregnancy [11]. According to Agarwal et al. [12], OS is also considered to be one of the key causes of defective gametes and non‐ or poorly developing embryos in assisted repro‐ ductive techniques (ART). A poor fertilization rate, impaired embryo development, and higher rates of pregnancy loss associated with increasing OS in male germ cells are among the adverse effects recorded [13]. A number of other studies have also confirmed significant pathological effects of OS on gametes, embryos, and subsequent implantation resulting in poor pregnancy outcomes. Sperm DNA damage [14] implicated as the cause of increased incidence of abortion [13], loss of plasma membrane fluidity that leads to decrease in vigor and ultimate immobilization, and decrease in mitochondrial potential that leads to apoptosis are among the pathological effects of OS on sperm reported [15]. While reduced quality, early developmental block, and retardation of embryos [11], high fragmentation, and lower blas‐ tulation rate that lead to a lower pregnancy rate [2] are among the pathological effects of OS reported on embryos. Considering these adverse effects of OS on reproduction, ameliorating strategies such as *in vivo* and *in vitro* supplementation of antioxidants have been suggested and implemented with improved results [15, 16].

#### **3.2. Antioxidants and their role in sperm cryopreservation**

Antioxidants can be classified as enzymatic and nonenzymatic antioxidants. Enzymatic anti‐ oxidants are also known as natural antioxidants; they neutralize excess ROS and prevent it from damaging the cellular structure. Enzymatic antioxidants are composed of superoxide dismutase (SOD), catalase, glutathione peroxidase (GPx), and glutathione reductase (GR), which also cause reduction of hydrogen peroxides to water and alcohol [6, 11]. Nonenzymatic antioxidants are also known as synthetic antioxidants or dietary supplements. The antioxi‐ dant system in the body is influenced by dietary intake of antioxidants, vitamins, and miner‐ als such as vitamin C, vitamin E, zinc, taurine, hypotaurine, and glutathione [6, 11].

In mammals, seminal plasma contains a number of antioxidants which include superoxide dismutase, catalase, glutathione peroxidase, free radical scavengers such as vitamins C and E, hypotaurine, taurine, and albumin [17]. The presence of these antioxidants in semen helps to counteract with the oxidants and protect the spermatozoa from damage. Semen dilution using extenders for the purpose of having more doses from a single ejaculate and cryopreservation, however, decreases the concentrations of natural components of antioxidants. The decrease in the components of antioxidants due to dilution coupled with an increase in production of ROS during cryopreservation exacerbates the condition of spermatozoa and further degrades its postthaw quality and fertilizing ability. To minimize the effect of oxidants on a diluted semen, researchers have tested the impact of adding antioxidants into extenders in many species of animals including bull and have observed improvement in the quality of postthaw spermatozoa compared with controls based on conventional andrological tests [8, 17].

There are numbers of antioxidants tested as supplements to mammalian sperm cryopreserva‐ tion, but perhaps the most frequently studied antioxidant is alpha‐tocopherol form of vitamin E. Vitamin E is a fat soluble vitamin that may directly quench the free radicals such as peroxyl and alkoxy (ROO•) generated during ferrous ascorbate‐induced LPO; thus, it is suggested as major chain breaking antioxidant and a protectant of LPO and polyunsaturated fatty acids (PUFAS) in cell membranes from oxidation [6]. Addition of natural antioxidants such as alpha‐ tocopherol and ascorbate has been reported to have protective effect on metabolic and cellu‐ lar viability of cryopreserved bovine spermatozoa [18, 19]. More recently, butylated hydroxyl toluene (BHT), a synthetic analogue of vitamin E, has been tested for its antioxidant effect on bulls [8] and buffalo bull spermatozoa [17]. These studies investigated the impact of adding BHT into semen extenders on postthaw semen quality based on conventional andrological tests such as motility and viability. Results of their study indicated improvement of semen quality following cryopreservation compared to untreated controls. A concentration range of BHT added between 0.5 and 1 mM for bull spermatozoa [8] and between 1 and 2 mM for buf‐ falo sperm [17] was reported to be optimum for cryopreservation and better postthaw sperm quality. BHT is a synthetic analogue of vitamin E that controls the auto‐oxidation reaction by converting peroxy radicals to hydroperoxides [17]. BHT has also been successfully tested to preserve liquid semen in other species of animals, such as turkey, to minimize cold shock damage in ram, boar, and goat spermatozoa [17]. Recently, BHT has also been tested in our laboratory as a supplement to both lecithin‐based and egg‐yolk‐based extenders for bull sperm cryopreservation [10]. Findings showed that supplementation of BHT improved general motil‐ ity, progressive motility, morphology, acrosome integrity, DNA integrity, and oxidative stress level of sperm at 0.5 mM/ml for lecithin‐based Bioxcel® and at 1–1.5 mM/ml of BHT for tris‐ and citrate‐egg‐yolk extenders compared with their controls. However, higher concentrations of 2.0 and 3.0 mM/ml of BHT had a detrimental effect compared with the control of all extend‐ ers evaluated and it was concluded that BHT supplementation at lesser concentrations (0.5–1.5 mM/ml) could improve frozen‐thawed bull sperm quality by reducing oxidative stress pro‐ duced during the freezing‐thawing procedures in either lecithin or egg‐yolk‐based extenders.
