**2. Semen cryopreservation**

technologies that have tremendously contributed to the genetic improvement and develop‐ ment of animal production especially, in the dairy cattle industry is artificial insemination (AI). AI is a process of depositing sperm manually into a female reproductive tract (usually, uterus, or cervix) for the purpose of achieving viable pregnancy through *in vivo* fertilization using a method other than natural mating. The first documented history of successful use of AI back‐ dates to 1780 by L. Spallanzani who experimented in a bitch that subsequently gave birth to three pups. Later in about 1900, research on AI continued in farm animals and subsequently E.I. Ivanoff who initially studied in horses became the first to successfully inseminate cattle and sheep [2]. Since then, AI has undergone tremendous advances in techniques and applica‐

AI provides a lot of advantages over natural breeding. It maximizes the lifespan reproductive potential of a given male as a single semen ejaculate can be diluted and used to inseminate sev‐ eral females. Other prominent advantages of using AI in farm animals include improvement of genetics through more accurate evaluation of breeder males and greater use of superior germplasm, control of sexually transmitted diseases, improved record keeping, and it avoids the cost and necessity of keeping breeder males in the farm. Although the need to detect females on estrus is one of the prominent disadvantages of AI considered, with the develop‐ ment and advances in other assisted reproductive techniques (ART) such as estrus synchro‐ nization and timed AI as well as heat detection aids, this disadvantage of AI is dwindling.

AI can be performed using either fresh or cryopreserved (frozen‐thawed) semen. Although the use of fresh semen in AI results in a higher success rate than using cryopreserved semen, it requires keeping males for semen collection in nearby place and immediate shipment of the semen for insemination; otherwise, the semen quality will quickly deteriorate. However,

**Figure 1.** Proper placement of insemination gun to deposit semen in the body of the uterus [61].

tions in a wide variety of species of animals and human.

104 Cryopreservation in Eukaryotes

Semen cryopreservation is a reproductive biotechnology used to preserve and store sperm at a low‐freezing temperature for a short or long period of time for various purposes such as in assisted reproduction technologies (ART), species or breed conservation and fertility treat‐ ment as in clinical medicine. As discussed earlier, this technique has played a significant role in the livestock industry by overcoming space, distance, and time limitations for the transport of genetically valuable sperm globally and use of AI. Sperm cryopreservation is also an integral component of human reproductive medicine, recognized as an efficient procedure for manage‐ ment of male fertility before therapy for malignant diseases, vasectomy, or surgical infertility treatments, to store donor and partner spermatozoa before assisted reproduction treatments and to ensure the recovery of a small number of spermatozoa in severe male factor infertility [4].

The observation made by Spallanzani in 1803 stated that sperm cooled with snow was not killed but rendered motionless until exposed to heat, after which they became motile for sev‐ eral hours, and the successful cattle insemination in the 1900s could be considered the initial triggers toward the discovery of the cryopreservation procedure. It was in 1940, A. S. Parkes and C. Polge developed a successful method for sperm freezing and storage at low tempera‐ tures (‐79°C) using dry ice [2]. The same researchers were able to identify glycerol, which is commonly used as a cryoprotectant up to now, as an important factor that helps to protect fowl sperm during the freezing and thawing process. Later in 1957, the freezing mechanism transformed from the use of dry ice to the use of liquid nitrogen contained in a large stainless steel or aluminum vacuum containers, pioneered by the American Breeders Association [2]. Packaging sperm using plastic tubes (straws) or glass ampules were also important adjunct discoveries in the history of development of successful cryopreservation protocol. From the onset of using frozen semen until about the 1970s, glass ampoules were used almost exclu‐ sively for packaging, while the straws (0.5 ml—medium straw; 0.25 ml—mini straw) have been the package of choice from the 1970 up to now. As shown in **Figure 2**, liquid nitrogen tank is used to freeze and store the sperm.

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

#### **2.1. Semen cryopreservation media**

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 have [2], include:


Despite vast research that has been conducted in the area of semen extender's composition and sperm cryopreservation, and remarkable improvements, yet it has remained inevitable for a big portion of the fresh semen to be damaged during cryopreservation and subsequently resulting in a significant decrease in its quality. This might conclude that the above‐men‐ tioned properties of a good semen extender are not good enough and the presence of many other unknown damaging factors that need to be identified and addressed as well. Among the other challenges that the sperm has to face during cryopreservation that appears to be overlooked is oxidative stress (OS). Traditional semen extenders lack specific components targeted to deal with oxidative stress or protect sperm from oxidative damage. As a result, recent studies have focused on finding supplements to semen extenders to protect sperm from oxidative stress as well as other supplements that would help to protect sperm damage in structure and function. In this chapter, we focus on antioxidants and fatty acids as supple‐ ments for sperm cryopreservation.
