**2. Epididymal spermatozoa**

When the spermatozoon leaves the testicle to the epididymis, it is a non-functional and infertile gamete. Only after the passage through the epididymis, it becomes mature and acquires progressive motility to become able to fertilize the oocyte. This maturation is complex and involves several factors, including the interaction of sperm with proteins that are synthesized in each epithelium region of the epididymis. Sperm passively migrates through the epididymis and after contact with the epididymal secretions get low-molecular weight and water-soluble compounds by an isovolumetric regulatory process. These compounds can be spent together with the cell water when the cells come into contact with hypo-osmotic fluids from accessory glands or genital female tract. The volume adjustment process serves to maintain the volume pattern into the sperm cell and to prevent angulation of the flagellum, which prevents the sperm to migrate efficiently in the female reproductive tract, being unable to fertilize oocytes. The channels responsible for this regulation are located in cytoplasmic droplets. The spermatozoa located in the cranial portion of the epididymis (caput) are considered immature to present an osmolyte content reduced insufficient for complete regulation of cell volume when exposed to hypotonic solutions [4]. The movement of the sperm through the epididymis is performed mainly by the contraction of smooth muscles of the wall of the Caput and corpus region. On the cauda, the smooth muscles of the epididymal duct is generally at rest until it is stimulated to contract at the time of ejaculation for the release of sperm, thus it is responsible for the protection and storage of sperm until ejaculation.

There are two moments prior to recovery of sperm that interferes directly in the success of the process. The first is the time from the death of the animal until the necropsy, and the second is the period of recovery of the gonads to obtain the gametes in the laboratory [5]. The higher the time of the gametes permanence in the cauda epididymis after death or after orchiectomy, the greater the damage to the sperm cell. At room temperature, motility is the first parameter affected. With the increase in the hours before recovery, there is a reduction in the percentage of moving spermatozoa, worsening from 24 hours [6, 7].

There are two moments for the recovery of spermatozoa from the epididymis:

**a.** After death

allows to maintain biological material at very low temperatures, indefinitely. The expansion of genetic material from high-value breeders became possible with the advent of semen cryopreservation. The main advantages of the technique are: portability for world trade because it is not necessary to purchase or move the males, and it also acts in the prevention of

Semen constitutes the union of sperm with seminal plasma, secreted by the male accessory glands. Sperms are produced in the testicles (gametogenesis), and maturation occurs in the epididymis when cells enter in the caput of epididymis, progress to the corpus, and finally reach the cauda region, where they are stored until the moment of ejaculation. Spermatozoa stored in the cauda region are generally of good quality and have a high level of maturity, being able to fertilize oocytes. The epididymis provides a favorable environment to retain the sperm with fertilizing capacity for several weeks. Therefore, the cauda epididymis is a major source of gametes of a breeder and in special cases they can be retrieved directly from it.

The sperm recovery from the epididymis is the last chance to use gametes from dead breeders, in addition to enabling the maintenance of a germplasm bank of animals of commercial interest or threatened with extinction [1]. A factor to be considered is the difficulty in collecting semen from wild species and the unexpected death of animals of zoological interest. In this case, the technic can be used to ensure the rescue of male gametes and the preservation by cryopreservation process for maintaining germplasm banks [2]. For animal production as livestock, the sperm recovery directly from the epididymis is a viable alternative when there is a sudden death of breeder of high commercial value, to increase their genetic stock or in cases of bulls unable to ejaculate for some reason [3]. It is the last alternative for future use of gametes from a breeder in assisted reproduction programs. In equine, this technique is important because there is a high incidence of accidental and unexpected death due to high incidence of colic or

severe traumatic accidents that compromise the reproductive life of the stallion.

understanding of the epididymis spermatozoa cryopreservation technique.

**2. Epididymal spermatozoa**

The first important point is the recovery of still viable sperm with good parameters of motility, concentration, and morphological defects as soon as possible after the male death. There is a time limit for this to successfully occur, usually dependent on temperature. The first step is getting the epididymis in the field, after the death of the animal. For handling ease, due to their anatomical location, the testes can be removed with them. Then both should be sent to the laboratory in suitable containers, with or without cooling. In the lab, the trained technicians perform gonads cleaning, epididymis isolation and spermatozoa recovery. Then it can be used in three different ways: first, shortly after harvesting; second, chilled, and third, after cryopreservation process. The last increases the availability time of gametes for application of assisted reproduction techniques. Next we will focus on the key topics relevant to the

When the spermatozoon leaves the testicle to the epididymis, it is a non-functional and infertile gamete. Only after the passage through the epididymis, it becomes mature and acquires

diseases capable of transmission through natural breeding.

122 Cryopreservation in Eukaryotes

**b.** After orchiectomy

The most common form of spermatozoa recovery from the epididymis is after death. It is indicated in cases of use of last spermatic reserve after breeder death. It is usually recommended when the breeder is found after dead in the field in cases of sudden death from a serious illness, accidents, poisoning and stress problems. It is worth mentioning that infectious diseases can contaminate tissues with pathogenic microorganisms, and in such cases the technique is not recommended to avoid contamination. Moreover, in some infectious diseases, there is an acceleration of tissue degradation, affecting the preservation of tissues and reducing the time for manipulation. One example is infection by *Clostridium chauvoei*, a bacterium when multiplied produces a toxin that causes injury to the host body as well as muscles and other tissues. The acute disease is considered highly lethal.

After death there is limited time to work before the occurrence of the degeneration of tissues (postmortem autolysis), damaging the quality of sperm. This time must be sufficient for catching the gonads in the field and transport to the laboratory. Trained technicians are required to perform rapidly obtaining of the spermatozoa, and then adding the medium to provide substrates necessary for maintenance of gametes.

The recovery after orchiectomy is usually performed in experimental works. Once is not common execute euthanasia in animals for research, the orchiectomy is an option to simulate the death for testis and epididymis. The interruption of blood supply to the testis and epididymis causes the same degenerative changes which occur after death. The orchiectomy is indicated in any situation where you do not want to eliminate the animal. In cases of unilateral testicular involvement, contralateral testicle can be tapped.

For wildlife, the difficulty of semen collection by conventional methods such as electroejaculation and artificial vagina turns recovery from epididymis interesting. Sperm can be obtained after orchiectomy, and the animal will stay alive, but will be unable to reproduce on their own. Both techniques are considered the last chance to use male gametes. The concentration of spermatozoa recovered is limited by storage capacity in the cauda epididymis of each species. The number of insemination doses is directly proportional to this concentration.

After recovery, the epididymis can be handled in two ways:


When an animal is found dead in the field, it was exposed to climatic conditions for hours. This ambient temperature accelerates tissue degeneration with loss of sperm viability in a given time period. This condition has been represented in previous studies to be closer to reality, to verify the time available and establish a window of opportunity to work. A large variation in ambient temperatures was reported successfully, between 18 and 24°C [2, 7–9]. At a temperature of 18–20°C, it is possible to recover viable gametes, With 41.25% of progressive motility by up to 30 hours after orchiectomy [7]. Thus, after the death of bulls exposed at Ambient temperature, the ideal time until sperm recovery is up to 30 hours. With the increase of the residence time of the gametes in the cauda epididymis after death or after orchiectomy, there is greater damage to the sperm cell. In general, motility is the first parameter affected.

On the other hand, in most studies the epididymis are kept at a refrigeration temperature of 5°C before recovery, which slows down the process of cell degradation increasing the time for collecting viable gametes. For bulls, the maintenance of refrigerated epididymis enables the achievement of viable sperm for up to 72 hours after the death [10]. By comparing the refrigerating temperature (4.9–6°C) with room temperature (21.5–17.9°C) for maintenance of the epididymis before harvesting in sheep, the highest temperature affects earlier some spermatic parameters such as the acrosome integrity, motility, concentration and morphology [11]. If possible, the testicles and epididymis should be transported to the laboratory chilled to increase the time for processing.

For spermatozoa recovery, The main techniques that can be used are:

#### **a.** Retrograde flow

#### **b.** Flotation

required to perform rapidly obtaining of the spermatozoa, and then adding the medium to

The recovery after orchiectomy is usually performed in experimental works. Once is not common execute euthanasia in animals for research, the orchiectomy is an option to simulate the death for testis and epididymis. The interruption of blood supply to the testis and epididymis causes the same degenerative changes which occur after death. The orchiectomy is indicated in any situation where you do not want to eliminate the animal. In cases of unilateral

For wildlife, the difficulty of semen collection by conventional methods such as electroejaculation and artificial vagina turns recovery from epididymis interesting. Sperm can be obtained after orchiectomy, and the animal will stay alive, but will be unable to reproduce on their own. Both techniques are considered the last chance to use male gametes. The concentration of spermatozoa recovered is limited by storage capacity in the cauda epididymis of each species. The number of insemination doses is directly proportional to this concentra-

When an animal is found dead in the field, it was exposed to climatic conditions for hours. This ambient temperature accelerates tissue degeneration with loss of sperm viability in a given time period. This condition has been represented in previous studies to be closer to reality, to verify the time available and establish a window of opportunity to work. A large variation in ambient temperatures was reported successfully, between 18 and 24°C [2, 7–9]. At a temperature of 18–20°C, it is possible to recover viable gametes, With 41.25% of progressive motility by up to 30 hours after orchiectomy [7]. Thus, after the death of bulls exposed at Ambient temperature, the ideal time until sperm recovery is up to 30 hours. With the increase of the residence time of the gametes in the cauda epididymis after death or after orchiectomy, there is greater damage to the sperm cell. In general, motility is the first pa-

On the other hand, in most studies the epididymis are kept at a refrigeration temperature of 5°C before recovery, which slows down the process of cell degradation increasing the time for collecting viable gametes. For bulls, the maintenance of refrigerated epididymis enables the achievement of viable sperm for up to 72 hours after the death [10]. By comparing the refrigerating temperature (4.9–6°C) with room temperature (21.5–17.9°C) for maintenance of the epididymis before harvesting in sheep, the highest temperature affects earlier some spermatic parameters such as the acrosome integrity, motility, concentration and morphology [11]. If possible, the testicles and epididymis should be transported to the laboratory chilled to

For spermatozoa recovery, The main techniques that can be used are:

provide substrates necessary for maintenance of gametes.

testicular involvement, contralateral testicle can be tapped.

After recovery, the epididymis can be handled in two ways:

**a.** Room temperature (about 19°C)

**b.** Chilled (4–5°C)

124 Cryopreservation in Eukaryotes

rameter affected.

increase the time for processing.

tion.

The methods to obtain gametes depend on the animal species, size of the epididymis and the experience of the laboratorist. The retrograde flow consists of cannulation to perfuse the lumen of epididymis with diluent, promoting the backflow of the cauda epididymis content [12]. This technique is used for large animals, which have bigger testicular size and larger epididymis. It has been used successfully in horses and cattle [6, 7]. Being a detailed technique, the time for extracting spermatozoa is wide. In general, the sperm collection technique by retrograde flow results in less contaminated samples and better sperm quality than other methods, and is more suitable [13].

For small animals, the flotation method is most appropriate because of the anatomical size of the epididymis. It consists of performing longitudinal and cross cuts in the cauda region of the epididymis, the fragments are deposited into a petri dish containing medium for release of sperm and later retrieval by filtration [14]. For the smallest mammal in the world, the shrew (*Tupaia belangeri*), this is the only technique applicable because of the epididymal size in this species [15]. The flotation method requires less technician experience, because of the facility of implementation it is also often used for large animals such as sheep [16] and bulls [5, 10]. One disadvantage of this practice is that the samples are usually contaminated with blood and cellular debris, as some blood vessels are also incised during the process. The recovered sperm concentration is lower, as some are stuck between the tissue fragments so they cannot swim through the diluent in the petri dish.

The spermatozoa collected from the epididymis are free of seminal fluid which is added by the accessory glands during ejaculation, and serves as a vehicle, stimulating sperm metabolism and provides the energy necessary for the spermatozoa to pass through the uterus [17]. For it is a gamete storage location, the concentration of spermatozoa in the epididymal tail is significantly higher than the concentration of semen after ejaculation. When sperms are retrieved from the epididymis, there is a lack of ejaculatory reflex, and thereby sperms have no contact with the plasma rich in electrolytes, fructose, ascorbic acid, various enzymes and vitamins. Because of this it is recommended to add seminal plasma or other medium that has the components necessary to maintain sperm viability after recovery. It is important that whatever the diluent, it must have composition substances favorable to sperm: macromolecules such as lipoproteins and phospholipids to act as stabilizers of the plasma membrane, nutrient source for sperm metabolism (sugars) and buffer to maintain the pH. For cryopreservation, it is indispensable to use a medium containing cryoprotectants.

Besides the absence of seminal plasma, another important characteristic of epididymal sperm is the presence of large amounts of medial and distal cytoplasmic droplets in the tail of the sperm [7, 10, 17, 18]. The cytoplasmic droplet is a small spherical mass of 2–3 m in diameter found in low amounts in the ejaculated spermatozoa as it is released during ejaculation, as shown in **Figure 1**. During the transit through the epididymis, the caudal migration of the cytoplasmic droplet occurs. The presence of distal cytoplasmic droplets is not considered a severe alteration because it does not interfere with the fertilizing capacity of the sperm.

Although it represents an alteration in the normal morphology of the spermatozoa, the presence of cytoplasmic droplets in epididymal sperm is a physiological finding. Sperms with a large number of distal cytoplasmic droplets tend to lose them after 15–30 minutes of incubation in a water bath (35°C) or after the agitation [17]. Therefore, it is recommended to keep the sperm from the epididymis in a water bath at 29 to 35°C for a period of 30–60 minutes, so that the cytoplasmic droplets are released spontaneously. With the release of the drops, the sperm changes the circular movement pattern for rectilinear.

**Figure 1.** Phase contrast microscopy (1000×) of a medial cytoplasmic droplet in bull epididymal sperm. During the transit through the epididymis the cytoplasmic droplet migrates along the middle piece of the sperm from the proximal to the medial and distal region.

The pair of testicles (right and left sides) of a male breeder has similar characteristics of dimensions and gamete production and reserve. It has been reported for bull and stallion [6, 19]. Bulls at reproductive age (between 3 and 7 years) have epididymis (caput, corpus and cauda) with an average weight of 34.2 g. Moreover, sperm motility parameters, morphology and concentration do not differ when compared with the epididymal spermatozoa recovered from right and left. The information becomes useful in cases of unilateral testicular involvement, assisting in the reproductive male prognosis and also in clarifying the normal male genital tract physiology.
