**3. Cryopreservation of epididymal sperm**

The first experiments arose with the need for establishment of a genetic bank for maintenance of the gametes for future use. Another reason for the cryopreservation of sperm from the epididymis is because there is a possibility that a bull could die before their semen is deposited in semen banks or at a time when only a small amount is in storage. While the use of frozen bovine semen has favored the establishment of semen banks which prolongs the usefulness of a bull and acts as a safety procedure in the event of an unexpected illness or death, not always the existing reserve is sufficient. The semen conservation methods in liquid form allow maintenance for only a few days, but it frozen in liquid nitrogen (N2L) it is possible to keep for years.

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

**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 proxi-

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

The first experiments arose with the need for establishment of a genetic bank for maintenance of the gametes for future use. Another reason for the cryopreservation of sperm from the

sperm changes the circular movement pattern for rectilinear.

mal to the medial and distal region.

126 Cryopreservation in Eukaryotes

genital tract physiology.

**3. Cryopreservation of epididymal sperm**

Sperm were recovery from the epididymis of a Jersey bull aged nine years after death and were frozen for the first time on November 26, 1953 [3]. The procedure was carried out as follows: The intact scrotum from the slaughtered bull was brought to the laboratory as soon after death as possible. The time for delivery varied between 1.5 and 3 hours. On arrival the scrotal skin and tunica vaginalis were incised and the testicle was handled as aseptically as possible after its removal. Spermatozoa were recovered from the epididymis by means of several incisions in each, following which the organ was squeezed, and the expressed fluid was scraped off with a scalpel and deposited in a small volume of diluent (pasteurized homogenized whole milk and antibiotics added). Epididymal fluid containing spermatozoa were added to milk diluent. An equal volume of 20% of glycerinated milk diluent was then added gradually in increasing amounts, over a period of 30 minutes, so that a final glycerol concentration of 10% by volume was present. The sample was then placed in a refrigerator for an equilibration period of 18 hours. Subsequently the sample was distributed into ampoules and frozen.

Although some characteristics are already previously mentioned, the epididymal spermatozoa resemble the ejaculated and may be successfully cryopreserved similarly. The protocols used for freezing and thawing interfere with sperm fertility rate, so different situations are tested to identify the best protocol and the best diluent used in this process. The absence of seminal plasma in epididymal spermatozoa seems to be a positive factor in the maintenance of membrane integrity during the cryopreservation process. Furthermore, the sperm membrane composition between individual breeders can affect the resistance to cryopreservation [20]. The post-thaw incubation of the spermatozoa from epididymis of cats with seminal plasma resulted in a lower total and progressive motility, and plasma membrane integrity than control [21]. Thus, if the objective is the cryopreservation, it is recommended to add a diluent itself instead of seminal plasma.

The protocols used for cryopreservation of conventional semen with the same diluents seem to be effective for epididymal spermatozoa. What differs is the pre-preparation of sperm samples. If there is a high percentage of cytoplasmic droplets in the tail of the spermatozoa after recovery, it must remain for a period of about 30–60 minutes in a water bath, for drops release. For this first step, the sperm must be diluted in a medium without cryoprotectants that present some toxicity when stay long in contact with the sperm cells. At the end of the process, a sperm sample should be evaluated under light microscope to see if there was the release of most of the cytoplasmic droplets. After this step, the sample must be centrifuged to remove any trace of the previous medium and other dirt, and then the cryopreservation diluent with cryoprotectants is added. The amount of diluent is based on the concentration of spermatozoa and the number of insemination doses.

The epididymal spermatozoa seem to be more resistant to the cryopreservation process than the ejaculated. In sheep, the sperms from the epididymis were more resistant to the stresses caused by freezing (osmotic variations, cryoprotectants toxicity and temperature variation) compared to the ejaculate [22].

The cryopreservation protocol in liquid nitrogen and the dilution with TRIS base-egg yolk are effective [8, 23]. For bulls, the process consists of first sample centrifugation to separate the spermatozoa from other contaminants and the recovery medium. The supernatant needs to be discarded, and the pellet resuspended with an extender consisting of Tris-egg yolk and 7% glycerol as cryoprotectant. The pre-freezing parameters of motility and the total cell number should be assessed to verify the effects of centrifugation and diluent. The straws are filled with a concentration of 20 million viable spermatozoa and sealed. For temperature stabilization, the doses were maintained for 3 hours in a semen cooling container (5°C), and then placed horizontally on a 6-cm high support in an expanded polystyrene box containing liquid nitrogen for 20 minutes. Finally, straws they are immersed in liquid nitrogen at a storage temperature of −196°C. Cryopreservation can also be performed in automatic machines with controlled temperature drop.

There is not yet a specific commercial diluent for epididymal sperm in any species. Thus, recent research was conducted in order to test and identify the best diluent in this case. Some results are mentioned below.

In sheep, the results of post-thaw viability and fertility after artificial insemination with Cryopreserved successfully epididymal spermatozoa show that the diluent with 20% egg yolk and 8% glycerol and base TES-Tris-fructose (TTF) was significantly more effective in maintaining the sperm viability [24]. Egg yolk base medium with 4% glycerol was used for epididymal sperm cryopreservation of domestic and wild cats (*Panthera tigris*) successfully, providing the application of this technology in genetic resources of banks of wild species of cats [25]. Also with the aim of maintaining a genetic resource bank of wild species, Cuvier's gazelle (*Gazella cuvieri*) spermatozoa can be cryopreserved using a diluent containing 18.5% raffinose with 20% egg yolk and 6% glycerol [26]. The concentration of glycerol varies among species and must be previously tested in each case.

The concentration of sugars and the type of carbohydrate added to the medium also affect the quality of post-thaw spermatozoa from the epididymis. In tests with epididymal sperm from wild deer (*Cervus elaphus hispanicus*), the use of monosaccharides in diluents for freezing, especially fructose, improves the maintenance of post-thaw viability compared to trisaccharides [27]. Furthermore, the addition of antioxidants such as cysteine, water-soluble vitamin analog or enzymes [28, 29] leads to improvement in total motility and post-thaw integrity in sperm plasma membranes of cat epididymal sperm.

Commercial extenders for Conventional bull semen (Botu-Bov® and Bovimix®) are viable options for cryopreservation of epididymal bull sperm. Both are effective in maintaining sufficient amounts of post-thaw viable spermatozoa for use in artificial insemination. When compared, these show no difference in sperm viability (movement, morphology and integrity of plasma and acrosomal membrane) after thawing [23]. These two extenders have egg yolk in its formulation and glycerol as a cryoprotectant.

The variations in response to cooling protocols and semen freezing must be attended. Individual male effects affect directly the longevity of spermatozoa during preservation. The mammals may be regarded as "good" or "bad" freezers, according to the characteristics of the sperm plasma membrane structure which is genetically determined and the survival predisposes to thermal stress. This fact allows researchers to sort the spermatozoa as resistant or susceptible to cryopreservation. The proportion of cells, which survive the cryopreservation protocol, is determined by the sensitivity to osmotic stress during cryopreservative addition and removal, and during cooling and rewarming. While there may be species differences in overall sperm sensitivity to cryopreservation, the ejaculate is heterogeneous with a variable resistance to osmotic stress among the cells. Nevertheless, even if we optimize the process and minimize the cell death, there will still be a proportion of cells which fail to survive. We need, therefore, to concentrate on the function of the surviving population [30].

After cryopreservation identified doses containing the spermatozoa can be deposited in canisters of cryogenic cylinder, indefinitely. Inside the cylinder, the semen is kept in liquid nitrogen (N2L), which preserves the frozen doses at a temperature of −196°C. The storage time is indefinite as long as the same is supplied periodically in order to maintain the N2L above the minimum required level, which should never be below 15 cm height. The cylinder should be stored and handled with utmost care, avoiding possible damage.
