**4. Epididymal sperm fertility**

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)

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

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

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

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

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

compared to the ejaculate [22].

128 Cryopreservation in Eukaryotes

temperature drop.

are mentioned below.

species and must be previously tested in each case.

sperm plasma membranes of cat epididymal sperm.

It is already proven the fertility potential of spermatozoa retrieved directly from the Epididymis in some species. The epididymal sperm, as well as the ejaculated, can be used in assisted reproductive technology (ART) as conventional artificial insemination, fixed time artificial insemination (FTAI), *in vitro* fertilization of oocytes (IFV) and intracytoplasmic sperm injection (ICSI). The viability of spermatozoa after cryopreservation depends on how well the quality is preserved throughout processing, storage and thawing procedures. High-quality semen is the one that will initiate a high percentage of pregnancies when properly used. The longest time to sperm recovery after death or after orchiectomy reduces the motility and other sperm parameters before freezing. Thus, this results in worse performance for fertilization and in consequence worse pregnancy outcomes.

*In vitro* fertilization is the most appropriate technique to use this material because it requires a lower concentration of viable sperm for embryo production, when compared to artificial insemination. The ability to *in vitro* and *in vivo* fertilization has been proven. Sperms collected from the epididymis tail of bulls and kept at 5°C have been used for *in vitro* production of embryos [10]. Under these conditions, it was viable to produce embryos *in vitro*, with blastocyst formation up to 9 days of development using Cryopreserved sperm from epididymides refrigerated for 24, 48 and 72 hours at 5°C. When the sperm was recovery up to 30 hours at ambient temperature (18–20°C) and then cryopreserved in liquid nitrogen successfully, it was also possible to produce viable embryos (blastocysts) of eight days from *in vitro* development. The total number of blastocysts and hatching rates were lower when the recovery of spermatozoa was performed 24–30 hours after orchiectomy. When the collection was performed 6–18 hours after orchiectomy the embryo production rate was approximately 30% [31]. This means that sooner the recovery and cryopreservation are performed, the better the results. The individual bull effect on embryo production is a relevant factor that can influence the success of IVF in those cases. Great individual variation in both post-thaw sperm parameters and embryo production between bulls can be observed. It is important to know the genetic background and the fertilization potential of sperm donors to maximize the success of IVF.

**Figure 2.** Flowchart of the recovery technique and sperm utilization from bulls epididymides, post-orchiectomy or postmortem when kept at ambient temperature; TM, total motility (adapted from: Bertol [34]).

Another option for use of gametes is fixed time artificial insemination (FTAI). This technique offers the following advantages: (1) does not require detection of estrus, (2) there is induction of the estrus and ovulation, (3) synchronizes births and (4) reduces the calving interval. It is already reported in the literature a pregnancy after FTAI with spermatozoa that remained for 30 hours in the epididymis at room temperature [31]. As already mentioned, recovery at room temperature is an important condition in gamete handling of epididymal reserve. After the death of a breeder, the first question we must ask is how long the animal is exposed to ambient temperature. If it was for a period of 30 hours, it would be possible to perform the retrograde flow technique for spermatozoa recovery from the cauda epididymis. If the evaluation of the total motility parameter (TM) in sperm after retrieval is greater than or equal to 40% of motile cells, it can be cryopreserved. This is important for future use without time limit in techniques of *in vitro* produce of embryos and artificial insemination. If it was less than 40% sperm must be used immediately after recovery. The flowchart of how to act in this situation is shown in **Figure 2**.

Regarding insemination with spermatozoa from the epididymis, the literature is scarce. The ability of frozen-thawed boar spermatozoa obtained from epididymides stored at 4°C for 1 day to produce piglets (three males and two females) after fallopian insemination or *in vitro* fertilization (IVF) of *in vitro* matured (IVM) oocytes was described [32]. This demonstrates the potential for *in vivo* fertilization of gametes. In sheep, insemination with the frozen/thawed epididymal sperm resulted in a lambing rate of 87.5%. Fourteen lambs (10 males, four females) were born from seven ewes. The average gestation length was 145.6 ± 1.1 days. Birth weights were 4.4 ± 0.4, 3.2 ± 0.7 and 3.6 ± 0.7 kg for single lambs, twins and triplets, respectively [18].

The technique of intracytoplasmic sperm injection to achieve fertilization, especially using retrieved epididymal spermatozoa from men with obstructive or non-obstructive azoospermia, has revolutionized the field of assisted reproduction in humans. The characteristics of postpartum development of children born after fertilization with spermatozoa from the epididymis did not differ from those from conventional methods. Children born from the intracytoplasmic injection of spermatozoa method (ICSI) with gametes from the epididymis were monitored and compared to those born from ICSI with spermatozoa from the ejaculated and by *in vitro* fertilization of embryos. The results showed that the method was successful, not to cause any fetal malformations, stillbirth or problems in the development of children [33]. These scientific findings have grounded the progress of biotechnology of reproduction. The possibility of recovery sperm from epididymis without ejaculation of animals and man, for later cryopreservation, has ensured the maintenance of important genetic source and the maintenance of compromised male in reproduction.
