**5. Conclusion**

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.

130 Cryopreservation in Eukaryotes

**Figure 2.** Flowchart of the recovery technique and sperm utilization from bulls epididymides, post-orchiectomy or

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

postmortem when kept at ambient temperature; TM, total motility (adapted from: Bertol [34]).

It is very important to have knowledge that it is possible to use the genetic reserve of a breeder even after his death. This avoids gamete wastage and an early loss of reproductive potential of a male of important genetic value. The application of this biotechnology should be proposed by the veterinarian at the time of the death of a high-value breeder. The owner of the animal should be aware that it is still possible to obtain the last descendants of the breeder. A basic protocol for the cryopreservation of epididymal sperm can be suggested as follows:


It is important to note that in all of these steps (1–10), an aliquot should be removed for sperm evaluation of some parameters such as motility, concentration and morphology. This is essential for identifying flaws in the sperm handling or viability changes by temperature drop during processing, osmotic stress and the formation and dissolution of extracellular ice crystals, which can impair the fertility of sperm. The success of epididymal sperm cryopreservation depends on the effectiveness of the process, including cautious handling of the sperm cells and the technical skills in the laboratory.
