**5.4 Ovum pick up/IVEP** *vs.* **superovulation/embryo transfer (ET)**

In the *in vivo* production of embryos, it is necessary to administer hormones so that superovulation (SOV) occurs and, subsequently, the transfer of the embryos. In Ovum Pick Up (OPU)/IVEP, however, obtaining oocytes and producing embryos do not require hormonal use. Furthermore, it is known that in *Bos indicus* animals, the number of embryos produced per aspiration session is higher than that of superovulation [67].

**Figure 3.**

*Schematic sequence of steps in the* in vitro *embryo production process (IVEP).*

The *in vitro* technique also allows for less spaced collections of oocytes from donors. In general, the minimum interval is 15–30 days, and there is no limit to the number of aspirations performed on the same cow [68]. On the other hand, SOV requires intervals of 40–60 days and should only be performed three or four times before a period of several months apart [69].

The production of embryos by SOV also does not allow pregnant cows, while in IVEP, this is possible. Follicular aspiration can be performed as long as the ovaries can be manipulated without being subjected to excessive traction. The process flow of *in vitro* and *in vivo* embryo production is shown in **Figures 3** and **4**, respectively.

### **5.5 Cryopreservation of** *in vitro* **produced embryos**

The cryopreservation of bovine embryos generated *in vivo* has protocols very well established and effective through a freezing process. However, despite the benefits obtained and the advantages of IVF already reported in previous topics, cryopreservation represents a challenge. The low cryotolerance of IVP embryos is a limiting factor for using the cryopreservation process associated with this process. IVP embryos are more susceptible to damage caused by cryopreservation when compared to those produced *in vivo*, as they present differences in morphological, metabolic, and chromosomal aspects of their structure [70].

The greater sensitivity of these embryos to low temperatures is mainly due to the greater accumulation of lipids in the cytoplasm [71]. Lipids, made up mostly of triacylglycerols, directly affect the survival of embryos during cooling, as they can undergo irreversible changes and severely compromise development. An alternative method to promote chemical delipidation of embryos and increase cryotolerance by decreasing lipid accumulation has been related [72]. Forskolin, for example, a compost derived from the Indian plant *Coleus forskohlii*, is able to promote intracellular lipolysis in swine [72] and bovine [73] embryos. When added to the medium at strategic periods of *in vitro* culture, this substance raises embryonic tolerance to levels that provide good pregnancy rates, even in *Bos indicus* embryos [73].

**Figure 4.** *Schematic sequence of steps in the* in vivo *embryo production process (SOV/ET).*

Among the cryopreservation methods, vitrification is the most used technique worldwide due to the speed of the process and its low cost [74]. On the other hand, direct transfer (DT), a technique used to simplify the *in vivo* postthawing rehydration step of embryos, has its main advantage the low concentration of cryoprotectants reducing embryotoxicity [75]. Also, DT eliminates the evaluation process before the transfer, thus becoming a more practical way than vitrification [76].
