![]() ![]() The alpaca ( Vicugna pacos ) is an important species for the production of fiber and food. The modifications we made to the previously published vitrification protocol (12)(13)(14) included the following: (1) replacement of glucose with galactose in the base medium, (2) replacement of fetal bovine serum with alpaca serum in all solutions containing serum, (3) use of a commercially available embryo holding medium for post-warming/pre-transfer temporary holding, (4) performing asynchronous embryo transfer (no more than 24 h asynchronous standard operating procedure for embryo transfer on this farm) as is common in pigs (22) and horses (23), and (5) an overnight in vitro culture period to enable post-warming observation of greater duration (because embryos that initially appear viable shortly after warming often fail to produce pregnancies after embryo transfer) this enables a more accurate in vitro assessment of post-warming embryo viability. We successfully employed a modified vitrification protocol to overcome the challenges to cryopreservation typically associated with large diameter and/or high lipid content embryos. Additionally, there are at least two factors that can be manipulated to increase the PVPR of embryos ˃ 300 Rev Mex Cienc Pecu 2020 11(4): 1142-1149 1143 µmØ one is to reduce their size by aspiring their blastocoelic liquid (BL), and the other is to induce a high temperature transfer index (TTI) to rapidly reach-196 ☌. At present time, embryos collected either the seventh or eighth day PO are ˃ 300 µmØ and are characterized to have a low post vitrification survival in order to increase their PVPR their EC might be punctured to make it permeable to cryopreserving solutions. The high PVPR of embryos ≤ 300 µmØ is due to an embryo capsule (EC) that is not fully developed yet and has a high permeability to cryo-preserving solutions. Embryos collected at the sixth day post ovulation (PO) are usually smaller or equal to 300 micrometers in diameter (≤ 300 µmØ) and can be routinely vitrified following simple protocols they have a higher post vitrification pregnancy rate (PVPR) when compared to large embryos which have more than 300 micrometers in diameter (˃ 300 µmØ). Several improvements to vitrification protocols have made possible to cryo-preserve embryos with different sizes since during the first decade after the year 2000, only small embryos were successfully vitrified. It consists in the dramatic reduction of temperature to levels close to-196 ☌, that allows the cryopreserving solution containing the embryo to pass from liquid to vitreous state. Vitrification is a cryo-preservation method often used in embryos obtained from mares or jennies. An additional objective is to ease the access to data about the solutions employed before and during the vitrification as well as the solutions used during the embryo warming and transfer steps. This study endeavors to provide information to help in the adaptation of vitrification protocols depending on the embryo size. In equids, the type of holding and transfer solutions used during the post vitrification step is an area scarcely explored. A high temperature transfer index (TTI) optimizes the embryo PVSPR. The type of embryo carrier, the size of the embryo and the volume of the vitrification solution vary between small and large embryos. Further knowledge is needed about the mixture of cryoprotectants either penetrating or non penetrating and the effect they exert over the embryos post vitrification survival and pregnancy rate (PVSPR). ![]() No experiments test the type of holding and washing solutions and to what extent they influence the embryo post vitrification survival. Big embryos, larger than three hundred micrometers (>300 µmØ) are recovered during or after the seventh PO day. Small equine embryos, equal or smaller than three hundred micrometers in diameter (≤ 300 µmØ) are collected before the seventh post ovulation (PO) day. ![]() Some embryos 300 microm (n = 19) did not produce embryonic vesicles. Transfer of morulae or blastocysts 300 microm were vitrified, thawed and transferred as in Experiment 1. No pregnancies were observed after transfer of blastocysts >300 microm (n = 3). Embryos were warmed in a two-step dilution and transferred into uteri of recipients. In a preliminary study (Experiment 1), embryos were exposed in three steps to vitrification solutions containing increasing concentrations of ethylene glycol and glycerol (EG/G) the final vitrification solution was 3.4 M glycerol + 4.6 M ethylene glycol in a base medium of phosphate-buffered saline. Experiments were conducted to determine viability of equine embryos in vivo after vitrification. ![]()
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