92 CRYOTIPS: A SIMPLE VITRIFICATION TECHNIQUE FOR SHEEP AND GOAT EMBRYOS

Abstract

Vitrification procedure for embryos has not been used massively because there is not a standard protocol according to species. We evaluated reproductive efficiency of vitrified embryos in plastic tips of micropipettes. Estrous cycles of 36 Criolla goats (donors, n = 10; recipients, n = 26) and 48 Merino sheep (donors, n = 10; recipients, n = 38) were synchronized by sponges containing 60mgof MAP (Progespon^®). At sponge removal (goats, Day 18; sheep, Day 14), recipients received 200 UI of eCG (Novormon^®, Syntex, Argentina). Sheep and goat donors were superovulated with a total of 80 (igpFSH (Folltropin V^®, Bioniche, Canada) every 12 h in 6 decreasing doses (18, 18, 14, 14, 8, 8) during the last 3 days of progestagen treatment. A dose of eCG (200 UI, Novormon^®) was applied at progestagen removal. Donors of both species were inseminated by laparoscopy with frozen/thawed semen (200 × 106 spz) 12 to 14h after the onset of estrus. Embryo recovery was done by surgical prepubic laparotomy, and uterine horns were flushed with PBS + 10% SFB at Day 8 after sponge withdrawal. Before vitrification, embryos were kept in commercial embryo holding medium (Syngro, Bioniche, USA) at 25°C. Vitrification solutions were prepared using holding solution (HS) contents, PBS + 20% FBS. Embryos were maintained at 25°C and processed according to the following procedures: (1) HS + 10% glycerol (G) for 5 min, (2) HS +10% G + 20% ethylene glycol (EG) for 5 min, and (3) HS + 25% G + 25% EG for 30 s. Embryos were aspirated in 1 μL of solution 3, into the center of a plastic micropipette tip (0.36 mm inner diameter), and plunged into a cryotube filled with liquid nitrogen. Before embryo transfer, tips were warmed with fingers for 10 s and immersed in 4 dilution steps during 5 min each with 2.5 mL of HS containing (1) 12.5% G+ 12.5% EG+ 0.5 M of sucrose (S), (2) 0.5 M S, (3) 0.25 M S, and (4) HS alone at 25°C. In both species, at Day 8 after sponge withdrawal, 2 embryos of similar stage were transferred. Under laparoscopy view, the tip of the uterine horn was exteriorized by a nontraumatic clamp, through a 1-cm-long midline incision. The embryos were placed into the uterine lumen after punction and the uterine horn was allowed to return to the abdomen (semi-laparoscopic technique). Pregnancy was diagnosed by ultrasonography (Aloka 500) at Day 28 after being transferred. Reproduction efficiency in both species is shown in Table 1. This technique proves to be simple and efficient for both ovine embryo stages but only for goat blastocysts and can be used by veterinarians in field conditions at a very low cost. Differences of morulae survival between species require further study.

Table 1.  Reproductive efficiency after vitrified/warmed embryo transfer in sheep and goats according to the embryo stage