Comparison of two vitrification processes on survival rates of ovine embryos.

FERNANDEZ J; BRUNO GALARRAGA, MARÍA MACARENA;; CATTANEO L; PRIETO C; ANTUÑA C; TARDIVO C; FONTANA B; BO G; GIBBONS A,

georgia

Conferencia; 44º Annual Conference of the International Embryo Transfer Society (IETS).; 2022

The objective of this study was to evaluate the effect of two vitrification and warming processes on the embryo survival rate of ovine embryos. The experiment was conducted at the Small Ruminants Reproduction Laboratory of the National Institute of Agricultural Technology, Bariloche, Argentina (41° S 70° W), during the breeding season. Twenty four adult multiparous Merino sheep were superovulated using intravaginal sponges (60 mg of MAP; Progespon®, Zoetis, Arg) for 14 days combined with 280 or 480 IU i.m. of recombinant equine chorionic gonadotrophin (reCG; FOLIREC®, Zoovet, Arg) at the end of progestagen treatment. The onset of estrus was detected by means of an adult teaser ram (24–48 h after sponge removal). All embryo donors were inseminated using laparoscopy with frozen/thawed semen (100×106 sperm per ewe). The embryos (n=107) were collected surgically 7 (D7) and 8 (D8) days after intravaginal sponge withdrawal. A total of 92 embryos suitable for vitrification (morulae=88; blastocysts=4) were randomly assigned to two vitrification processes at 25 ºC: V1 (n=47), three successive solutions: i) basic medium (BM) (Flushing medium, Dipla flash plus®, Serendipia, Arg) supplemented with 5% FBS + 10% glycerol (G) + 0.5 M sucrose for 5 min; ii) BM + 10% G + 12.5% ethylene glycol (EG) + 0.5 M sucrose for 5 min; iii) BM + 25% G + 25% EG + 0.5 M sucrose for 30 s; V2 (n=45), using the same solutions as for V1 but without sucrose. The embryos were then loaded in 1 µl final solution into a plastic micropipette tip (Paralwall, Arg) and plunged into liquid nitrogen. For thawing, the micropipette tips were warmed between the thumb and middle fingers for 10 s and the vitrified embryos were randomly assigned to two warming processes: D1: three dilution steps: (i) BM + 12.5% G+ 12.5% EG + 0.5 M sucrose; (ii) BM + 0.5 M sucrose and (iii) BM + 0.25 M sucrose, for 5 min each, to allow the removal of cryoprotectants. Then, the embryos were introduced in two BM solutions for 2.5 min each at 25 °C; D2: two dilution steps: (i) BM + 0.5 M sucrose and (ii) BM + 0.25 M sucrose, for 5 min each. Finally, the embryos were introduced in two BM solutions for 2.5 min each at 39 ºC. Therefore 4 groups were formed: V1D1 (n=24), V1D2 (n=23), V2D1 (n=24), and V2D2 (n=21). Embryos were cultured in TCM 199 at 39 ºC and a 6.5% CO2 atmosphere for 48 h according to the different groups. Embryo survival rate data for vitrification and warming processes were analyzed using the Chi-square test. Embryo survival rate was greater for embryos in the V1D1 (37.5 %) and V1D2 (43.5%) processes than in the V2D2 (14.3%) process (P