Gel-Loading Tip Vitrification of In Vitro-Matured Bovine Oocytes and Subsequent Embryo Production by IVF and Nuclear Transfer

Abstract

T h e p u r p o s e o f t h i s s t u d y w a s t o cryopreserve bovine oocytes for subsequent blastocyst production by in vitro fertilization (IVF) and somatic cell nuclear transfer (SCNT). A vitrification procedure using g e l -l o a d i n g t i p s a s c o n t a i n e r s w a s a p p l i e d t ocryopreserve in vitro-matured and denuded oocytes. In Experiment 1, oocytes were vitrified-warmed in vitrification solution (VS) containing 25, 28, 31, or 40% e th y le ne gl ycol (EG) an d 1 . 0 M su crose . T he proportions of survived oocytes that appeared to be morphologically normal after warming, and cleaved oocytes after IVF were lower with 25% EG-based VS when compared with 28-40% EG-based VS. Blastocyst yields 8 days after IVF of oocytes vitrified-warmed in 28 and 31% respectively) were not significantly different from those of the fresh control group (32%). Day-7 blastocysts derived from vitrified oocytes were composed of a smaller number of inner cell mass (ICM) and trophectoderm cells than the fresh Day-7 blastocysts. In Experiment 2, oocytes vitrifiedwarmed in 31% EG-based VS were subjected to enucleation and SCNT. The proportions of oocytes fused and cleaved in the vitrified group were comparable to those in the fresh control group. Blastocyst yields 6 and 7 days after SCNT of vitrified oocytes were lower than those of control oocytes, but 8 days after the SCNT, the difference became statistically comparable (45 versus 58% in control group). ICM and trophectoderm cell numbers in Day-7 blastocysts derived from vitrified oocytes were smaller than those of control blastocysts due to a slower developmental rate. In conclusion, bovine oocytes cryopreserved by vitrification in gelloading tip were capable of developing into blastocysts after conventional IVF and SCNT, with slightly smaller cell numbers and a slower developmental rate.

Cryodevices developed for minimum volume cooling vitrification of bovine oocytes

“…Vajta et al ( 1998 ) reported that a very high cooling rate (>20,000°C/min) can be achieved in an open pulled straw (OPS), resulting in a 13% blastocyst yield from post‐warmed bovine MII oocytes and a calf birth, followed by a few minor modifications as superfine OPS (Isachenko et al, 2001 ) and sealed pulled straw (Chen et al, 2001 ). Conventional disposable plastic tools were successfully used for the MVC vitrification of bovine oocytes, as reported with the gel‐loading tip (Tominaga et al, 2005 ) or flexipet‐denuding pipette (Morató et al, 2008 ). Because of the big market for human ART, a closed system using CryoTip® (Fujifilm Irvine Scientific) has been commercialized and is widespread in infertility clinics (Kuwayama, Vajta, Ieda, et al, 2005 ; VerMilyea & Brewer, 2017 ).…”

Contribution of spermatozoal centrosomes to the microtubule-organizing centre in Antarctic minke whale (Balaenoptera bonaerensis)

“…Nevertheless, the poor availability of bovine ovaries in our laboratory and a daily limit on the number of treated oocytes (less than 10) forced us to use the cryopreserved oocytes in the present study. The gel-loading tip vitrification procedure, characterized by an ultra-rapid cooling rate (Tominaga & Hamada, 2001), was found to be a promising method for cryopreservation of bovine oocytes after some modifications (Tominaga et al, 2005). However, an adverse effect of using vitrified oocytes and/or re-frozen spermatozoa on sperm aster formation cannot be ruled out.…”

“…After maturation culture, oocytes were freed from cumulus cells by vortex-mixing and pipetting, and only oocytes which had extruded the first polar body were cryopreserved according to the method described by Tominaga et al (2005) with a few modifications. Briefly, groups of five oocytes were exposed to 3% (v/v) ethylene glycol (EG; Wako Pure Chemical Industries, Osaka, Japan) and 20% FBS in TCM199 for 12-15 min at 37 • C and then transferred to a vitrification solution composed of 31% EG, 1.0 M sucrose (Wako) and 20% FBS in TCM199 at the same temperature.…”

“…Cumulus-oocyte complexes (COCs) were recovered from abattoir-derived bovine ovaries and cultured in TCM199 medium (Gibco-BRL, Grand Island, NY) supplemented with 5% (v/v) fetal bovine serum (FBS; Equitech Bio, Ingram, TX), 0.002 AU/ml FSH (Antrin; Denka Pharmaceutical, Kawasaki, Japan) and 1 µg/ml estradiol-17β (Sigma Chemical, St Louis, MO) for 22 h at 38.5 • C in 5% CO 2 in air, at a density of 30-50 COCs per 750 µl of medium under 250 µl of mineral oil in 4-well dish (Tominaga et al, 2000). After maturation culture, oocytes were freed from cumulus cells by vortex-mixing and pipetting, and only oocytes which had extruded the first polar body were cryopreserved according to the method described by Tominaga et al (2005) with a few modifications. Briefly, groups of five oocytes were exposed to 3% (v/v) ethylene glycol (EG; Wako Pure Chemical Industries, Osaka, Japan) and 20% FBS in TCM199 for 12-15 min at 37 • C and then transferred to a vitrification solution composed of 31% EG, 1.0 M sucrose (Wako) and 20% FBS in TCM199 at the same temperature.…”