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ABSTRACT. The effect of addition of linoleic acid-albumin (LAA) to culture medium before freezing on the survival rate of bovine 16-cell embryos after freezing-thawing was investigated. Embryos were incubated in CR1aa containing LAA (0.25 mg/ml) for 4 days after insemination. A conventional slow cooling method was used, in which embryos were cooled at a rate of 0.3°C/min to 30°C in medium supplemented with 1.5 M ethylene glycol and 0.2 M trehalose. The developmental rate to the blastocyst stage of thawed embryos that had been cultured with LAA-containing medium before freezing was higher than that of these cultured without LAA (P<0.05). However, with fresh, non-frozen, embryos that were incubated under the same culture conditions (with and without LAA), no such difference was found.-KEY WORDS: bovine 16-cell embryo, cryopreservation, linoleic acid-albumin.J. Vet. Med. Sci. 62(4): 465-467, 2000 follicles (2-5 mm in diameter) using an 18-gauge needle connected to a 5 ml syringe (Terumo, Tokyo, Japan). COCs were washed 3 times with a maturation medium (TCM199: Gibco BRL, Life Technologies Inc., Grand Island, NY, U.S.A.) containing 5% (v/v) of newborn calf serum (NBCS: Nakashibetu, Japan), 0.002 AU/ml of FSH (Antrin: Denka Pharmaceutical Inc., Kawasaki, Japan) and 1 µg/ml of estradiol-17β (Sigma Chemical Co., St. Louis, MO, U.S.A.). For oocyte maturation in vitro, 20-35 COCs were transferred into the maturation medium in a 4-well multidish (Nunclon, Roskilde, Denmark) and kept for 22 hrs at 38.5°C under 5% CO2 in air. Frozen-thawed semen from Japanese black bulls was used for in vitro fertilization. An aliquot of semen, which was thawed in a water bath at 37°C for 30 sec, was transferred into Brackett-Oliphant (BO) medium [1] supplemented with 5 mM caffeine (Sigma) or theophylline (Sigma) without BSA. In some experiments, Percol (Pharmacia) gradient centrifugation was performed to separate live spermatozoa [13]. The sperm suspension was washed twice by centrifugation at 500 × g for 5 min. The resulting sperm pellet was resuspended in 100 µl of the BO medium containing 5 mg/ml BSA (Sigma), 5 µg/ml heparin (NovoHeparin 100, Novo Industry A/S, Osaka, Japan) and 2.5 mM caffeine or theophylline to give a final concentration of 5 × 10 6 sperm/ml. An aliquot of sperm suspension was introduced into the BO medium containing 25-35 matured oocytes covered with mineral oil (Nacalai Tesque Inc., Kyoto, Japan). Six hours after insemination (the time of insemination = Day 0), cumulus cells were removed from the oocytes in Ca -and Mg-free Dulbecco's modified phosphate buffered saline (D-PBS; Nissui Pharmaceutical Co., Ltd., Tokyo, Japan) containing 0.025% hyarulonidase (Sigma) by vortexing for 1.5 min. Denuded zygotes were then cultured in CR1aa supplemented with 3 mg/ml fatty acid-free BSA (Sigma) in a 4-well multidish at 38.5°C under 5% CO 2, 5% O2 and 90% N2.Many calves have been produced using embryo transfer of bovine blastocysts after freezing-thawing. However, embryos that are produced in vitro are more sensitive to the f...

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.