Mucci N, Aller J, Kaiser G G, Hozbor F, Cabodevila J, Alberio R H
Laboratorio de Producción in vitro de Embriones, Departamento de Producción Animal, INTA, CC 276 (7620) Balcarce, Argentina.
Theriogenology. 2006 May;65(8):1551-62. doi: 10.1016/j.theriogenology.2005.08.020. Epub 2005 Oct 17.
The present study investigated the effect of estrous cow serum (ECS) during culture of bovine embryos on blastocyst development and survival after cryopreservation by slow freezing or vitrification. Embryos were derived from in vitro maturation (IVM) and in vitro fertilization (IVF) of abbatoir-derived oocytes. At Day 3, embryos were cultured in three different media: Charles Ronsenkrans medium + amino acids (CR1aa; without bovine serum albumin (BSA)) + 5% estrous cow serum (CR1-ECS), CR1aa + 3 mg/mL BSA (CR1-BSA) or CR1aa + 5% ECS + 3 mg/mL BSA (CR1-ECS-BSA). At 7.5 d post-insemination (PI), blastocyst yield and quality were evaluated; blastocysts and expanded blastocysts from each media were cryopreserved by Open Pulled Straw (OPS) vitrification method or slow freezing (1.5 M ethylene glycol, EM). Total blastocyst yield did not differ among CR1-ECS, CR1-BSA and CR1-ECS-BSA (30.9, 33.1 and 32.9%, respectively, P < 0.05). Embryo survival (hatching rate) was higher in vitrified versus slow-frozen embryos (43% versus 12%, respectively, P < 0.01), and in embryos cultured in CR1-BSA (40.3%) compared with those cultured in serum-containing media (CR1-ECS, 21.5% and CR1-ECS-BSA, 19.8%; P < 0.01).
(a) it was possible to produce in vitro bovine embryos in serum-free culture medium without affecting blastocyst yield and quality; (b) serum-free medium produced the best quality embryos (in terms of post-cryopreservation survival); and (c) vitrification yielded the highest post-cryopreservation survival rates, regardless of the presence of serum in the culture medium.
本研究调查了发情母牛血清（ECS）在牛胚胎培养过程中对慢速冷冻或玻璃化冷冻后囊胚发育及存活的影响。胚胎来源于屠宰场采集的卵母细胞的体外成熟（IVM）和体外受精（IVF）。在第3天，胚胎在三种不同培养基中培养：查尔斯·罗恩斯克兰斯培养基+氨基酸（CR1aa；不含牛血清白蛋白（BSA））+5%发情母牛血清（CR1 - ECS）、CR1aa + 3 mg/mL BSA（CR1 - BSA）或CR1aa + 5% ECS + 3 mg/mL BSA（CR1 - ECS - BSA）。在授精后7.5天（PI），评估囊胚产量和质量；来自每种培养基的囊胚和扩张囊胚通过开放式拉长细管（OPS）玻璃化方法或慢速冷冻（1.5 M乙二醇，EM）进行冷冻保存。CR1 - ECS、CR1 - BSA和CR1 - ECS - BSA之间的总囊胚产量无差异（分别为30.9%、33.1%和32.9%，P < 0.05）。玻璃化冷冻胚胎的存活率（孵化率）高于慢速冷冻胚胎（分别为43%对12%，P < 0.01），并且与在含血清培养基中培养的胚胎相比，在CR1 - BSA中培养的胚胎存活率更高（40.3%）（CR1 - ECS为21.5%，CR1 - ECS - BSA为19.8%；P < 0.01）。
（a）在无血清培养基中培养体外牛胚胎并影响囊胚产量和质量是可行的；（b）无血清培养基产生的胚胎质量最佳（就冷冻保存后的存活率而言）；（c）无论培养基中是否存在血清，玻璃化冷冻产生的冷冻保存后存活率最高。
