Outgrowths and capsule derived from aggregated cloned equine embryos

GAMBINI A,; JARAZO J; DE STEFANO A; KARLANINAN F; SALAMONE D

Vancouver

Congreso; International Congress on Animal Reproduction (ICAR); 2012

International Congress on Animal Reproduction (ICAR)

The formation of the embryonic capsule seems to be necessary for pregnancy to be established in the equine. It is reported that an in vivo development is necessary to allow normal capsule formation, and remains unclear if cloned equine embryos cells are capable to produce capsular material on in vitro culture. Our aim was to establish culture conditions to allow in vitro development of equine cloned embryos beyond day 7, and to evaluate the ability of these embryos to produce outgrowths and capsule. Oocyte collection, maturation, cloning, and activation procedures were performed as described by Lagutina et al. (2007 Theriogenology 67, 90–98). After activation, zona free reconstructed embryos (REs) were cultured in DMEM/F12 with 5% of FBS in well of well system by the aggregation of three REs per well until day 7. Cloned equine blastocysts were maintained in DMEM/F12 with 10% of FBS from day 7 until their collapse (approximately day 17). Blastocysts development and morphological characteristics were observed every 24 h. After their collapse, they were picked up using 18G needles and then cultured in petri dish as tissue samples in DMEM/F12 medium containing 10% FBS, 1% ATB and 1 ul/ml ITS in 5% CO2 in humidified air at 39C. Outgrowths were obtained from embryos, and they were allowed to grow for 20–30 days, then fixed. A total of nine cloned equine blastocysts were used for this experiment. Embryo size means per day are detailed bellow: Day 7, 110.50 lm ± 31.45; Day 8, 157.14 lm ± 44.16; Day 9, 230.32 lm ± 67.79; Day 11, 517.44 lm ± 276.10; Day 12, 777.81 lm ± 401.11; Day 13, 1218.20 lm ± 523.71; Day 14, 1702.22 lm ± 917.93; Day 15, 2242.27 lm ± 893.44 and day 16, 2884.84 lm ± 1068.31. All blastocysts were able to allow primary embryo outgrowth formation. We did not see, under light microscopy, a clear and confluent capsule as in in vivo embryos on in vitro zona-free equine cloned embryos neither at day 7, nor during culture until day 17. However, a structure morphologically identical to an equine 608 Abstracts embryo capsule was found floating in the culture medium in six of nine explants, two to five days after embryo collapse. In conclusion, in vitro culture of equine embryos in DMEM/F12 allows embryo development beyond day 7. The high capability of day 17 collapsed embryos to produce outgrowths, show the elevated grade of proliferation of equine embryos cells in DMEM/F12 medium. Further experiments should be done to clarify if this capsule-like structure formed by cloned equines embryos has the same molecular characteristic of in vivo embryo capsule, and to know if the addition of uterus secreted molecules on in vitro culture medium it is necessary for a normal capsule formation at physiological times. Key Words: Capsule, equine, embryo, outgrowth, explant