Fig 1.
Experimental setup of FLI supplemented to oocyte maturation medium.
Cumulus oocyte complexes (COCs) were matured in oocyte maturation medium (OMM) with (n = 1475) or without (n = 1475) FLI. FLI was supplemented at the dosage of FGF2 (40ng/ml), LIF (20ng/ml), and IGF1 (20ng/ml). For analysis of transzonal projections, COCs were collected at 6 h, 12 h, 18 h, 24 h after placement into OMM. At 24 h, maturation stage was recorded. Following fertilization, a subset of putative zygotes (day 1) was collected for lipid content quantification. Cleavage (at least one cellular division) was recorded on day 3 and development to the blastocyst stage was recorded on days 7 and 8. On day 7 or 8, blastocyst stage embryos were either collected for quantification of lipid content or slow frozen, thawed, and subjected to a TUNEL assay to analyze apoptosis. IF = immunofluorescent labeling.
Fig 2.
Experimental setup of FLI supplemented to embryo culture medium.
Cumulus oocyte complexes (COCs) were matured in oocyte maturation medium (OMM). Following fertilization, putative zygotes were placed in culture with (n = 1,930) or without (n = 2,104) FLI. FLI was supplemented at the dosage of FGF2 (40ng/ml), LIF (20ng/ml), and IGF1 (20ng/ml). Cleavage (at least one cellular division) was recorded on day 3 and development to the blastocyst stage was recorded on day 7 and 8. Day 7 or 8, blastocyst stage embryos were either collected for cell counting, quantification of lipid content, membrane integrity analysis, quantification of reactive oxygen species, or slow frozen, and subjected to TUNEL assay to analyze apoptosis, membrane integrity analysis, or quantification of reactive oxygen species post-thaw. IF = immunofluorescent labeling.
Fig 3.
FLI Supplementation to culture medium improves embryo development.
Bovine oocytes (n = 1,066) were of different qualities. (A) Example of oocytes classified as poor quality (470) or good quality (596) (B) Proportion of zygotes that underwent at least one cellular division. (C) The extent of the effect of FLI supplementation in culture medium on development to blastocyst stage varies based on oocyte quality/source. Asterisk (*) indicates statistical differences (P < 0.05). Values are LSMEANS ± SEM.
Fig 4.
Representative images of bovine blastocysts showing the actin filament staining.
(A) Grade 1 blastocyst stage embryos displayed sharp staining of actin filaments and cells are clearly organized as inner cell mass or trophectoderm. (B) Grade 2 shows less distinct outlining of the cells and some breaks in the cytoskeleton structure. (C) Grade 3 is characterized by large gaps in actin staining and some actin within the cytoplasm. Actin filaments were visualized in 101 fresh (n = 52 control, n = 49 +FLI) and 52 (n = 22 control, 30 +FLI) thawed embryos.
Fig 5.
FLI supplementation advances transzonal projection dissociation.
(A) The effect of the addition of FLI to oocyte maturation medium on the number of TZP at 6, 12, 18, and 24hr. (n = 198; 100 control and 98 +FLI). (B) The images are representative of TZP dissociation at 24hr of maturation. Arrows indicate intact TZP. Asterisk (*) indicates statistical differences (P < 0.05).
Table 1.
In vitro development of bovine embryos supplemented with and without FLI during maturation.
Fig 6.
FLI supplementation during oocyte maturation decreases lipid content at the blastocyst stage.
(A) Lipid content was compared at the zygote (n = 239; 109 controls and 120 +FLI) and blastocyst stage (n = 222; 116 controls and 106 +FLI). (B) Blastocyst stage bovine embryos illustrating Nile red staining by fluorescence microscopy used to determine lipid content. Asterisk (*) indicates statistical differences (P < 0.05).
Table 2.
Hatching rates post-thaw of bovine embryos supplemented with and without FLI during maturation following cryopreservation by slow-freezing.
Table 3.
In vitro development and number of nuclei in bovine embryos supplemented with and without FLI in culture.
Fig 7.
FLI supplementation during embryo culture improves cryopreservation.
(A) The effect of the addition of FLI to culture medium and post-thaw hatching rate (n = 117; 51 control and 66 +FLI). (B) Images of bovine embryos post-thaw subjected to the TUNEL assay (green) and fluorescence imaging. Asterisk (*) indicates statistical differences (P < 0.05).