(74) The Combination of CCL14 Chemokine and FLI During In Vitro Maturation Enhances In Vitro Development of Bovine Embryos

Paula Mangiavacchi¹; Kiho Lee^1,2; Bethany K. Redel³

1. Division of Animal Science, College of Agriculture Food and Natural Resources, University of Missouri, 920 East Campus Drive, Columbia - MO, USA.

2. National Swine Resource and Research Center, University of Missouri Columbia - MO, USA.

3. United States Department of Agriculture - Agriculture Research Service, Plant Genetics Research Unit, Columbia - MO, USA.

Abstract Text: FLI supplementation, composed of FGF2, LIF and IGF1, is a well-established strategy to improve in vitro embryo production in different species, including pigs and cattle. However, in vitro conditions are still considered to be suboptimal. Chemokines have been shown to play an important role in reproductive events, such as ovulation and embryo implantation. Among them, CCL14 acts in the regulation of trophectoderm development and in trophoblast migration, which is essential for the pre-implantation embryo development. Notably, CCL14 and its receptor CCR1 have been identified in bovine oocytes and early embryos. This study was aimed to evaluate the effect of different concentrations of CCL14 (10, 50 and 100ng) in the presence or absence of the components FLI in a chemically defined maturation system, on oocyte maturation, cleavage, blastocyst percentage and total number of cells. Bovine oocytes were obtained from a commercial supplier and allocated into eight experimental groups, combining the presence or absence of FLI with varying concentrations of CCL14. After 22 hours of in vitro maturation in 5% CO₂ at 38.5ºC, oocytes were denuded, and nuclear maturation was assessed or fertilized with 1 × 10⁶ sperm cells/ml for 18 hours in 5% CO₂ at 38.5ºC. Embryo development was assessed up to D7 and D8, when blastocyst percentages were evaluated and collected for CDX2 staining and subsequent cell count. All data were analyzed using one-way ANOVA with Tukey’s post hoc test in GraphPad Prism8 to assess statistical significance. The results obtained in oocyte maturation demonstrate a trend of improvement in the groups with FLI and CCL14, especially in the treatment with 100ng CCL14 (77%, p=0.19). This demonstrates a possible synergistic action between FLI and CCL14, enhancing oocyte competence. Similar trends were observed for cleavage, with the group FLI plus 100ng CCL14 demonstrating the highest developmental outcomes, compared with this same supplementation in the absence of FLI (79% vs 62% p=0.14), and compared with no FLI group (79% vs 61%, p< 0.05). Also, the FLI group with 100ng CCL14 also increased blastocyst percentages on both D7 and D8, compared to the group without FLI (31% vs. 18%, p< 0.05) and to the group without FLI plus 50ng CCL14 (31% vs. 12%, p< 0.01). Among the groups supplemented with CCL14 in the absence of FLI, the treatment with 100ng also showed improved blastocyst percentages on D7 compared to the other groups (30% vs. 18%, p< 0.05), indicating a potential standalone benefit at this dose. CDX2 immunostaining showed that supplementation with FLI and CCL14 influences cell allocation in bovine blastocysts. Although the FLI and 50ng group presented the most balanced ratio between ICM and TE (1:3), this treatment resulted in low developmental outcomes. The group FLI and 100ng of CCL14 had a high number of CDX2+ cells (81%), suggesting enhanced trophectoderm development. In contrast, groups without FLI had an imbalanced ICM:TE ratio (1:8) and lower developmental competence, despite a high percentage of CDX2+ cells (up to 88%). In conclusion, supplementation with FLI and 100ng of CCL14 improves oocyte maturation, cleavage, and blastocyst development, as well as cell allocation within the embryo, supporting its potential as a valuable strategy in bovine in vitro embryo production. Further research is warranted to explore the expression of related genes and pathways, as well as the specific role of CCR1 in cumulus-oocyte complexes and early embryos.