Interleukin-6 supplementation improves bovine conceptus elongation and transcriptomic indicators of developmental competence†

Abstract

A high incidence of pregnancy failures occurs in cattle during the second week of pregnancy as blastocysts transition into an elongated conceptus. This work explored whether interleukin-6 supplementation during in vitro embryo production would improve subsequent conceptus development. Bovine embryos were treated with 0 or 100 ng/mL recombinant bovine interleukin-6 beginning on day 5 post-fertilization. At day 7.5 post-fertilization, blastocysts were transferred into estrus synchronized beef cows (n = 5 recipients/treatment, 10 embryos/recipient). Seven days after transfer (day 14.5), cows were euthanized to harvest reproductive tracts and collect conceptuses. Individual conceptus lengths and stages were recorded before processing for RNA sequencing. Increases in conceptus recovery, length, and the proportion of tubular and filamentous conceptuses were detected in conceptuses derived from interleukin-6-treated embryos. The interleukin-6 treatment generated 591 differentially expressed genes in conceptuses (n = 9-10/treatment). Gene ontology enrichment analyses revealed changes in transcriptional regulation, DNA-binding, and antiviral actions. Only a few differentially expressed genes were associated with extraembryonic development, but several differentially expressed genes were associated with embryonic regulation of transcription, mesoderm and ectoderm development, organogenesis, limb formation, and somatogenesis. To conclude, this work provides evidence that interleukin-6 treatment before embryo transfer promotes pre-implantation conceptus development and gene expression in ways that resemble the generation of a robust conceptus containing favorable abilities to survive this critical period of pregnancy.

Keywords: cow; elongation; embryokine; in vitro embryo production; interleukin-6; pregnancy.

Graphical Abstract

Figure 1

Supplementation with IL6 increases ICM and total cell numbers in expanded blastocysts. Immunofluorescence staining for CDX2 (TE marker) and DAPI (DNA intercalating agent) was used to determine total cell numbers (A), TE cell number (B), ICM cell number (C), and the ICM:TE ratio (D) in control and IL6-treated (100 ng/mL) blastocysts harvested at day 7.5 post-fertilization. Individual marks represent the number for each blastocyst examined (n = 28–34). The bar within treatment group represents the mean. Treatment differences are indicated with an asterisk (*) (P < 0.05).

Figure 2

Supplementation with IL6 improves conceptus recovery and size. Conceptuses developed from control and IL6-treated IVP embryos were collected at day 14.5 (10 embryos/recipient; 5 recipients/treatment). Shown are the percentage conceptuses recovered (A), conceptus length (B), distribution of conceptus lengths with median and upper/lower quartiles (C), and the distribution of ovoid, tubular, and filamentous conceptuses (D). Treatment differences are indicated with an asterisk (*) (P < 0.05).

Figure 3

Transcriptome analysis of day 14.5 conceptuses. (A) Principal component analysis using 12 980 genes quantified. Included are the ovoid (n = 6/treatment) and tubular (n = 4/treatment) conceptuses from control (n = 10) and IL6-treated (n = 10) IVP blastocysts before transfer. (B) Heat map of the 100 genes with the lowest adjusted P value (false discovery rate) for the comparison of IL6-treated embryos versus controls.