No animals were used for this work. All studies were completed on slaughterhouse-derived materials from a commercial slaughterhouse that followed humane slaughter practices according to USDA guidelines. Reagents were purchased from ThermoFisher Chemical Company (Waltham, MA), unless otherwise specified.
In vitro Embryo Production
Bovine blastocysts were produced by in vitro maturation, fertilization and culture procedures described previously [5, 30]. In brief, cumulus-oocytes complexes (COCs) were extracted from slaughterhouse-derived ovaries (Brown Packing, Gaffney, SC). For fertilization, COCs were exposed to live spermatozoa (1 x 106 spermatozoa/ml medium) from pooled semen from four Holstein bulls (donation from Select Sires, Plain City, OH, USA) using a biphasic gradient (40 and 80% [v/v] BovipureTM; Nidacon; Spectrum Technologies, Healdsburg, CA, USA). After 14-18 hours at 5% CO2 in air at 38.5°C, presumptive zygotes were denuded by gentle pipetting and placed in groups of ~25 in 50 µl SOF-BEI drops under light mineral oil and incubated in 5% CO2, 5% O2 and 90% N2 in humidified air at 38.5°C . The day of fertilization was designated as day 0. The embryos were cultured in SOF-BEI until day 7, 8, 9, or 10, as specified for each experiment.
In most studies, treatments were administered to existing drops by addition of 2 µl of concentrated recombinant bovine IL6 (KingFisher Biotech, St. Paul, MN, USA) prepared in SOF-BEI medium. The control treatment was composed of carrier only (1% [w/v] bovine serum albumin [BSA]). Embryos were then maintained in their original drops until harvested for analysis. In one study, day 7 blastocysts were collected from their existing drops, and individual blastocysts were placed into 50 µl SOF-BEI containing 0 or 100 ng/ml IL6. In another study, embryos that had been exposed to IL6 treatments beginning on day 5 were removed from their drops on day 7, washed twice in SOF-BEI and placed into non-IL6-treated drops (50 µl SOF-BEI; 2-7 embryos/drop).
A stock solution of 100 mM AZD1480 (JAK 2 inhibitor; S2162; Selleck Chemicals, Houston, TX, USA) was prepared with DMSO as the carrier (stored at -80°C). Blastocysts were removed from their original drops on day 7 or 8 post-fertilization and placed into 50 µl SOF-BE1 containing either 3 µM AZD1480 or carrier only (0.003% DMSO). Blastocysts were collected and processed for immunofluorescence after 24 or 48 h.
Procedures were completed as described previously, with some modifications [3, 5]. Differential staining for TE and ICM cells in blastocysts was completed as described previously  using mouse anti-Caudal Type homeobox 2 (CDX2; Biogenex, San Ramon, CA, sold ready-to-use), anti-mouse IgG (Alexafluor 488; 1:200), and 4,6-diamidino-2-phenylindole (DAPI; 1 µg/ml) .
For CDX2, NANOG and GATA6 co-staining studies, blastocysts were permeabilized with 0.5% Triton-X in Dulbecco’s Phosphate Buffered Saline (DPBS) for 30 min, then blocked with 10% [v/v] horse serum for 1 h. Due to antibody overlap, two rounds of primary and secondary antibody incubations were completed. First, blastocysts were incubated with rabbit anti-GATA6 (Cell Signaling Technology, Danvers, MA; 5851T; 1:500) and mouse anti-NANOG (eBioscience; 14-5768-82; 1:200) for 1 h at room temperature, then blastocysts were incubated with donkey anti-rabbit IgG (Alexafluor 555; 1:200) and donkey anti-mouse IgG (Alexafluor 647; 1:200). Blastocysts were then incubated with mouse anti-CDX2 antibody (same as above) for 1 h at room temperature and finally exposed to donkey anti-mouse IgG (Alexafluor 488; 1:500).
For pSTAT3Y705 co-staining with NANOG or GATA6, blastocysts were incubated with 70% [v/v] ethanol for 5 min at room temperature then were blocked with 10% horse serum containing 0.5% Triton-X for 1 h at room temperature. Blastocysts were then incubated for 1 h at room temperature or overnight at 4°C with either rabbit anti-pSTAT3Y705 (Cell Signaling Technologies; 9145T; 1:100) and mouse anti-NANOG (same as above; 1:200), or with mouse anti-pSTAT3Y705 (Santa Cruz Biotechnology, Dallas, TX; sc-8059; 1:200) and rabbit anti-GATA6 (same as above; 1:500). After washing, blastocysts were incubated with donkey anti-mouse IgG (Alexafluor 488 or 647; 1:200) and anti-rabbit IgG (Alexafluor 555; 1:200).
After each of these staining procedures, embryos were incubated with 1 µg/ml DAPI for 5 minutes at room temperature then washed in PBS-PVP and flattened on a glass slide lined with petroleum jelly. Staining was visualized with an Eclipse Ti-E inverted microscope equipped with an X-cite 120 epifluorescence illumination system and DS-L3 digital camera. Images were captured with NIS-Elements Software (Nikon Instruments, Melville, NY), and cell counting was completed with the cell counter plugin in the program FIJI (ImageJ) . Some embryos presented in figures may appear larger than others. This is a result of variable pressure applied to each coverslip as embryos were “flattened”. All embryos were imaged at 20X magnification. A representative sampling of regular, expanded and hatched blastocysts was selected on day 8. At day 9, only expanded hatched blastocysts were selected for analysis, and at day 10, only hatched blastocysts were selected. In some studies where blastocysts were only stained with anti-CDX2 antibody and DAPI, CDX2+:DAPI+ nuclei were considered TE, while CDX2-:DAPI+ nuclei were considered ICM cells. This method and anti-CDX2 antibody are commonly used to determine ICM and TE cell numbers in bovine blastocysts [32-35].
All analyses were completed using the Statistical Analysis System (SAS for Windows; SAS Institute Inc., Cary, NC, USA). Three to four replicates were completed for most studies. For all analyses, embryo was considered the experimental unit. Replicate was considered a random, independent variable. Differences in cell number were analyzed by least-squares ANOVA, using the general linear model (Proc GLM). In one study, ICM cell number data were cube-root transformed before analysis as they contained a right-tail distribution. Individual comparisons were partitioned further by using the Probability of Difference (PDIFF) test in SAS. Statistical significance was determined at P ≤ 0.05.