Clean up the environment

rm(list = ls())

Load libraries for data reading, plotting, statistical analysis

library(readxl)       # For reading Excel files
library(ggpubr)       # For creating publication-ready plots
library(dplyr)        # For data manipulation
library(rstatix)      # For statistical tests and adding significance markers
library(knitr)        # For displaying tibbles in a nicely formatted table
library(kableExtra)   # For enhancing table formatting

Load data

# Load pig information and measurement of anemia status
PigInfo <- read_excel("Data/Data.xlsx", sheet = "PigInfo")
Anemia <- read_excel("Data/Data.xlsx", sheet = "Hb_and_Hct")

Plot Hct and Hb

Fig1 <- Anemia %>% 
            filter(!PigID %in% c(19, 24)) %>% ## Remove the two piglets where the blood in the heparin tube heavily coagulated
            inner_join(PigInfo, by = "PigID") %>% 
            mutate(Diet = factor(Diet, levels = c("ALAC", "WPI", "SF"))) %>%
            ggboxplot(y = "Hct", x = "Diet", 
                      fill = "Diet", palette = c("turquoise3","purple", "orange"),
                      xlab = "", ylab = "Hct", add = "jitter") 
Fig2 <- Anemia %>% 
            filter(!PigID %in% c(19, 24)) %>% ## Remove the two piglets where the blood in the heparin tube heavily coagulated
            inner_join(PigInfo, by = "PigID") %>% 
            mutate(Diet = factor(Diet, levels = c("ALAC", "WPI", "SF"))) %>%
            mutate(Hb = Hb/10) %>% # Hb was in g/L, convert unit
            ggboxplot(y = "Hb", x = "Diet", fill = "Diet", 
                      palette = c("turquoise3","purple", "orange"),
                      xlab = "", ylab = "Hb (g/dL)",  add = "jitter") 
Fig.all <- ggarrange(Fig1, Fig2, common.legend = TRUE)
Fig.all

# Save the figure in pdf:
ggsave(plot=Fig.all, height=4, width=7.5, dpi=300, filename="SI Figure 2/SI.Fig2.pdf", useDingbats=FALSE)

Statistical analysis via Tukey HSD

Anemia %>% filter(!PigID %in% c(19, 24)) %>% ## Remove the two piglets where the blood in the heparin tube heavily coagulated
             inner_join(PigInfo, by = "PigID") %>% 
             mutate(Diet = factor(Diet, levels = c("ALAC", "WPI", "SF"))) %>%
             tukey_hsd(Hb ~ Diet) %>%
             kable() %>% # Print the tibble in a nicely formatted table
             kable_styling(bootstrap_options = c("striped", "hover")) # Enhance table formatting with kableExtra
term group1 group2 null.value estimate conf.low conf.high p.adj p.adj.signif
Diet ALAC WPI 0 -1.26083 -32.25921 29.73755 0.994 ns
Diet ALAC SF 0 -13.32532 -52.97859 26.32795 0.683 ns
Diet WPI SF 0 -12.06449 -50.60054 26.47156 0.718 ns 
Anemia %>% filter(!PigID %in% c(19, 24)) %>% ## Remove the two piglets where the blood in the heparin tube heavily coagulated
              inner_join(PigInfo, by = "PigID") %>% 
              mutate(Diet = factor(Diet, levels = c("ALAC", "WPI", "SF"))) %>%
              tukey_hsd(Hct ~ Diet) %>%
              kable() %>% # Print the tibble in a nicely formatted table
              kable_styling(bootstrap_options = c("striped", "hover")) # Enhance table formatting with kableExtra
term group1 group2 null.value estimate conf.low conf.high p.adj p.adj.signif
Diet ALAC WPI 0 -0.0916667 -7.173774 6.990441 0.999 ns
Diet ALAC SF 0 -4.8000000 -13.859463 4.259463 0.396 ns
Diet WPI SF 0 -4.7083333 -13.512548 4.095882 0.390 ns 
sessionInfo()
## R version 4.2.2 (2022-10-31)
## Platform: x86_64-apple-darwin17.0 (64-bit)
## Running under: macOS Big Sur ... 10.16
## 
## Matrix products: default
## BLAS:   /Library/Frameworks/R.framework/Versions/4.2/Resources/lib/libRblas.0.dylib
## LAPACK: /Library/Frameworks/R.framework/Versions/4.2/Resources/lib/libRlapack.dylib
## 
## locale:
## [1] en_US.UTF-8/en_US.UTF-8/en_US.UTF-8/C/en_US.UTF-8/en_US.UTF-8
## 
## attached base packages:
## [1] stats     graphics  grDevices utils     datasets  methods   base     
## 
## other attached packages:
## [1] kableExtra_1.3.4 knitr_1.45       rstatix_0.7.2    dplyr_1.1.3     
## [5] ggpubr_0.6.0     ggplot2_3.5.1    readxl_1.4.3    
## 
## loaded via a namespace (and not attached):
##  [1] tidyselect_1.2.0  xfun_0.40         bslib_0.5.1       purrr_1.0.2      
##  [5] carData_3.0-5     colorspace_2.1-0  vctrs_0.6.5       generics_0.1.3   
##  [9] viridisLite_0.4.2 htmltools_0.5.6.1 yaml_2.3.7        utf8_1.2.4       
## [13] rlang_1.1.2       jquerylib_0.1.4   pillar_1.9.0      glue_1.6.2       
## [17] withr_3.0.1       lifecycle_1.0.4   stringr_1.5.1     munsell_0.5.1    
## [21] ggsignif_0.6.4    gtable_0.3.5      cellranger_1.1.0  ragg_1.2.6       
## [25] rvest_1.0.3       evaluate_1.0.1    labeling_0.4.3    fastmap_1.1.1    
## [29] fansi_1.0.6       highr_0.10        broom_1.0.5       scales_1.3.0     
## [33] backports_1.4.1   cachem_1.0.8      webshot_0.5.5     jsonlite_1.8.8   
## [37] abind_1.4-5       farver_2.1.1      systemfonts_1.0.5 textshaping_0.3.7
## [41] gridExtra_2.3     digest_0.6.33     stringi_1.7.12    cowplot_1.1.1    
## [45] grid_4.2.2        cli_3.6.2         tools_4.2.2       magrittr_2.0.3   
## [49] sass_0.4.7        tibble_3.2.1      tidyr_1.3.0       car_3.1-2        
## [53] pkgconfig_2.0.3   xml2_1.3.5        rmarkdown_2.28    svglite_2.1.2    
## [57] httr_1.4.7        rstudioapi_0.15.0 R6_2.5.1          compiler_4.2.2