########################################################################################## ### Analyses: relationships between sperm morphology, swimming velocity and stored ATP ### ########################################################################################## rm(list=ls()) ### load raw data files: "Experimental males summary data.txt" and ### "Microplate sample repeatability.txt" summarydata <- read.table(file.choose(),header=T) # note that missing values = NA wellrepeats <- read.table(file.choose(),header=T) ######################################################################################### ### Microplate sample repeatability ##################################################### ######################################################################################### ### description of variables for file "Microplate sample repeatability.txt" ### Sample - an identifer for each males' samples ### WellNumber - an identifer for the repeat wells for each males' samples. Samples were run in triplicate. ### BioUnit - the bioluminescent value detected in the well. ### Analyses of well repeatability ###################################################### ### repeatability of microplate samples measurements (methods from Lessels & Boag 1987) repeatwell <-read.table(file.choose(),header=T) repeatwell$WellNumber <- as.factor(repeatwell$WellNumber) str(repeatwell) rep1 <- aov(repeatwell$BioUnit ~ repeatwell$Sample) summary(rep1) # Df Sum Sq Mean Sq F value Pr(>F) # repeatwell$Sample 38 537417734 14142572 7.411 5.64e-14 *** # Residuals 78 148843557 1908251 # --- # Signif. codes: 0 ‘***’ 0.001 ‘**’ 0.01 ‘*’ 0.05 ‘.’ 0.1 ‘ ’ 1 # approx repeatability = 0.90 (n for this is 3) # calculated repeatability = # S2a = (MSa-MSw)/n # S2a = (14142572-1908251)/2 = 6117160.5 # S2 = MSw # s2a/(s2+s2a) # 6117160.5/(1908251+6117160.5) # 6117160.5/(8025411.5) = 0.76 ######################################################################################### ### Relationships between sperm morphology, swimming velocity and stored ATP ######################################################################################### ### description of variables for file "Experimental males summary data.txt" ### MaleID - unique identifier for each experimental male ### Line - the selection line identity of the male ### meanVSL - mean straight line velocity of all sampled sperm from a male ### meanVAP - mean average path velocity of all sampled sperm from a male ### meanVCL - mean curvilinear velocity of all sampled sperm from a male ### meanPC1 - mean principle component 1 value of all sampled sperm from a male (dervived from VAP, VSL & VCL) ### mean10VSL - mean straight line velocity of the fastest 10% of sampled sperm from a male ### mean10VAP - mean curvilinear velocity of the fastest 10% of sampled sperm from a male ### mean10VCL - mean curvilinear velocity of the fastest 10% of sampled sperm from a male ### mean10PC1 - mean principle component 1 value of the fastest 10% of sampled sperm from a male (dervived from VAP, VSL & VCL) ### meanhl - mean head length of all sampled sperm from a male ### meanml - mean midpiece length of all sampled sperm from a male ### meantail - mean tail length of all sampled sperm from a male ### meantl - mean total length of all sampled sperm from a male ### spermconc - sperm concentration (million) measured by Neubauer chamber ### averageATP - average ATP value from sampled sperm from a male (across the triplicate wells) ### averageATPnmols - average ATP value (nanomoles) from sampled sperm from a male (triplicate wells) ### standardisedATPnmolpermillionsperm - average ATP value (nanomoles) per million sperm from a male ### Analyses of sperm morphology, swimming velocity and stored ATP ################################## ##################################################################################################### ### Pearson's correlations between sperm components cor.test(summarydata$meanhl,summarydata$meantl) # r2 = 0.46 cor.test(summarydata$meanml,summarydata$meantl) # r2 = -0.28 cor.test(summarydata$meantail,summarydata$meantl) # r2 = 0.88 ### sperm morphology summary data across all males - included in Table S1 ### covariance function co.var <- function(x,na.rm=TRUE) 100*(sd(x,na.rm=na.rm)/mean(x,na.rm=na.rm)) mean(summarydata$meanhl) # 11.19um sd(summarydata$meanhl) # 0.62 range(summarydata$meanhl) # 9.49 - 12.99 co.var(summarydata$meanhl) # 5.55 mean(summarydata$meanml) # 30.47um sd(summarydata$meanml) # 4.47 range(summarydata$meanml) # 16.11 - 39.80 co.var(summarydata$meanml) # 14.67 mean(summarydata$meantail) # 24.72um sd(summarydata$meantail) # 9.28 range(summarydata$meantail) # 9.55 - 46.74 co.var(summarydata$meantail) # 37.55 mean(summarydata$meantl) # 66.38um sd(summarydata$meantl) # 7.23 range(summarydata$meantl) # 49.57 - 79.76 co.var(summarydata$meantl) # 10.90 mean(summarydata$meanfl) # 55.19um sd(summarydata$meanfl) # 6.97 range(summarydata$meanfl) # 39.12 - 68.33 co.var(summarydata$meanfl) # 12.63 ### mean sperm swimming velocity summary data across all males - included in Table S2 mean(summarydata$meanVSL) # 39.08 sd(summarydata$meanVSL) # 15.83 range(summarydata$meanVSL) # 0.091 - 83.12 co.var(summarydata$meanVSL) # 40.51 mean(summarydata$meanVCL) # 53.53 sd(summarydata$meanVCL) # 16.91 range(summarydata$meanVCL) # 0.75 - 89.83 co.var(summarydata$meanVCL) # 31.58 mean(summarydata$meanVAP) # 42.89 sd(summarydata$meanVAP) # 15.76 range(summarydata$meanVAP) # 0.26 - 87.42 co.var(summarydata$meanVAP) # 36.75 ### mean sperm swimming velocity summary data from fastest 10% of sperm across all males - ### included in Table S2 mean(summarydata$mean10VSL) # 69.67 sd(summarydata$mean10VSL) # 19.06 range(summarydata$mean10VSL) # 0.90 - 114.92 co.var(summarydata$mean10VSL) # 27.36 mean(summarydata$mean10VCL) # 83.81 sd(summarydata$mean10VCL) # 15.41 range(summarydata$mean10VCL) # 7.40 - 117.72 co.var(summarydata$mean10VCL) # 18.39 mean(summarydata$mean10VAP) # 73.18 sd(summarydata$mean10VAP) # 18.44 range(summarydata$mean10VAP) # 2.59 - 117.12 co.var(summarydata$mean10VAP) # 25.20 ### Relationships between swimming velocity and sperm components ### (fastest 10% of sperm) across all males - included in Table S3. ### calculate flagellum length, flagellum:head and midpiece:tail summarydata$meanfl <- summarydata$meanml + summarydata$meantail summarydata$meanfhratio <- summarydata$meanfl/summarydata$meanhl summarydata$meanmtratio <- summarydata$meanml/summarydata$meantail ### head length m1 <- lm(mean10PC1~meanhl, data=summarydata) summary(m1) ### midpiece length m1.2 <- lm(mean10PC1~meanml, data=summarydata) summary(m1.2) ### tail length m1.3 <- lm(mean10PC1~meantail+I(meantail^2), data=summarydata) summary(m1.3) ### total length m1.4 <- lm(mean10PC1~meantl+I(meantl^2), data=summarydata) summary(m1.4) ### flagellum: head m1.5 <- lm(mean10PC1~meanfhratio+I(meanfhratio^2), data=summarydata) summary(m1.5) ### midpiece: tail m1.6 <- lm(mean10PC1~meanmtratio+I(meanmtratio^2), data=summarydata) summary(m1.6) ### Relationship between midpiece length and stored ATP ### Log ATP ATP <- log(summarydata$standardisedATPpermillionsperm) m2 <- lm(ATP ~ meanml, data=summarydata) summary(m2) Call: lm(formula = ATP ~ meanml, data = summarydata) Residuals: Min 1Q Median 3Q Max -1.22598 -0.41876 -0.09896 0.37508 1.50833 Coefficients: Estimate Std. Error t value Pr(>|t|) (Intercept) 6.82475 0.75061 9.092 7.41e-11 *** meanml -0.08364 0.02542 -3.290 0.00225 ** --- Signif. codes: 0 ‘***’ 0.001 ‘**’ 0.01 ‘*’ 0.05 ‘.’ 0.1 ‘ ’ 1 Residual standard error: 0.6334 on 36 degrees of freedom (144 observations deleted due to missingness) Multiple R-squared: 0.2312, Adjusted R-squared: 0.2098 F-statistic: 10.83 on 1 and 36 DF, p-value: 0.002246 ### Relationship between swimming velocity and stored ATP m3 <- lm(ATP ~ mean10PC1, data=summarydata) summary(m3) Call: lm(formula = ATP ~ mean10PC1, data = summarydata) Residuals: Min 1Q Median 3Q Max -1.49382 -0.46071 -0.04876 0.28140 2.00703 Coefficients: Estimate Std. Error t value Pr(>|t|) (Intercept) 4.55918 0.43062 10.587 1.32e-12 *** mean10PC1 -0.05537 0.12692 -0.436 0.665 --- Signif. codes: 0 ‘***’ 0.001 ‘**’ 0.01 ‘*’ 0.05 ‘.’ 0.1 ‘ ’ 1 Residual standard error: 0.7205 on 36 degrees of freedom (144 observations deleted due to missingness) Multiple R-squared: 0.005259, Adjusted R-squared: -0.02237 F-statistic: 0.1903 on 1 and 36 DF, p-value: 0.6653 ### Relationship between model for swimming velocity and ATP per unit midpiece length ### length adjusted ATP content adjATP <- summarydata$standardisedATPpermillionsperm / summarydata$meanml adjATP <- log(adjATP) m4 <- lm(adjATP ~ mean10PC1, data = summarydata) summary(m4) Call: lm(formula = adjATP ~ mean10PC1, data = summarydata) Residuals: Min 1Q Median 3Q Max -1.74066 -0.50953 -0.02787 0.32824 2.22052 Coefficients: Estimate Std. Error t value Pr(>|t|) (Intercept) 1.23304 0.47756 2.582 0.014 * mean10PC1 -0.06746 0.14076 -0.479 0.635 --- Signif. codes: 0 ‘***’ 0.001 ‘**’ 0.01 ‘*’ 0.05 ‘.’ 0.1 ‘ ’ 1 Residual standard error: 0.799 on 36 degrees of freedom (144 observations deleted due to missingness) Multiple R-squared: 0.00634, Adjusted R-squared: -0.02126 F-statistic: 0.2297 on 1 and 36 DF, p-value: 0.6346 ### References Lessels CM & Boag PT (1987) Unrepeatable repeatabilities: a common mistake. Auk 104:116-121.