DOI: 10.5061/dryad.mpg4f4r99
title: Reproductive output of old males is limited by seminal fluid, not sperm number

description: Male reproductive senescence is typically characterised by a decline in
 the number of sperm produced and transferred by old males, a phenomenon
 that may be exacerbated in polygynous species where males mate-multiply.
 However, males also transfer seminal fluid to females, and little is known
 about its role in modulating male reproductive senescence. Here, we
 explore the contributions of sperm and seminal fluid towards male
 reproductive senescence in a series of sequential matings, using
 Drosophila melanogaster. As expected, old males produce fewer offspring
 than young males. However, this pattern is not driven by sperm limitation:
 old males have more sperm and transfer similar numbers to females,
 compared to young males. Instead, females storing fewer sperm of old males
 compared to that of young males, over a long-term, drives male
 reproductive senescence. We are able to mitigate the age-related decline
 in male reproductive output by supplementing females with the seminal
 fluid of a young male, before she mates with an old male. Similarly, we
 alleviate the reduction in reproductive output across sequential matings
 by supplementing females with seminal fluid. Our findings highlight that
 seminal fluid, rather than sperm number, limits reproductive success in
 old or multiply mating males, highlighting its under-appreciated role in
 reproductive ageing.
description: We conducted experiments and colelcted data directly through
 observations.
description: # Reproductive output of old males is limited by seminal fluid, not sperm
 number Note: In the dataset, the labels for Experiment ID are contrasting
 the paper: in the dataset, Experiment B is actually experiment C from the
 paper, and experiment C in the dataset is experiment B in the paper.
 [https://doi.org/10.5061/dryad.mpg4f4r99](https://doi.org/10.5061/dryad.mpg4f4r99) ## Description of the data and file structure 1\. BG.csv : csv data file for plotting batemans’s gradient in R code 2\. Stripping simple.rmd: R code needed to run all analysis (all data provided on OSF) 3\. Stripping: data from experiments A and C on reproductive output 4\. gfp_f.csv : data on sperm numbers inside females after dissection of females, to be used in R code. Also has data on the numbers of offspring produced by females over 24 hours of egg laying, in a male’s mating sequence, for experiment B. 5\. gfp_m.csv : data on sperm numbers inside male SV and size of male Accessory glands from experiment B 6\. latency.csv: data on male mating latency and copulation duration across three experiments 7\. Prominence.csv: data on the relationship between prominence on find maxima and estimated sperm counts, to determine the most suitable prominence for counting sperm using the find maxima function 8\. Repeatability.xlsx : repeatability in sperm counts data 9\. Sample sizes.xlsx : sample sizes from all three experiemnts 10\. Survival_population.csv : data for survival curves of males in the three lines used. 11\. Stripping-simple.html: HTML output of full code ### Files and variables File: BG.csv Description: Variables • ID: ID of male • MS: Mating success • RS: Reproductive success • Age: Age of male • Experiment: Experiment A B or C • Treatment: Old or young male File: gfp_f.csv Description: Variables • Rep: Block • Female_number: Female rank in mating sequence • Male_ID: Male ID • Female_ID: Female ID • Duration: Mating duration • Age: Male age • F1_counts: Number of offspring produced • Freezing: When female was frozen-immediately or after 24 hours of egg laying • Total30: Total sperm from immediately frozen females • SR: Sperm in seminal receptacle • Total24: Total sperm in females frozen after 24 hours • Fertility: Female fertility (1/0) • Total_matings: Mating success of males • Fertility_F1: Female fertility File: gfp.clean.csv Description: Data for GFP males Variables: • Male_ID: male ID • Rep: Replicate/block • Age: Old or young age groups • N_matings: mating success of males • Sperm_total: total sperm in male accessory glands • AG_mean: mean accessory gland size area of males • Squared: squared number of sperm File: gfp_m.csv Description: Data for GFP males Variables • Male_ID: Male ID • Rep: Block • Age: Male age • N_matings: Mating success • Sperm_total: Total sperm in male seminal vesicle • AG_mean: Mean size or area of accessory glands File: Prominence.csv Description: Find maxima prominence setting determination Variables • Image_ID: Image ID • Prominence: Prominence set on find maxima • Count: Sperm counts for a given prominence estimated by find maxima • Sex: Sex of individual from whom sperm was obtained (Male or female) storage File: latency.csv Description: Mating latency, duration, success data Variables • Line: GFP, sot, or dah line • F_num: Female rank • Male_ID: Male ID • Latency: Mating latency • Duration: Copulation duration • Male_Age: Male age File: Read_me.docx Description: Read me document File: sample_sizes.xlsx Description: Final sample sizes used Variables • Age: old or young males • SF: whether seminal fluid was provided with sot males or not provided • Female_number: female rank in mating sequence • N: Sample size • F1:counts: average F1 counts of offspring numbers produced • Sd: standard deviation of offspring numbers produced • Se: standard error of offspring numbers produced • CI: confidence interval of offspring numbers produced • Experiment: A, B, or C (not that labels of experiments B and C are swapped compared to the paper) • Duration: Mating duration File: repeatability.xlsx Description: Repeatability of find maxima and manual sperm counts on ImageJ/FIJI Variables Sheet 1 • ID: Image ID • Manual_KS: manual sperm count by Krish Sanghvi using cell counter • Fiji_threshold: counts estimated automatically using thresholding in FIJI • Manual_tara: manual counts by Tara • Manual_Becky: Manual counts by Becky • Fiji_findmaxima: Automated sperm counts estimated using find maxima plugin • Fiji_findmaxima_sharpen: automated find maxima counts on sharpened images • Manual_KS_pixelated_czi: manual counts done by KS Sheet 3: • Image_ID: image ID • Find_maxima (5000 prominence): estimated sperm counts automated using find maxima plugin • Manual: sperm counts done manually Sheet 2: Notes File: stripping.csv Description: Offspring produced by females for experiments Variables • Row: Row ID • Rep: Block • Block: Block within experiment • Age: Male age • SF: Sot experiment C (sot) or dah experiment A (No) • Treatment: Overall treatment • Female_number: Female rank in mating sequence • sot_duration: son of tudor male mating duration • lag: Lag between sot and dah mating in experiment C • Latency: dah male mating latency • Duration: dah male copulation duration • Total_matings: Mating success • Male_ID: Male ID • Female_ID: Female ID • F1_counts: Numbers of offspring produced • Fertility: Fertile or not • F_num.adjusted: Mating success of male • Prev_current_fert: Whether previous mating was fertile or not (not used in analysis) • 0_1: Whether previous meting was infertile but next mating was fertile (not relevant) File: stripping_simple.Rmd Description: RMD file for analysis of data File: survival_population.csv Description: Survival of males from three lines used Variables • ID: Male ID • Born: Male birth date • Died: Death date • Censor: Whether death was observed or not • Line: sot, dah or gfp line used • Lifespan: Lifespan of male File: stripping-simple.html Description: HTML R markdown file of analysis ## Code/software R studio, R v4 Access information Other publicly accessible locations of the data: • [https://osf.io/5z7m3/](https://osf.io/5z7m3/) Data was derived from the following sources: • Open science framework