Authors:
Melissah Rowe¹*, Daniel M. Hooper², Antje Hofgaard³, Laura L. Hurley⁴, Callum S. McDiarmid⁴, Ioanna Pataraia¹, Jan T. Lifjeld⁵, and Simon C. Griffith⁴

1 Department of Animal Ecology, Netherlands Institute of Ecology (NIOO‐KNAW), 6700 AB, Wageningen, The Netherlands.
2 Institute for Comparative Genomics and Richard Gilder Graduate School, American Museum of Natural History, New York, NY 10024, USA.
3 Electron Microscopical Unit for Biological Sciences, Department of Molecular Biosciences, University of Oslo, 0316 Oslo, Norway.
4 School of Natural Sciences, Macquarie University, Sydney, NSW 2109, Australia.
5 Natural History Museum, University of Oslo, 0318 Oslo, Norway.

• Author for correspondence: m.rowe@nioo.knaw.nl

In this study, we gather raw data on sperm size to investigate sperm morphology (sperm length) in the Australian Estrildidae grassfinch, the red-browed finch (Neochmia temporalis temporalis) and Lesser red-browed finch (Neochmia temporalis minor). Our results show that the red-browed finch has highly atypical sperm morphology.

Access this dataset on Dryad

Description of the data and file structure

Sperm size data

Sperm size data were obtained using using SpermSizer v1.6.6 (McDiarmid et al. 2021).

SpermSizeData.csv - data on sperm size for the Red-browed finch and Lesser red-browed finch.
Dataset includes the following variables:

• Species = species common name
• Male ID = a unique identifier for each male bird
• Sampling location = location of the sampled indiviual, includes town, state, and country information.
• wild/captive = whether the bird was wild or captive (captive born)
• Sampling date = date of sampling in forat DD/MM/YYYY
• Sperm head length = average length of the sperm head in µm
• Sperm midpiece length = average length of the sperm midpiece in µm
• Sperm flagellum length = average length of the sperm flagellum in µm
• Total sperm length = average length of the entire sperm cell in µm
• Standard deviation of total sperm length = the standard deviation of length measure in µm
• Number of sperm cells measured = total number of unique sperm cells measured to obtain sperm size data

For the red-browed finch, we obtained measures for the length of the sperm head (including the acrosome, nucleus, and midpiece, as it was not possible to distinguish the midpiece using brightfield microscopy) and flagellum. In these cases, midpiece length is represented in the data as NAs. For the lesser red-browed finch, we obtained measures for the length of the sperm head (acrosome and nucleus), midpiece length, and flagellum length. For both species, total sperm length was calculated as the sum of head and flagellum lengths.

Sperm swimming speed data (folder)

Sperm swimming speed data were collected from captive male red-browed finches (maintained at the Netherlands Institute of Ecology, NIOO-KNAW) by computer-assisted-sperm-analysis (CASA) using the Sperm Class Analyzer ® (SCA, Microptic, Barcelona). All buffers and equipment were preheated to 40°C (avian body temperature). Sperm recordings were captured at 50 frames per second using negative phase contrast microscopy at 100x magnification with a Basler acA1300-200uc camera connected to a Nikon E200 microscope. All recordings were completed within 1-minute of sample loading. All recordings were later visually examined, and any tracking or detection errors (e.g. detection of cell debris) were deleted manually. We considered sperm immotile when with VCL < 20.0 μm/s (based on analysis of dead sperm videoed using the same capture settings to identify the kinematic profile of drifting sperm). From these analyses, we obtained data on curvilinear velocity (VCL), straight-line velocity (VSL), average path velocity (VAP) of the sperm cells.

SpermSwimmingSpeedData.zip - folder containing sperm swimming speed data for 6 male red-browed finches provided as individual .csv files. Each file includes the following variables:

• Field = Field of view during CASA analysis
• Sperm number = individual ID for each cell identified by CASA.
• Head X = X coordinate for identified cell
• Head Y = Y coordinate for identified cell
• First Frames = frame number, between 0 - 50, identifies which frame the cell is identified in
• Area = size in pixels of identified cell
• Elongation = sperm elongation (Length-Width)/(Length+Width)
• VCL = curvilinear velocity (m/s). Time-averaged velocity of a sperm head along its actual curvilinear path, as perceived in two dimensions in the microscope.
• VSL = straight-line (rectilinear) velocity (µm/s). Time-averaged velocity of a sperm head along the straight line between its first detected position and its last.
• VAP = average path velocity (µm/s). Time-averaged velocity of a sperm head along its average path. This path is computed by smoothing the curvilinear trajectory according to algorithms in the SCA Motility module.
• STR = straightness. Linearity of the average path, VSL/VAP.
• LIN = linearity. The linearity of a curvilinear path, VSL/VCL.
• WOB = wobble. A measure of oscillation of the actual path about the average path, VAP/VCL.
• ALH = amplitude of lateral head displacement (µm). Magnitude of lateral displacement of a sperm head about its average path.
• BCF = beat-cross frequency (Hz). The average rate at which the curvilinear path crosses the average path.
• Velocity = Classification of velocity into cateogries static, slow, medium, and rapid. static = < 20 µm/s, slow = 20-50 µm/s, medium = 51-100 µm/s, rapid = > 100 µm/s.
• Progression = Classification of sperm motility as 'motile' or 'progressive' based on default settings. Sperm is classified as progressive when motile AND when STR > 70.
• IsHyperactive = Classification as hyperactive 'True' or 'False'. Based on default parameters on CASA. Not considered in avian sperm motility analysis.
• IsMucusPenetration = Classification as Mucus Penetration 'True' or 'False'. Based on default parameters on CASA. Not considered in avian sperm motility analysis.
• IsCircular = Classification as circular track 'True' or 'False'. Based on default parameters on CASA. Not considered in avian sperm motility analysis.
• IsRoundCell = Classification as Round Cell 'True' or 'False'. Based on default parameters on CASA. Not considered in avian sperm motility analysis.
• IsValid = Classification as valid track 'True' or 'False'. Based on default parameters on CASA. Not considered in avian sperm motility analysis.
• IsSperm = Classification as sperm cell 'True' or 'False'. Based on default parameters on CASA. Not considered in avian sperm motility analysis.

SEM images

Scanning electron microscopy (SEM) was used to image sperm cells from the red-browed finch and the lesser red-browed finch, as well as the Crimson finch and Double-barred finch. Sperm were adhered to a glass coverslip, dehydrated using a grade ethanol series and critical point dried. Coverslips were sputter-coated with 4-6 nm platinum using a Cressington 308R coating system, and sperm cells imaged using a Hitachi S-4800 Field Emission Scanning Electron Microscope operated at 5.0 kV.

SEM_images_for_other_finches.zip contains SEM images of sperm from the Lesser red-browed finch, Crimson finch, and Double-barred finch. Includes images presented in Rowe et al. and additional images obtaind during the study.
SEM_images_for_Red-browed_finch.zip contains SEM images of sperm from the Red-browed finch. Includes images presented in Rowe et al. and additional images obtaind during the study.

TEM images

Transmision electron microscopy (TEM) was used to image sperm cells from the red-browed finch. Fixed sperm samples were washed 3x in 1x PHEM, postfixed with 1% OsO4 + ferricyanide followed by 3 washes in 1x PHEM. Samples were then stained with 1% UA and finally washed another 3x with 1xPHEM. Samples were then dehydrated in a graded ethanol series at room temp followed by infiltration with a EPON (without DMP30): ETOH 1:1, rotating over 2 days at room temperature. Subsequently, samples were further infiltrated with 100% EPON (with 1.5% DMP-30) and incubated overnight (rotating) at room temperature. Following infiltration, samples were placed in moulds and polymerized over two days at 60°C. Finally, ultrathin sections (80 nm) were obtained with a Leica ultramicrotome equipped with a diatome diamond knife and observed using a JEOL 1400plus transmission electron microscope operated at 120 kV.

TEM_images_for_Red-browed_finch.zip contains TEM images of sperm cells from red-browed finch. Includes images presented in Rowe et al. and additional images obtained during the study.

Testis mass and Body mass data

Body mass (grams) was measured using an electronic balance (+/- 0.01 mg). The left testis was snap frozen immediately after collection, and we later thawed the left testis and measured testis length and width (+/- 0.01 mm) using digital callipers. From these dimensions we calculated the mass of the left testis, estimated combined testes mass by doubling this value, and estimated relative testes mass for each male (i.e., as testes mass/body mass x 100).

Red-browed_finch_testis_and_body_mass_data.csv contains testis mass and body mass data for the five males examined in this study. Of these five males, only four were reproductively active at the time of sampling, and thus only these four males were used in our estimation of average relative testes mass.

Phylogenetic tree

RAxML_bipartitions.240612.wg.raxml.10k_thin.tre phylogentic tree file used in the analyis in Rowe et al.

Digital images of sperm

Digital images of sperm from the red-browed finch and lesser red-browed finch captured using brightfield microscopy. Images were captured using various camera and microscope systems. Raw images are provided for all males sampled during the study; However, not all sperm cells imaged could be measured to obtain data on sperm length.

LesserRedBrowedFinchSperm.zip compressed file containing raw images of sperm cells from the lesser red-browed finch.
RedBrowedFinchSperm.zip compressed file containing raw images of sperm cells from the red-browed finch.

Code/Software

For the red-browed finch, we tested for differences in sperm size (sperm head length, flagellum length, and total sperm length) among the different populations sampled using an ANOVA (see below) using R v4.4.4.1 (R Core Team 20204) and R studio (Posit Team 2023).

Differences among populations?
m1<-aov(DATA$SpermTotalLengthDATA$Population)
summary(m1)
m2<-aov(DATA$SpermFlagellumLengthDATA$Population)
summary(m2)
m3<-aov(DATA$SpermHeadLength~DATA$Population)
summary(m3)

R Core Team. 2024. R: A Language and Environment for Statistical Computing. R Foundation for Statistical Computing, Vienna, Austria.
Posit team. 2023. RStudio: Integrated Development for R. Posit Software, PBC, Boston, MA.

Version changes

2-July-2025: added additional raw data associated with the project.
Added SpermSwimmingSpeedData.zip containing folder containing sperm swimming speed data for 6 male red-browed finches provided as individual .csv files.
Added SEM images for other finches and SEM images for Red-browed finches folders containing raw SEM images, including files included in Rowe et al. and additional images obtained during the study.
Added TEM images for Red-browed finch folder containing TEM images of sperm cells from red-browed finch. Includes images presented in Rowe et al. and additional images obtaind during the study
Added Red-browed finch testis and body mass data.csv
Added RAxML bipartitions.240612.wg.raxml.10k_thin.tre phylogentic tree file used in the analysis in Rowe et al.
Added LesserRedBrowedFinchSperm.zip and RedBrowedFinchSperm.zip containing raw digital images of sperm.