DOI: 10.5061/dryad.z8w9ghxfm
title: Frequency-dependent viscosity of salmon ovarian fluid has biophysical implications for sperm-egg interactions

description: Gamete-level sexual selection of externally fertilising species is usually
 achieved by modifying sperm behaviour with mechanisms thought to alter the
 chemical environment in which gametes perform. In fish this can be
 accomplished through the ovarian fluid, a substance released with the eggs
 at spawning. While its biochemical effects in relation to sperm energetics
 have been investigated, the influence of the physical environment in which
 sperm compete remains poorly explored. Our objective was therefore to gain
 insights on the physical structure of this fluid and potential impacts on
 reproduction. Using soft-matter physics approaches of steady-state and
 oscillatory viscosity measurements, we subjected salmon ovarian fluids to
 variable shear stresses and frequencies resembling those exerted by sperm
 swimming through the fluid near eggs. We show that this fluid, which in
 its relaxed state is a gel-like substance, displays a non-Newtonian
 viscoelastic and shear-thinning profile, where the viscosity decreases
 with increasing shear rates. We concurrently find that this fluid obeys
 the Cox-Merz rule below 7.6 Hz and infringes it above, thus indicating a
 shear-thickening phase where viscosity increases provided it is probed
 gently enough. This suggests the presence of a unique frequency-dependant
 structural network with relevant implications on sperm energetics and
 fertilisation dynamics.
description: Sample collection and preliminary measurements
 Wild anadromous Atlantic salmon were collected in early September
 from a fish ladder at Grand Falls (48° 55' N, -55° 39' W) during
 their up-stream spawning migration on the Exploits River (Newfoundland,
 Canada). Following previous protocols (Rooke et al., 2019), fish were
 transferred to covered, outdoor tanks next to the river, and experienced
 ambient temperatures and light. Over two weeks in early November, females
 were assessed for ovulation using gentle abdominal pressure, fish were
 then anaesthetised using a solution of 2ml/L clove oil, measured for
 length and weight, and stripped of eggs after drying the urogenital pore.
 Each female’s eggs (and associated ovarian fluid) were kept in sealed
 glass jars, enclosed with bubble wrap, and placed in a cooler of wet ice
 for transport to the laboratory. Each egg batch was separated from its
 ovarian fluid using a fine mesh net (Purchase & Rooke, 2020)
 within 10 hours of stripping. For each ovarian fluid we recorded volume
 and weight to deduce density, followed by pH and conductivity.
 Rheological characterization of ovarian fluid
 The mechanical properties of many soft biological materials are
 neither purely viscous (liquid-like) nor purely elastic (solid-like), and
 these rheological properties correlate strongly with their function (Storm
 et al., 2005). Structured fluids often do not flow until they reach a
 critical stress level, below which a material is considerable elastic, and
 above which the structure of the material breaks down and starts to flow.
 Two experiments were performed to define how the ovarian fluid’s polymeric
 structure (and related physical properties that in turn would affect sperm
 swimming activity) can be modulated, depending on swimming sperm flagellar
 beat frequency. Specifically, we tested ovarian fluid ‘behaviour’, both
 under steady shear (i.e., “flow curves”) and under small-amplitude
 oscillatory shear (SAOS). The former examines the viscoelastic response of
 the ovarian fluid by continuous deformation and breakup of internal
 networks, while the latter can probe weaker internal structures (Bird et
 al., 2021; Markovitz, 1981). A preliminary rheological analysis (n= 5
 fish) was conducted to assess different fluid preservation methods (see
 supplementary material). Each frozen sample was thawed at room temperature
 for 1hr prior to analysis, and measurements were made using 1.5 mL
 aliquots. All the analyses were performed in the Soft Matter Lab at
 Memorial University using an MCR 301 rheometer, equipped with a cone-plate
 (CP50-0.5, 50 mm diameter plate and cone angle, Anton Paar GmbH, St.
 Albans, UK) system. Ovarian fluid samples were individually filtered
 through a 200 mm sieve to remove any particulates (e.g., coagulated blood,
 ovarian tissue) that could influence the rheological measurements.
 Pipetted fluid was equilibrated for three minutes at the plate temperature
 of 6oC, allowing for homogenous sample relaxation from any uncontrolled
 pre-shear imposed on the fluid during loading. 
 Steady-state shear properties Samples were
 tested for their resistance to flow in order to measure their viscosity
 under a specific rate of deformation. To obtain a flow curve, the shear
 stress was measured for a range of shear rates (), from 10 to 500 s-1 in 50
 equally spaced steps. The resultant shear stresses of the ovarian fluid
 were measured to determine the apparent viscosity ηa, which was averaged
 across three aliquots per female (n= 11) and plotted as a function of the
 shear rate. Among each of the three ovarian fluid
 aliquots per fish, a run with distilled water was performed as a control.
 For distilled water (pure Newtonian fluid), a theoretical positive
 relationship between shear stress and shear rate should be linear and the
 fit line should pass through zero. When the profiles of water runs were
 fitted, a positive intercept (typical for these kind of measurements) of
 0.0133 Pa was concluded to be low-shear rate instrumental noise. It
 subtracted from all the water and ovarian fluid samples as standardization
 (shear stress - 0.0133 Pa)/(shear rate), creating a small change in
 values. A comparison of individual ovarian fluid viscosity profiles with
 distilled water for each of the instrumental replicates allowed us to
 assess variability among females. The apparent
 viscosity of ovarian fluid decreased with increasing shear rates, in
 contrast with water whose apparent viscosity (ηa= 0.00151 ± 0.00003 Pa·s)
 was independent of shear rate. The apparent viscosity at s−1 was roughly
 10 times the viscosity of water but returned comparable at  s−1 (see Results). For
 three females the ovarian fluid samples had apparent viscosities ηa in the
 order of 0.003 Pa·s at 10 s−1, showing no meaningful differences with the
 rheological behaviour of water at the same shear rate. Likely, these
 samples were contaminated by urine and/or water during stripping of
 gametes and for these reasons were not included in the main results. The
 remaining 11 flow curves were globally fitted to the form ηa, which is a
 simple equation incorporating an elastic component, the yield stress
 which must be
 overcome before there is flow, and a viscous component , which represents
 the viscosity at very high shear rates. This simple form was arrived at
 when fits to a more complicated formula, the Herschel-Bulkley equation
  (Herschel, 1926) resulted in power laws n that were very close to
 unity. Small Amplitude Oscillatory Sweeps
 To preserve finer polymeric structures and obtain a dynamic
 profile that informs about the viscous and elastic components, we
 subjected the ovarian fluid to small-amplitude oscillatory shear. For
 these measurements, a sinusoidal deformation (was imposed on the sample at
 a fixed frequency and a maximum amplitude (Schoff
 & Kamarchik, 2005). Measurements were
 performed for a range of frequencies (ω), from 0.01 to 500 rad· s−1 in 24
 equally spaced logarithmic increments. The storage modulus, and the loss
 modulus, were obtained as a function of frequency (ω). The modulus of the
 complex viscosity η* was obtained from the relation
 |h*| º [(G’)2 +
 (G’’)2]1/2/w,                                                                                                                             while the damping factor (or loss factor) tan d º G”/ G’ represents the ratio between viscous and elastic contributions to the viscoelasticity. Applicability of the Cox-Merz rule. The Cox-Merz rule, an empirical method to rationalize steady shear and oscillatory rheological data (Cox & Merz, 1958), was used to compare the two different rheological analyses adopted in our study. A strong correlation between two independent methodologies is a good consistency check. This rule states that the apparent viscosity (ηa = σ/) at a specific shear rate is equal to the complex viscosity at a specific oscillatory frequency (w). When the rule is obeyed, rheological properties of a fluid can be described by either oscillatory or steady-state shear experiments (“Engineering Properties of Foods,” 2014). Statistical analyses All ovarian fluid measurements and fish morphological data (mean ± SD, 95% CI and Coefficient of Variation (CV%)) were summarised using the descriptive statistics function in GraphPad Prism, version 8.0.0, (GraphPad Software, San Diego, California USA). Rheometer reads were first standardized for instrumental error and the model fits were applied as described above. Subsequently, the average values of G’ and G” (dependent variables) across all the sampled females were pair-wise compared trough t-tests at specific frequencies (independent variables) of interest within two shear stress ranges, 0.001 to 0.105 and 0.105 to 1 rad· s−1, to double-check their uniformity within the plateau region and/or alternatively the prevalence of either the viscous or the elastic component of the ovarian fluid in this dimensional range. Normality of the residuals was ensured through D’Agostino-Pearson test followed by Shapiro-Wilk test (P= 0.2174 and 0.4697, respectively). Throughout the analyses, the statistical significance threshold used was a= 0.05.
description: Microsoft Excel