# Frequency-dependent viscosity of salmon ovarian fluid has biophysical implications for sperm-egg interactions

## Data files

## Jun 27, 2022 version files 81.09 KB

## Abstract

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.

## Methods

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.

## Usage notes

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