# A simulated marine heatwave impacts European sea bass sperm quantity, but not quality

Benjamin Geffroy, Leydy Sandoval-Vargas, Myriam Boyer-Clavel, Maritza Pérez-Atehortúa, Stephane Lallement, Iván Valdebenito Isler

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Benjamin Geffroy, Leydy Sandoval-Vargas, Myriam Boyer-Clavel, Maritza Pérez-Atehortúa, Stephane Lallement, et al.. A simulated marine heatwave impacts European sea bass sperm quantity, but not quality. Journal of Fish Biology, 2023, 10.1111/jfb.15327. hal-03974878

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# A simulated marine heatwave impacts European sea bass sperm quantity, but not quality

Benjamin Geffroy<fnref n="1" /> | Leydy Sandoval-Vargas<fnref n="2" /> | Myriam Boyer-Clavel<fnref n="3" /> | Maritza Pérez-Atehortúa<fnref n="2" /> | Stephane Lallement<fnref n="1" /> | Iván Valdebenito Isler<fnref n="2" />

**Correspondence**

Benjamin Geffroy, MARBEC University of Montpellier, CNRS, Ifremer, IRD, Palavas-Les-Flots, France.

Email: benjamin.geffroy@ifremer.fr

**Funding information**

Climat AmSud, Grant/Award Number: 21-CLIMAT-14

**Abstract**

Rapid environmental changes will be the major challenge that most biota will have to deal with in the near future. Extreme events, such as marine heatwaves, are becoming more frequent and could be spatially uniform at a regional scale for a relatively long period of time. To date, most research studies on heatwaves have focused on sessile organisms, but these extreme events can also impact mobile species. Here, a 3-week marine heatwave was simulated to investigate its effects on the male reproductive performance of a Mediterranean Sea emblematic species, the European sea bass *Dicentrarchus labrax*. Males from the control condition (c. 13°C) produced significantly more sperm than those exposed to a relatively warm thermal treatment (c. 16°C). Nonetheless, neither the percentage of motile spermatozoa nor most of the other sperm motility parameters were significantly affected by the rearing temperature over the whole period. Overall, the results of this study suggest only moderated effects of a potential winter heatwave on the reproductive performance of male European sea bass.

**KEYWORDS**

global warming, Mediterranean Sea, reproduction, sperm quantity

## 1 | INTRODUCTION

According to the Intergovernmental Panel on Climate Change technical summary (Arias et al., 2021), the global mean of sea surface temperature (SST) has increased by 0.88°C since the beginning of the 20th century. One might consider that this increase would only have minor effects on fish, and would argue that species would have sufficient time to adapt. In reality, this relatively slow increase of the mean hides drastic effects linked to the intensity of marine heatwaves that could last for several days. Indeed, surface ocean marine heatwave days (defined as days exceeding the 99th percentile in SST relative to 1995–2014) are expected to increase by a factor 5–10 by 2100 (Arias et al., 2021). Nowadays, the effect of global warming is already preoccupying, and it affects various functions related to fish physiology (McKenzie et al., 2021), such as gametogenesis and the capacities of fish to successfully reproduce (Alix et al., 2020).

Yet, most of the work has been conducted on females, whereas less is known regarding males (Alix et al., 2020). In addition, the few studies investigating the effects of warming on milt quality and quantity have been performed in freshwater fishes with contrasting results (Alix et al., 2020). For instance, Ashton et al. (2019) detected that milt quantity decreased in the Burbot, *Lota lota* (L. 1758) as the temperature increased (from 2 to 6°C during 21 days), though they did not quantify to what extent. Sperm quantity decreased by a factor of 2 in the common bream, *Abramis brama* (L. 1758), when exposed to +3°C for 10 days (Targońska et al., 2014). On the contrary, *Gambusia holbrooki* males (Girard, 1859) exposed to high temperature (30°C) produced about three times more sperm than those kept in colder water (18°C) (Adriaenssens et al., 2012). Regarding sperm characteristics, sperm motility of the European grayling, *Thymallus thymallus* (L. 1758) decreased with temperature (Lahnsteiner & Kletzl, 2012). Interestingly, only 1°C of difference was sufficient to trigger the change in

<footnote n="1">¹MARBEC University of Montpellier, CNRS, Ifremer, IRD, Palavas-Les-Flots, France</footnote>

<footnote n="2">²Nucleus of Research in Food Production, Faculty of Natural Resources, Catholic University of Temuco, Temuco, Chile</footnote>

<footnote n="3">³Montpellier Ressources Imagerie, Biocampus, University of Montpellier, CNRS, Montpellier, France</footnote>
<figure><img src="image_2.png" /><figcaption>**FIGURE 1** Temperatures experienced by adult males *Dicentrarchus labrax* from 17 February 2022 to 11 March 2022. Natural temperature is indicated in blue, and relatively warm temperature is indicated in red. Natural temperature, Warm temperature</figcaption></figure>

motility (Lahnsteiner & Kletzl, 2012), whereas an increase of temperature of 5°C did not change the motility in the sperm of brown trout *Salmo trutta* (L. 1758) (Lahnsteiner & Leitner, 2013).

This study aimed to investigate the effect of high temperature exposure within the natural spawning period (i.e., between January and March, Abascal et al., 2007; Fauvel et al., 1999) of a marine emblematic fish species for the Mediterranean Sea: the European sea bass *Dicentrarchus labrax* (L. 1758). Specifically, the authors tested the effects of an exposure to +3°C over 3 weeks on (a) milt volume production, (b) sperm motility parameters and (c) survival of spermatozoa.

## 2 | MATERIALS AND METHODS

The study was conducted in February and March 2022 in the Aquaculture Experimental Station Ifremer, Chemin de Maguelone, France. A total of 21 three-year-old males *Dicentrarchus labrax* were randomly divided into two groups in fibreglass tanks (5 m³) with a continuous supply of sea water (300 l h⁻¹). One group was kept at natural water temperature (around 13°C), and the other one was kept at c. +3°C for 3 weeks (Figure 1), thanks to a thermostat (Aquavie ICE 3000, Connaux, France). From 11 February 2022, the temperature was gradually increased at a rate of ±0.5°C day⁻¹ during 6 days in the warm condition to reach the desired temperature at the start of the experiment: 17 February 2022 = Day 0. Then, the temperature was manually adjusted each day to follow the natural decrease of temperature observed over time. Temperature was automatically recorded in each tank every 30 min (thanks to a HOBO temperature data logger) during the 23 days of experiment. There were no significant differences in fish weight based on the treatment (natural temperature: 3.5 ± 0.25 kg vs. warm: 3.8 ± 0.19 kg; t-test P-value = 0.3). Fish were fed daily with commercial diet containing 51% protein, 16% fat and 1% fibre. Sperm samples were collected on different

fish after 16 and 23 days of rearing at the two different temperatures: natural (n = 6 at 16 days and n = 4 at 23 days) and warm (n = 6 at 16 days and n = 5 at 23 days).

Milt samples were collected by manual stripping, taking care of avoiding urine contamination. The ejaculate volume of each male was measured directly during the milt collection using 1 or 5 ml syringes. Each syringe was immediately placed at 4°C until being processed in less than 1 h. To assess sperm concentration, milt samples were diluted at a ratio of 1:2000 (1:20 and 1:100) with formaldehyde-buffered saline. Then, the spermatozoa were counted using a Thoma haemocytometer under an optical microscope at ×400 magnification. The results are expressed as spermatozoa × 10⁹ ml⁻¹. Sperm motility was assessed using a CASA (Computer-Assisted Sperm Analysis) system (IVOS II, Hamilton Thorne, Beverly, MA, USA). Milt samples (2 µl) were activated with 1000 µl of seawater solution containing bovine serum albumin 2%, to prevent the sperm sticking to the slide (Pérez *et al.*, 2016). Immediately after sperm activation, 3 µl of the sperm subsample was loaded onto a standard four-chamber slide (Leja Products B.V., Nieuw Vennep, The Netherlands). This was done readily because the duration of sperm motility is particularly low in the European sea bass, tipically between 40 and 50 s (Abascal *et al.*, 2007; Fauvel *et al.*, 1999). The following motility parameters were recorded before 10 s: percentage of total motile sperm, curvilinear velocity (VCL, µm s⁻¹), straight-line velocity (VSL, µm s⁻¹), average path velocity (VAP, µm s⁻¹), linearity (LIN, %), progression (PROG, %), amplitude of lateral head (ALH, µm) displacement, straightness index (STR, %) and wobble (WOB, %). Measurements of sperm kinetic were carried out in triplicate on each of the 21 individuals. In addition, a viability test was carried out using the fluorescence LIVE/DEAD Sperm Viability Kit (Molecular Probes, Invitrogen, Eugene, OR, USA) and flow cytometry on the fish producing sperm after 23 days (11 March). A milt sample (0.5 µl) was incubated with 0.75 µl of SYBR-14 and 0.75 µl of propidium iodide at 10°C in the dark for 15 min. Thereafter, samples were centrifuged at 160 g for 5 min, suspended in phosphate-buffered saline and analysed with a FACS Cantoll flow cytometer (BD Bioscience, Franklin Lakes, NJ, USA). Forward and side scatter areas were used to select the cells of interest. Then forward scatter area and forward scatter time-of-flight were crossed to remove doublets allowing an analysis on singulet cells. To consider these repeated measurements on the same individual, all data were analysed with linear mixed-effects models, with individuals as random variables and both treatment (warm or natural) and time (16 or 23 days) as fixed factors. All statistical analyses were performed with R (R Core Team, 2022), and mixed-effects models were performed using the lme4 package (Bates *et al.*, 2015). Plots were done with ggplot2 (Wickham, 2016) and data were given as mean ± S.E.

## 2.1 | Compliance with ethical standards

All fish were handled in accordance with the Universities Federation for Animal Welfare Use and Care Committee Handbook on the Care and Management of Laboratory Animals (http://www.ufaw.org.uk/pubs.htm#Lab) and following the guidelines for animal
Ejaculate Volume (ml)

<figure><img src="image_3.png" /><figcaption>**FIGURE 2** Ejaculate volume of adult males European sea bass *Dicentrarchus labrax* collected after 16 and 23 days (16d and 23d) and from the two thermal treatments (natural and warm). Each grey point represents the volume sampled on each individual, whereas the blue and the red points represent the mean ± S.E. of the natural and the warm treatments at each time, respectively. \* indicates a significant difference between treatments where *P* < 0.05</figcaption></figure>

experimentation established by Directive 2010-63-EU of the
European Union and the corresponding French legislation.

3 | RESULTS

The water temperature of both treatments is shown in Figure 1. Usu-
ally, the mean temperature experienced by European sea bass during
the winter is considered to be 13°C in the same geographical zone
(Abascal et al., 2007; Fauvel et al., 1999). Here, the monitoring of the
fish from 1 January 2022 to 11 March 2022 was in agreement with
those previous findings, with a mean natural temperature of 13.1
± 0.1°C. Overall, fish kept in warm temperatures produced signifi-
cantly (P < 0.05) less volume of sperm when compared to those kept
in natural temperatures (Figure 2). Regarding sperm concentration, no
significant differences were found among thermal treatments or
between experimental days. The percentage of motile spermatozoa
was not significantly affected by the rearing temperature or experi-
mental days (Figure 3). For fish kept in warm temperatures, the mean
values of spermatozoa velocities (VCL, VSL and VAP) were not signifi-
cantly different from those kept in controlled condition. The thermal
treatment and the experimental time did not affect the indices of
sperm motility (LIN, PROG and WOB). Only the ALH was significantly
lower in sperm samples of fish kept in warm than those in natural
temperatures (Figure 3). The STR variable (VSL/VAP) was significantly
higher for warm than control fish (Figure 3). Because only two fish
produced sperm after 23 days at warm temperature, the authors only
compared sperm viability to two others randomly chosen fish kept at

natural temperature. The mean values of sperm viability were close to
65%, with no significant differences between the treatments.

4 DISCUSSION

The ongoing climate crisis is causing several changes in environmental
conditions, including a well-described increase in water temperature.
The reproduction of aquatic organism occurs in a limited temperature
range (Farhadi & Harlioglu, 2018; Miranda *et al.*, 2013), and for this
reason, one might wonder about the potential effects of climate
change on gamete quality. For the European sea bass, the usual tem-
perature experienced in the winter is around 13°C in the western
Mediterranean zone, and this study shows that an increase of 3°C
over a period of 3 weeks reduced significantly the milt volume pro-
duced. In river lamprey *Lampetra fluviatilis* (L. 1758) exposed to three
different controlled thermal regimes (7, 10 and 14°C) during
6 months, only 30% of males produced milt when temperatures were
increased to 10 and 14°C (Cejko *et al.*, 2016). Nevertheless, there
were no significant differences on the average milt volume, sperm
concentration and total sperm production between the three regimes
(Cejko *et al.*, 2016). It is likely that the observed decrease in milt
volume in the present experiment would involve alterations in the
endocrine regulation of the hypothalamus-pituitary-gonadal axis. High
water temperatures for *Cyprinodon variegatus* (Lacepède, 1803)
showed an increase, in males and females, of gene transcripts encoding
gonadotropin-inhibiting hormone (gnih) and gonadotropin-releasing
hormone-3 (gnrh3), as well as decreased beta-subunits of follicle-
stimulating hormone (fshβ) and LH hormone (lhβ) only in males (Bock
et al., 2021). In *D. labrax*, GnIH was shown to decrease the plasma
levels of luteinizing hormone LH (Paullada-Salmero *et al.*, 2016). LH
increases the production of testicular steroids (11-ketotestosterone,
KT-11 or 17α, 20β-dihydroxy-4-pregnen-3-one, DHP), which together
are clearly involved in the regulation of spermiation in fish. Nonethe-
less, the mechanisms of action of these hormones on milt hydration
and sperm migration to the sperm duct or increase in milt volume are
still unclear (Schulz *et al.*, 2010).

Regarding sperm concentration, there were no significant differ-
ences between temperatures or experimental periods. It should be
noted that the sperm concentration recorded in this study was lower
than that reported by Fauvel et al. (1999) in the same species. Those
authors recorded a pic (60 × 10⁹ spermatozoa/mL) in February, which
then decreased with the advancement of the reproductive period
(Fauvel et al., 1999). Because this study was performed in March, it is
likely that the lower concentration observed here was only linked to
the time-point of sperm collection. Nevertheless, it is important to
pinpoint that the sperm concentration of this species shows a great
variation between studies (5.5 to 144 × 10⁹ spermatozoa ml⁻¹)
(Sorbera et al., 1996; Rainis et al., 2003). Here, the values obtained are
higher than those reported by Asturiano et al. (2001) and similar to
those obtained by Felip et al. (2006).

To date, the effects of temperature changes on sperm motility
parameters are scarce in fish species. In this respect, Cejko *et al.*
<figure><img src="image_4.png" /></figure>

**FIGURE 3** Measurements of sperm characteristics using the CASA (Computer-Assisted Sperm Analysis) system (IVOS II, Hamilton Thorne, Beverly, MA, USA). Percentage of total motile sperm, curvilinear velocity (VCL, µm s⁻¹), straight-line velocity (VSL, µm s⁻¹), average path velocity (VAP, µm s⁻¹), linearity (LIN, %), progression (PROG, %), amplitude of lateral head (ALH, µm) displacement, straightness index (STR, %) and wobble (WOB, %), as well as sperm concentration, are represented. Each ejaculate was collected after 16 and 23 days (16d and 23d) and from the two thermal treatments (natural and warm). Each grey point represents the value for one individual, whereas blue and red points represent the mean ± S.E. of the natural and the warm treatments at each time, respectively. \* indicates a significant difference between treatments where P < 0.05

(2016) detected higher sperm performance [motility, progressive motility (PM) and velocities] in river lamprey maintained at 7°C in comparison to that maintained at 14°C. On the contrary, in *Gambusia holbrooki*, the VSL was significantly higher when the fish were reared at 30°C compared to 18°C. In this study, no significant differences were found for the most important kinetic parameters. Regarding motility, the spermatozoa of the warm treatment presented a mean value that tended to be below that of the natural

treatment, and both were lower than those reported by Abascal et al. (2007) in the same species, where fresh milt presented 86.67% motility and sperm velocity values similar to those of this study. Mitochondria is important for the activation and sustainability of motility and progressive movement of spermatozoa due to the production of ATP that can be altered using oxidative stress leading to increased reactive oxygen species (ROS) (Figueroa et al., 2017; Sandoval-Vargas et al., 2021). ROS has been reported to affect the
spermatozoa, especially due to the presence of a double lipid membrane. The rise in oxidative stress in aquatic species is increased by environmental factors, such as temperature changes, oxygen levels, salinity, pesticides, among others, and because sperm have a low content of cytoplasm, it is the seminal plasma that provides the main defences against ROS (Cabrita et al., 2014). Therefore, the low level of seminal plasma in the spermatozoa from the warm treatment probably activated the mitochondrial function before obtaining the samples due to oxidative stress. This explains why the STR index is higher, because the spermatozoa probably managed to go in a straight line with a higher speed at the beginning, but not constantly. This could also explain why they have presented a lower ALH. Confidently, these spermatozoa presented their highest peak of ATP immediately when activated and did not experience a stop of PM, which was rather almost immediate. Several kinetic parameters such as total motility, PM, VCL, VSL VAP, LIN and WOB had been proposed as good indicators of fertility and hatching rates (Gallego et al., 2013). Changes in the pattern of STR and ALH parameters have not been significantly related to the sperm function so far.

Regarding sperm viability, there were no significant differences when the fish were reared 3°C above the natural temperature. This might be due to the fact that the maximum temperature evaluated in this study was not above the maximum critical temperature for the species. Sperm membrane damages are usually related to more stressful factors such as cryopreservation or oxidative stress (Sandoval-Vargas et al., 2021).

It is evident that warmer-than-optimal temperatures may alter the reproductive capacity in some fish species. In this study, males' *D. labrax* kept at 16°C showed significantly lesser milt volume and lower values of ALH than those males reared at 13°C, whereas the STR variable was significantly higher in the warm temperature. It is likely that the difference in the range of temperature evaluated was not enough to lead to drastic impairment of the sperm quality. It is important to stress that the best indicators of sperm quality are not necessarily the motility nor the ability to fertilize an oocyte, but it also relies on the capacity to contribute in the obtention of a high percentage of live and hatched embryos (*Pérez-Atehortúa* et al., 2022). Therefore, future studies should also include this parameter to ensure that sperm quality is not affected by such an increase in temperature. Anticipating potential effects of warming at long term is of vital importance, and more studies are needed to really capture what will happen regarding fish reproduction in this context of global warming.

## AUTHOR CONTRIBUTIONS

Benjamin Geffroy: data generation, data analysis, manuscript preparation, funding. Leydy Sandoval-Vargas: data generation, data analysis, manuscript preparation. Myriam Boyer-Clavel: data generation. Maritza Pérez-Atehortúa: manuscript preparation. Stephane Lallement: data generation. Iván Valdebenito Isler: data generation, data analysis, manuscript preparation, funding.

## ACKNOWLEDGEMENTS

The study was supported by the Climat AmSud fund (WARMFISH project - grant no. 21-CLIMAT-14). We thank the "Palavas Team" for taking care of the fish and Alain Vergnet for providing the HOBO temperature data logger.

## CONFLICT OF INTEREST

The authors declare that they have no conflict of interest.

## ORCID

Benjamin Geffroy https://orcid.org/0000-0001-6120-1103

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