Links between mitochondrial operate, whole-animal metabolic price, telomere dynamics and swimming efficiency in minnows

Experimental overview

This experiment was carried out beneath the jurisdiction of a UK Home Office venture licence (PP6899400). We selected to conduct this research on wild Eurasian minnows [Phoxinus phoxinus (Linnaeus 1758)], as a result of they adapt effectively to captive circumstances (authors’ private observations), are comparatively good swimmers and carry out effectively in swim tunnel protocols (Holthe et al., 2009; Rubio-Gracia et al., 2020). The wild minnows have been captured by dip-netting on 19 February 2020 within the River Endrick, Scotland (56°03′41″N, 4°22′28″W). A complete of 238 suitably sized minnows have been transferred to aquarium services on the University of Glasgow, the place they have been held in round tanks (circumference 283 cm, complete quantity 325 litres) and equipped with a relentless turnover of aerated, copper-free water that had undergone sediment filtration, de-chlorination and UV sterilisation. During this time, minnows have been fed each day advert libitum utilizing frozen bloodworm; initially being held at ∼11°C, however then step by step elevated to ∼14°C by the beginning of the experiment in August 2020. On 19 February 2020, all minnows have been anaesthetised (with a 25 mg l⁻¹ benzocaine answer) and measured for physique fork size (±0.1 mm), with the vast majority of people (n=126) falling inside a measurement class of 25–35 mm, which, in keeping with Frost (1943), would age them at roughly 1 yr previous at the moment. Therefore, we used people on this measurement class for our swim efficiency trials, so on 9 March 2020, 81 of those minnows (chosen at random from the 126 people) have been once more anaesthetised (25 mg l⁻¹ benzocaine answer) and have been tagged on their dorsal floor (within the space between the gill plate and the dorsal fin) utilizing Visible Implant Elastomer (VIE; Northwest Marine Technology Inc.), with every fish being given a novel four-tag mixture utilizing three colors in complete. The tagged minnows have been then held in the identical round tanks till the beginning of the experiment, which started on 18 August 2020 (the delay was brought on by the lockdown restrictions surrounding the Covid-19 pandemic). In complete, 54 of the VIE tagged minnows have been used on this experiment and have been randomly assigned to teams of three fish. Each minnow spent a complete of 29 days within the experiment; see Fig. 1 for a abstract of the experimental timeline. In teams of three, minnows have been examined twice for vital swimming velocity (U_crit) on day 1 after which 1 week afterward day 8. Fish have been then allowed to recuperate for ∼14 days earlier than their commonplace (SMR) and most metabolic charges (MMR) have been measured between days 21 and 23. Then after an additional week of relaxation, fish have been euthanised on day 29 and tissues have been sampled for mitochondrial assays, muscle histology and telomere measurement. When not present process trials to measure swimming velocity or metabolic price, fish have been held in mesh baskets (one basket per group of three fish) that have been suspended inside a single tank (100×100×40 cm) that was held at a relentless temperature of 14°C and have been fed advert libitum on frozen bloodworm. There was no mortality of the minnows all through the experiment.

Measurement of vital swimming velocity

To quantify extended swimming efficiency, every minnow was examined for U_crit on its experimental day 1 and once more on day 8. U_crit checks have been performed in a 90 l Steffensen-type swim tunnel (Loligo Systems, Tjele, Denmark) at 14°C. The working part of the tunnel measured 66×20×20 cm (size×top×width) and an exterior chiller was used to keep up the water temperature at 14°C (the identical as within the holding tanks). Fish have been examined in teams of six, with three fish present process their first trial (day 1) and three fish present process their second trial (their day 8). Testing the swimming efficiency of shoaling species individually versus in a gaggle can have an effect on their U_crit rating, partly due to the stress of being held individually (Remen et al., 2016; Zeng et al., 2024); due to this fact, three random non-experimental minnows from the inventory inhabitants have been used to make up numbers if solely three focal fish have been being examined, e.g. for the primary and final trials. Therefore, fish have been all the time examined in a gaggle of six, whereas the U_crit scores have been calculated for every fish individually.

Fish have been fed within the morning of the day previous to a trial, that means that they’d been fasted for ∼24 h. The group of six fish was added to the swim tunnel within the afternoon previous to the trial and allowed to acclimate in a single day at a sluggish water velocity of two.5 cm s⁻¹, which roughly corresponded to 0.5 physique lengths per second (BL s⁻¹), because the imply±s.e.m. fork size of the examined minnows was 5.18±0.60 cm. Minnows are naturally gregarious fish, and so the six fish shaped a college and oriented into the circulate. On the day of testing, room lighting was switched on a minimum of 1 h previous to a trial beginning (with every trial commencing at 10:30 h ±20 min). During the trial, water velocity was incrementally elevated by 2.5 cm s⁻¹ (i.e. 0.5 BL s⁻¹) each 5 min till every fish was exhausted to the purpose of now not with the ability to keep place inside the water column and thus ceasing swimming. These 5 min increments have been routinely utilized in different research on small fish (Seebacher et al., 2016; Frenette et al., 2019; Thambithurai et al., 2019). A fish was recognized as having reached this level if it fell sideways in opposition to the again grid of the swim tunnel and remained there for a minimum of 5 s. Trials have been noticed by two scorers by way of a slit in a sheet of opaque black materials, with one observer recording the time and water velocity (BL s⁻¹) at which the person had reached the purpose of exhaustion, whereas the opposite observer shortly eliminated every exhausted fish through a hatch on the rear of the swim tunnel and transferred them to a bucket of aerated water. This course of was repeated till all six of the minnows had reached the purpose of exhaustion. The fork size of every fish was then measured (±0.1 mm) – utilizing a small plastic bag full of water to softly restrain the fish – to calculate a particular BL s⁻¹ measurement for every particular person. The two separate measurements of fork size (on the 2 separate days that U_crit was measured) have been extremely correlated (Spearman’s rho=0.97, N=54, P<0.001), thus displaying that this was an acceptable methodology for acquiring fork size with out the necessity for anaesthesia. The six fish then remained within the bucket of aerated water (for not more than 15 min) to make sure that they’d resumed regular swimming behaviour previous to being returned to their respective mesh holding basket.

Critical swimming velocity was then calculated as per Dalziel et al. (2012) utilizing the next method: U_crit=U_i+(U_ii×t_i/t_ii), the place U_i is the best velocity (BL s⁻¹) at which a person was capable of swim for a full 5 min increment, U_ii is the incremental velocity enhance (0.5 BL s⁻¹), t_i is the period of time {that a} fish was capable of swim at its closing velocity previous to reaching exhaustion (min), and t_ii is the period of time spent at every velocity (i.e. 5 min). There was good repeatability between the primary and second U_crit measurements (Spearman’s rho=0.61, N=54, P<0.001); due to this fact, the imply of each measurements was used for the following analyses.

Estimation of metabolic charges

The most metabolic price (MMR) and commonplace metabolic price (SMR) (see Table 1) of every particular person was measured utilizing automated intermittent-flow respirometry (Killen et al., 2021). The respirometry setup consisted of 9 80 ml glass cylindrical chambers submerged inside an aerated ambient water tub, held at 14°C (13.97±0.10°C, imply±s.d.). Each chamber had its personal mixing circuit, consisting of (1) the chamber, (2) a probe vessel containing a single oxygen probe and (3) a circuit of gas-impermeable tubing. Each of the 9 oxygen probes have been hooked up to a Firesting O₂ 4-channel oxygen sensor (FSO2-4, PyroScience, Aachen, Germany; as much as 4 probes per sensor) and have been set to measure oxygen content material each 2 s. A peristaltic pump (set to 100 rpm) was used to flow into water by way of the blending circuits. Throughout the measurement interval, every circuit alternated between flush cycles (lasting 3 min) and closed cycles (lasting 8 min). During the closed cycles, the automated flush pumps have been switched off to stop contemporary aerated water from getting into the circuits, thus permitting the speed of oxygen depletion (i.e. the speed of oxygen uptake by the fish) to be measured.

In preparation, fish weren’t consumed the day on which they have been transferred to the respirometry chambers (so had not been fed because the earlier morning, to make sure that their intestine could be empty). At round 16:00 h, fish have been uncovered to a 3-min exhaustive chase protocol, previous to being transferred to their respective respirometry chamber. This protocol assumes that the following price of oxygen uptake by the fish is at its most, and could be captured after switch to the chamber (Killen et al., 2015); it has been proven to yield the identical outcomes as when MMR is measured by an incremental swimming take a look at (Killen et al., 2017b; Zhang et al., 2020; however see additionally Raby et al., 2020). Therefore, the best price of oxygen uptake by the fish in the course of the first closed cycle of the respirometer was used as our estimate of MMR (mg O₂ h⁻¹) and was calculated based mostly on a 1 min rolling regression of the speed of oxygen depletion; see Table S1 for additional particulars. Fish then remained of their chambers in a single day till round 09:30 h the next morning with the intention to calculate SMR. During this time, the speed of oxygen uptake (mg O₂ h⁻¹) was calculated individually for every of the closed cycles, and SMR was estimated because the imply of the bottom twentieth percentile of those charges (with the primary 5.5 h of oxygen measurements being excluded from the SMR calculations). The AS for every fish was then calculated because the distinction between its MMR and SMR. Fish have been then faraway from their chambers and anaesthetised to permit correct measurement of physique mass (±0.001 g), which was then used within the subsequent calculations of mass-corrected residual metabolic charges (see Fig. 2). The two earlier measurements of physique fork size (taken on the identical days because the U_crit measurements) have been for the aim of the U_crit (BL s⁻¹) calculations, however solely this closing measurement of physique mass was used for the following analyses.

Tissue sampling

When a given particular person had reached experimental day 29 (see Fig. 1), it was euthanised with a 50 mg l⁻¹ answer of benzocaine and its muscle tissue was sampled for varied measurements. The muscle mass from one aspect of the fish was sampled (tissue mass vary: 47–219 mg) for mitochondrial evaluation (see ‘Measurement of mitochondrial respiration and ROS production rates’) and instantly transferred to 2 ml of ice-cold mitochondrial respiration buffer MiR05 (0.5 mmol l⁻¹ EGTA, 3 mmol l⁻¹ MgCl₂, 60 mmol l⁻¹ lactobionic acid, 20 mmol l⁻¹ taurine, 10 mmol l⁻¹ KH₂PO₄, 20 mmol l⁻¹ HEPES, 101 mmol l⁻¹
d-sucrose, 1 g l⁻¹ important fatty acid free BSA; pH 7.3). A subsample of muscle mass from the opposite aspect of the fish was taken for the histological evaluation of the relative proportion of cardio (slow-twitch) and anaerobic (fast-twitch) muscle fibres inside the muscle mass of that particular person (see ‘Estimation of aerobic versus anaerobic muscle’). To achieve this, a transverse cross-section of myotome muscle was sampled, guaranteeing that it contained each the dorsal (epaxial) and ventral (hypaxial) muscle mass. Muscle sections have been first fastened in a ten% formaldehyde answer for 3 days after which transferred to 70% ethanol for long-term storage. The remaining tissue from the second aspect of the fish was then flash-frozen and transferred to a −70°C freezer for long-term storage, for the following measurements of citrate synthase (CS) exercise and telomere size (see under).

Measurement of mitochondrial respiration and ROS manufacturing charges

Preparation of the mitochondrial tissue samples (a mix of pink and white muscle) adopted the identical protocol as outlined in Dawson et al. (2022). Following dissection and the speedy switch to MiR05 buffer (as outlined in ‘Tissue sampling’), the tissue was first minced within the buffer with microsurgical spring scissors, adopted by six rounds of mild homogenisation utilizing a Dounce homogeniser (Cole-Parmer PTFE Tissue Grinder, Cambridgeshire, UK) at 100 rpm.

Mitochondrial operate was analysed utilizing high-resolution respirometry (Oxygraph-2k with O2k-Fluorescence module, Oroboros Instruments, Innsbruck, Austria), following modified protocols by Dawson et al. (2022) and Dawson and Scott (2022). The protocols have been performed at 14°C with steady stirring, with samples consisting of 30 mg of tissue in a closing quantity 2 ml of respiration buffer. The price of ROS manufacturing was measured following the protocol of Dawson et al. (2018), utilizing the O2k-FluoRespirometry module within the presence of Ampliflu Red (15 µmol l⁻¹), and after the addition of a recognized amount of exogenous H₂O₂ within the presence of horseradish peroxidase (3 U ml⁻¹).

The respiration charges of the mitochondria have been measured when oxygen consumption had stabilised for a minimum of 5 min, following the addition of the substrates malate (2 mmol l⁻¹) and pyruvate (5 mmol l⁻¹), adopted by ADP (5 mmol l⁻¹), then glutamate (10 mmol l⁻¹) and eventually succinate (25 mmol l⁻¹). This price of respiration within the presence of all substrates represents the utmost price of oxidative phosphorylation (hereafter termed OXPHOS capability), as it’s occurring beneath circumstances the place each Complex I and Complex II are capable of function with an extra of substrates and ADP. Therefore, OXPHOS capability can be utilized as a proxy of the utmost capability for mitochondrial ATP manufacturing. Cytochrome c (10 mmol l⁻¹) was then added to evaluate the standard of the mitochondrial preparations, as a result of respiration within the presence of cytochrome c is a measure of mitochondrial membrane integrity (Kuznetsov et al., 2004).

Next, oligomycin (an inhibitor of F₁F₀ ATP synthase) was added to measure leak state respiration (the oxygen consumption that’s used to offset the leakage of protons throughout the internal mitochondrial membrane moderately than to generate ATP; hereafter termed LEAK). Finally, antimycin A (an inhibitor of complicated III) was added to evaluate non-mitochondrial (i.e. background) oxygen consumption, which was then subsequently subtracted from all respiration measurements.

Measurement of citrate synthase exercise

CS exercise is a generally used biomarker for mitochondrial quantity (Larsen et al., 2012; McLaughlin et al., 2020), thereby permitting us to raised management for mitochondrial quantity within the measurements of OXPHOS capability. The maximal price of CS exercise was measured in our tissue samples at 14°C utilizing a SpectraMax Plus 384 spectrophotometer (Molecular Devices), following a protocol just like that of Dawson et al. (2020). The sub-sample of frozen muscle tissue was floor in liquid nitrogen after which homogenised utilizing a Power Gen 125 homogeniser (Fisher Scientific) in 20 volumes of chilly homogenisation buffer [100 mmol l⁻¹ KH₂PO₄, 1 mmol l⁻¹ EGTA, 1 mmol l⁻¹ EDTA, 0.1% Triton X-100 at pH 7.2 and 1 mmol l⁻¹ phenylmethylsulfonyl fluoride (PMSF)]. These diluted homogenates have been then centrifuged at 1000 g for five min at 4°C and the supernatant was collected. The change in absorbance was measured in triplicate at 412 nm inside the linear vary of the assay over time, utilizing the next assay circumstances: KH₂PO₄ 100 mmol l⁻¹, pH 8.0, acetyl-CoA 0.15 mmol l⁻¹, 5,5′-dithiobis-2-nitrobenzoic acid 0.15 mmol l⁻¹ and oxaloacetate 0.5 mmol l⁻¹. Our management was measured within the absence of oxaloacetate to verify that our measured exercise price was particular to CS. Using an extinction coefficient (ε) of 14.15 mmol⁻¹ cm⁻¹, the enzyme exercise was expressed in µmol per gram of tissue per minute. The among-triplicate repeatability (%CV) of the CS measurements was 3.87±0.4%, indicating a excessive diploma of repeatability.

Calculation of mitochondrial traits

Our measurements of OXPHOS capability have been divided by CS exercise (hereafter OXPHOS/CS) with the intention to standardise for mitochondrial quantity within the samples (Dawson et al., 2018). The respiratory management ratio (RCR) was calculated as RCR=(OXPHOS capability/LEAK); from this we then calculated the OXPHOS coupling effectivity (OxCE) as OxCE=1–(1/RCR). This represents the proportion of consumed oxygen that’s dedicated to OXPHOS (moderately than to offset the proton leak) and might thus be used as a proxy for the effectivity with which the mitochondria are utilizing oxygen to transform gasoline into ATP. It has the benefit over RCR as being a extra intuitive and constrained variable because it ranges between 0 (least environment friendly) and 1 (most effective). Note that OxCE is also referred to as P–L management effectivity (Gnaiger, 2020; Roussel et al., 2023) and internet phosphorylation effectivity (Shama et al., 2016; Dawson et al., 2024), and is typically thought-about an abbreviation of oxidative management effectivity. Therefore, we ended up with two closing measures of mitochondrial respiration that have been utilized in subsequent analyses: OXPHOS/CS (i.e. a measure of the mitochondrial capability for ATP manufacturing, correcting for mitochondrial quantity) and OxCE (i.e. the effectivity with which the mitochondria use oxygen when producing ATP).

Estimation of cardio versus anaerobic muscle

In order to determine what quantity of a person’s muscle part consisted of cardio (slow-twitch) versus anaerobic (fast-twitch) fibres, we adopted an immunohistochemistry (IHC) method, which utilised the Anti-Skeletal Muscle Myosin Antibody F59, as this has beforehand been proven to label cardio muscle strongly and anaerobic muscle faintly in zebrafish (Devoto et al., 1996; Barresi et al., 2000). The muscle samples have been embedded longitudinally in wax, with 2.5 µm sections being minimize and incubated in a single day at 37°C. The IHC was carried out on a DAKO Autostainer Link 48, and warmth induced antigen retrieval was achieved utilizing a sodium citrate buffer (pH 6). The major antibody was F59 (Santa Cruz catalogue ref. sc-32732; diluted 1:200) and the secondary antibody was RTU Dako Envision mouse HRP (catalogue ref. K4001), with each antibodies being incubated for 30 min at room temperature. Visualisation was then achieved utilizing Dako DAB+Substrate (catalogue ref. K3468) with a ten min incubation. The sections have been counterstained utilizing Gill’s Haematoxylin for 27 s, earlier than being dehydrated by way of graded alcohols, after which cleared and mounted utilizing an artificial resin.

Images of the IHC slides have been captured utilizing a Leica DFC 450C digital digicam mounted on a Leica M165 FC stereomicroscope, together with LAS v4.4 software program (Leica Microsystems). ImageJ software program (National Institutes of Health, Bethesda, MD, USA) was used to tell apart between the strongly stained cardio muscle fibres and the weakly stained anaerobic muscle mass fibres; see Fig. S2 for an illustrative picture. The total space of the muscle part was outlined and measured (in variety of pixels) utilizing the freehand choice device. In most species of fish, cardio muscle fibres are situated in a wedge-shaped area on the lateral finish of the horizontal myoseptum (that separates the dorsal and ventral muscle mass) (Devoto et al., 1996). Therefore, with the intention to distinguish between true cardio muscle on this wedge-shaped area (that was clearly seen within the captured photographs) and residual F59 staining in deeper areas of the muscle part, the freehand choice device was once more used to stipulate and measure (in pixels) this wedge-shaped area of cardio muscle. From that, the proportion of the muscle part that consisted of cardio (slow-twitch) fibres was then calculated, with the rest being anaerobic (fast-twitch) fibres.

Measurement of telomere size

Although absolute telomere lengths could differ amongst tissues, research have discovered sturdy correlations in telomere size between completely different tissues in a variety of vertebrate taxa: birds (Reichert et al., 2013), lizards (Rollings et al., 2019) and fish (Debes et al., 2016). In this research we extracted genomic DNA from every particular person utilizing the frozen subsample of floor muscle tissue utilizing the Qiagen Puregene Tissue Kit following the producer’s protocol, however with one further step to take away lipid contamination. For this extra step, following the lysis step of the Puregene protocol, 10 ml of chloroform was added to the lysate, which was then combined by inversion and centrifuged at 4500 rpm for 10 min. The supernatant (containing the DNA) was fastidiously pipetted off and the chloroform step was then carried out on that supernatant for a second time, earlier than persevering with with the precipitation step of the Puregene protocol. Following DNA extraction, a complete of 10 µg DNA was then digested in a single day at 37°C utilizing the restriction enzymes HinfI, HaeII and RsaI (New England Biolabs, Hitchin, UK). Standardising every pattern to a closing DNA content material of 10 µg required differing volumes of DNA. Therefore, to then standardise each quantity and focus, the digestion step was adopted by an ethanol precipitation step (0.1×the ultimate quantity of sodium acetate 3 mol l⁻¹ and a couple of.5×the ultimate quantity of 100% ethanol). The ensuing DNA pellets have been resuspended in 13 µl DNA Hydration Solution (supplied with the Puregene Tissue Kit) after which saved at −20°C till telomere size measurement.

The imply telomere size of every particular person was measured utilizing telomere restriction fragment (TRF) evaluation (Mender and Shay, 2015) utilizing a protocol just like that of Haussmann and Mauck (2008). In temporary, samples (i.e. 13 µl digested DNA) have been run on a 0.8% agarose gel utilizing a Chef DRII pulsed-field gel electrophoresis system (Bio-Rad) and beneath the next circumstances: 3 V cm⁻¹, swap time 0.5 to 7.0 s for 19 h at 14°C in 0.5% TBE buffer (Life Technologie). Following electrophoresis, the gel was dried utilizing a Slab Gel dryer mannequin GD 2000 (Hoefer) pre-hybridised in Church and Gilbert’s Hybridization Buffer (VWR) for 1 h at 37°C after which hybridised in a single day at 37°C with a radioactive ³²P-labelled oligonucleotide probe (CCCTAA)₄, which binds the three′ single strand overhang telomere sequence. The gel was then positioned on a phosphor display screen (Amersham Biosciences) for 4 days. Following this era, the phosphor display screen was scanned utilizing a Typhoon Variable Mode imager (Amersham Biosciences). ImageJ software program was then used to visualise the telomeres, which might then be measured by evaluating them with a radioactively ³²P-labelled molecular marker (Quick loading 1 kb Extend DNA Ladder, New England Bioscience), which had additionally been run on the gel.

Statistical evaluation

We first examined the relationships between physique mass and every of SMR, MMR and AS utilizing regression analyses. Both physique mass and the corresponding metabolic variables have been log₁₀-transformed for the MMR and AS regressions as this improved the normality of the residuals from these analyses. In distinction, log transformation didn’t enhance normality within the physique mass/SMR regression and so each variables have been left unlogged for that evaluation (see Fig. 2). The residuals generated from these three regression equations have been then used to create three new variables, residual commonplace metabolic price (rSMR), residual maximal metabolic price (rMMR) and residual cardio scope (rAS), with these new variables permitting us to distinguish people with increased than anticipated metabolic charges for his or her physique mass (i.e. these with optimistic residuals) from these with decrease than anticipated metabolic charges (these with adverse residuals). rMMR and rAS have been extremely colinear (r²=0.997, P<0.001, n=54), and so solely rMMR was used as a variable within the subsequent statistical analyses.

A abstract of all of the measured traits is introduced in Table 2. Data have been analysed with a complete of 5 linear mixed-effects fashions utilizing R (model 4.5.1 in R Studio model 2025.05.1) and the glmmTMB package deal (McGillycuddy et al., 2025). Model 1 examined whether or not U_crit assorted as a operate of physique mass, rSMR, rMMR, OXPHOS/CS, OxCE or the proportion of cardio fibres inside its muscle mass. Model 2 examined whether or not rSMR assorted as a operate of physique mass, rMMR, OXPHOS/CS, OxCE or the proportion of cardio fibres inside its muscle mass. Model 3 examined whether or not rMMR assorted as a operate of physique mass, rSMR, OXPHOS/CS, OxCE or the proportion of cardio fibres inside its muscle mass. Model 4 examined whether or not charges of mitochondrial ROS manufacturing (ROS_OXPHOS) assorted as a operate of physique mass, rSMR, rMMR, OXPHOS/CS or OxCE. Lastly, mannequin 5 examined whether or not telomere size assorted as a operate of physique mass, ROS_OXPHOS, rSMR, rMMR, OXPHOS/CS or OxCE. See Table S2 for a abstract of the 5 statistical fashions. In order to acquire extra parsimonious fashions, we additionally utilized mannequin choice to every mannequin utilizing the Bayesian data criterion (BIC), whereby fastened phrases have been sequentially faraway from the mannequin (offering that this resulted in a relative discount of the BIC rating) till solely vital phrases remained; see Table 3 for a abstract of those slimmed fashions. All 5 fashions included a random intercept time period for cohort ID to account for non-independence amongst people of the identical experimental group (see Tables 4 and S3 for a abstract of the random slopes within the full and slimmed fashions, respectively).

Continuous variables have been imply centred with an ordinary deviation of 1 utilizing the size () operate, with the intention to permit comparability between coefficients. Owing to logistical limitations on our pattern sizes, interplay phrases weren’t included as they might have made the fashions over-parameterised they usually lacked any apparent organic plausibility. All fashions have been examined for singularity and any fashions with a singular match have been refitted with a specified gamma prior (Chung et al., 2013) utilizing the glmmTMB package deal. Residuals from our linear mixed-effects fashions have been examined utilizing a Shapiro–Wilk take a look at, which confirmed normality in all 5 fashions.