# Long chain polyunsaturated fatty acid (LC-PUFA) composition of fish sperm: nexus of dietary, evolutionary, and biomechanical drivers

Deepali Rahi Roy, Koushik Roy, Stéphane Panserat, Vlastimil Stejskal, Jan Mraz, Giovanni M Turchini

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## ► To cite this version:

Deepali Rahi Roy, Koushik Roy, Stéphane Panserat, Vlastimil Stejskal, Jan Mraz, et al. Long chain polyunsaturated fatty acid (LC-PUFA) composition of fish sperm: nexus of dietary, evolutionary, and biomechanical drivers. Progress in Lipid Research, 2024, 96, pp.101305. 10.1016/j.plipres.2024.101305. hal-04799817

HAL Id: hal-04799817

https://hal.science/hal-04799817v1

Submitted on 29 Nov 2024

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Distributed under a Creative Commons Attribution 4.0 International License
1 Full Title:

2 Long chain polyunsaturated fatty acids (LC-PUFA) composition of fish sperm: nexus of dietary,

3 evolutionary, and biomechanical drivers

4 Running Title:

5 EcoEvoBio sperm

6 Author list:

7 Deepali Rahi Roy¹, Koushik Roy*¹, Stephane Panserat², Vlastimil Stejskal¹, Jan Mraz¹, Giovanni M.

8 Turchini³

9 Affiliation:

10 ¹Faculty of Fisheries and Protection of Waters, University of South Bohemia in České Budějovice, Na

11 Sádkách 1780, 370 05, Ceske Budejovice, Czech Republic.

12 ²INRAE, Université de Pau Et Des Pays de L'Adour, NuMeA, Aquapôle, 64310, Saint-Pée-Sur-

13 Nivelle, France

14 ³School of Agriculture, Food and Ecosystem Sciences, Faculty of Science, The University of

15 Melbourne, VIC 3010, Australia

16 *Correspondence:

17 Dr. Koushik Roy, Ph.D.

18 Na Sádkách 1780

19 Faculty of Fisheries and Protection of Waters,

20 University of South Bohemia in České Budějovice,

21 Ceske Budejovice 370 05,

22 Czech Republic.

23 Email: kroy@frov.jcu.cz

24 Acknowledgment

25 The study was funded by Ministry of Education, Youth and Sports of the Czech Republic, project

26 CENAKVA LM2023038. The authors also thank the reviewers and editor for improving the article.

27 Conflict of interest statement: Authors declare no conflict of interest.

28 Data availability statement: Data made available on Mendeley Data.
29 Long chain polyunsaturated fatty acids (LC-PUFA) composition of fish sperm: nexus of dietary,
30 evolutionary, and biomechanical drivers

31 ABSTRACT

32 Long-chain polyunsaturated fatty acids (LC-PUFA) like arachidonic acid (ARA, 20:4n-6),
33 eicosapentaenoic acid (EPA, 20:5n-3), and docosahexaenoic acid (DHA, 22:6n-3) constitute one-third
34 to half of fish sperm lipids. Fish sperm is rich in phospholipid (PL) classes—primarily
35 phosphatidylcholine, phosphatidylethanolamine, and sphingomyelin. DHA is generally the most
36 abundant LC-PUFA in each PL class, followed by competition between ARA and EPA. While the
37 total n-6: n-3 PUFA ratio does not correlate significantly with sperm biomechanics, LC-PUFAs do.
38 DHA positively influences sperm biomechanics, while ARA and EPA may reduce motility duration
39 despite enhancing velocity. Fish sperm maintains a lower (≤1) total n-6 PUFA per unit of n-3 PUFA
40 but keep a higher (>1) ARA per unit EPA. A weak dietary influence on sperm EPA and DHA exists
41 but not on ARA. The DHA: EPA ratio in fish sperm is often >1, though values <1 occur. Certain
42 species cannot fortify DHA sufficiently during spermatogenesis, particularly in lineages diverging
43 through whole genome duplications. Fish genomes from Devonian, Cretaceous, or Jurassic eras show
44 sperm ARA: EPA ratios ≥1, while Paleozoic or Triassic lineages show ratios ≤1, likely due to
45 evolutionary shifts in prostaglandin pathways. DHA-rich PL bilayers provide unique packing and
46 fusogenic properties, with ARA/EPA-derived eicosanoids guiding sperm rheotaxis/chemotaxis,
47 modulated by DHA-derived resolvins. Docosapentaenoic acid (DPA, 22:5n-3) sometimes substitutes
48 for DHA in fish sperm. While testes biosynthesize PL and LC-PUFA de novo, some features may
49 carry over to sperm. As diet impacts sperm, sometimes limited corrective capacity risks unintended
50 selection in hatchery-bred fish. Knowledge gaps are discussed.

51 **Keywords:** Fish Sperm; Lipidomics; Reproductive Fitness; Evolutionary Adaptations; Motility
52 Mechanisms; Aquaculture Animal Nutrition.
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# 1. Introduction (fish and fish sperm)

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In terms of fatty acids composition, fish are what they eat [1], even though, to some extent, they have undergone natural evolution of their fatty acid metabolism to counteract deficiencies or imbalances of fatty acids of physiological importance [2, 3]. There is ample literature available on fatty acid composition and metabolism in freshwater and marine fish and their dietary requirements at different ontogeny stages [4, 5]. Also, literature regarding differences in fatty acid composition, abundance, and variable capacity to synthesize polyunsaturated fatty acids (PUFA) *de novo*, also referred to as fatty acid bioconversion, among freshwater and marine fish is available. Evolution of desaturase and elongase enzymes and a series of promoters regulating their gene expression have been widely studied [1, 3, 6]. At the whole fish level, the broad fatty acid metabolism, including anabolic and catabolic processes, bioconversion, and deposition, has been abundantly researched [4, 7-10]. Fatty acids, in addition to being an important energy source, are also fundamental structural components of the cell membranes and, as such, contribute to cell flexibility, membrane fusion capacities, membrane functionality, and integrity, and for free moving cells like sperm, their swimming capabilities. Sperm is designed in a way that accomplishes its sole function, which is to swim, reach, and diffuse part of its membrane (fertilize) with the egg to transfer nuclear materials. PUFA is known to improve motility kinetics, membrane fusion, and fertilization capacity of fish sperm [11, 12]. Although the understanding of fatty acid stoichiometry on human sperm quality has been reviewed [13-15], such understanding on evolutionarily primitive fish sperm is scarce [16].

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In fish sperm, most of the energy required to sustain motility could be derived from pre-stored ATP during the quiescent state of the spermatozoa [17-19]. The importance of stored glycogen in fish spermatozoa is still not adequately described, but glucose and tricarboxylic acids are *well established* energy substrates in fish sperm [20-23]. *Triacylglycerol* catabolism or β-oxidation of fatty acids is used almost exclusively for the regeneration of ATP levels after motility has ended or intra-cellular glucose, glycogen, or tricarboxylic acid reserves are depleted [22]. *Among fatty acids*, the shorter-length SFA are easiest metabolic fuels [24]. Compared to saturated fatty acids (SFA), the mitochondrial β-oxidation of monounsaturated fatty acids (MUFA) and polyunsaturated fatty acids (PUFA) require 1-2 extra set of enzymes [24]. For example, mitochondrial preparations from rainbow trout red muscle show 14:0 or 16:0 fatty acids, having ~100% β-oxidation rate (relative to pyruvate), whereas PUFA (18:2n-6, 18:3n-3, 22:6n-3) have only 18-58% β-oxidation rate [25]. In fish sperm *too*, shorter chain (≤16) saturated fatty acids (14:0, 16:0) or monounsaturated fatty acids (16:1) is reported to be depleted as metabolic fuel in common carp and Eurasian perch sperm, compared to other fatty acids [19, 26]. Post-motility shifts in the ARA: EPA ratio (i.e., a marker for eicosanoid actions) and DHA: EPA ratio has suggested the potential utilization of EPA as eicosanoid precursors or prone to β-oxidation in Eurasian perch sperm [19]. The β-oxidation contributes energy [24], while eicosanoids (lipid-based signaling molecules) modulate self-defense, inflammatory/ anti-inflammatory responses, chemotaxis/ sperm rheotaxis, and fertilization [27]. Eicosanoids may influence sperm motility and thus fertilization event in humans as well. Such as the series-2 and 3 prostaglandins (PG) containing two and three double bonds, produced from 20:4n-6 (ARA) and 20:5n-3 (EPA), respectively. For example, PGF1α binds with high affinity to the calcium channel of sperm (CatSper), in human sperm important for motility [28, 29]. Within hermaphrodite body (*Caenorhabditis elegans*), sperm-guiding PG have been reported to guide and help fertilize egg, present within the same body [30]. Despite carp and trout sperm proving a 'guided behavior' under exposure to ovarian fluids [31, 32], the externally fertilizing fish sperm's self-guiding PG synthesis capabilities (under lack of ovarian fuluid PG exposure [28]) is a black box. This is mainly due to a lack of inter-disciplinary exchanges between lipidologist, nutritionists and sperm biologists. In this review, we have created such inter-disciplinary exchange.
The long chain (≥20 carbon) polyunsaturated fatty acids (LC-PUFA) in human sperm, which has a long motility duration from several hours to day(s), a paradox exists between LC-PUFA's energetic use versus non-energetic use [13, 14, 33]. Meanwhile, LC-PUFA in shortly motile fish sperm (limited to minutes or <1 minute [34]) most likely serves non-energetic purposes, as time is limited for catabolism of LC-PUFA. Non-energetic purposes of sperm LC-PUFA, such as bio-mechanical (structural and fluidity) and signaling (eicosanoids), are equally crucial for fertilization. Elevated glycolysis or rapid burst of energy does not always ensure fertilization by fish sperm. The highest swimming velocities in a fish sperm can be associated with reduced motility duration and poor fertilization rates. Rather, the intra-cellular or seminal fluid lipid levels are correlated with the fertilization capacity [35]. Fish spermatozoa contain abundant lipids (e.g., 17±2% of fresh sperm), characterized by high levels of phospholipids (approximately 60% of total lipid). LC-PUFA such as docosahexaenoic acid (22:6n-3, DHA) can make up to 1/3rd of fish sperm lipid and, together with arachidonic acid (20:4n-6, ARA) and eicosapentaenoic acid (20:5n-3, EPA) can exceed half of fish sperm lipid [19]. Our current review focuses on these typical fatty acid compositions, which are essential for structural fluidity, membrane fusion and eicosanoid responses during swimming in an external environment (mostly). The present review helps us to understand this in greater detail across dietary treatments and fish phylogeny. We targeted the three most abundant LC-PUFA in fish sperm for data exploration: ARA, EPA, and DHA (hereinafter mentioned as LC-PUFA). We have also considered the total omega-6 series PUFA (n-6 PUFA) and total omega-3 series PUFA (n-3 PUFA) ratio. Our scientific questions were to understand: (1) correlation of sperm LC-PUFA with sperm biomechanics; (2) dependency of fish sperm LC-PUFA, in its entirety or stoichiometrically, on fatty acid composition of food, or (3) if some sperm LC-PUFA does not have much plasticity to change and independent of dietary effects. There, we explored if sperm LC-PUFA may have an evolutionary or genetic background.

## 2. Methods of literature review and synthesis

### 2.1. Data collection and arrangement

For data analysis, the keywords (“fish” OR “freshwater” OR “marine” OR “teleost”) AND (“sperm” OR “spermatozoa” OR “semen” OR “milt”) AND (“fatty acid” OR “PUFA biosynthesis” OR “fads” OR “elovl”) AND (“food” OR “fish feed”) were searched. Web of Science (WoS), Scopus, ScienceDirect, and Google Scholar online databases were used for the literature search. Only articles published in peer-reviewed journals were included in the present study. A total of 34 currently available research articles reporting fish sperm fatty acid composition were obtained.

The following standards were set to select only relevant articles for the present study: (1) The articles presenting fatty acid composition of either whole semen (spermatozoa including seminal fluid) or centrifuged pellet (spermatozoa excluding seminal fluid) were included. Fatty acid composition of only seminal fluid (without spermatozoa) was not considered for analysis [11]. (2) The articles presenting fatty acid composition at only matured stage of spermatozoa were considered, immature or maturing spermatozoa fatty acid profile were not covered. The data on the fatty acid composition of testicular, immature, maturing spermatogonia, and spermatids were not selected [36]. (3) Articles representing fatty acid composition from whole spermatozoa were considered. For example, a study reporting only the fatty acid composition within the phospholipid fraction of spermatozoa was excluded [12]. (3) Data from articles presenting the fatty acid data expressed as ‘percentage of total lipids’ were collected. Fatty acid quantification data from non-WoS and non-Scopus indexed journals were excluded [37]. Finally, 24 peer-reviewed research articles (published in the year 1991 to 2023) were selected, and authors unpublished data on three fish species (*Esox lucius*, *Oreochromis niloticus*, and *Sander lucioperca*) were supplemented to strengthen the dataset. Among all the studies, sperm
fatty acids with a carbon chain from 8 to 24 were reported. The fatty acids were detected using gas chromatography/gas-liquid chromatography in all the included studies. The data used were fatty acids methyl esters (FAME) share over total FAME, as generated by gas chromatography techniques and reported in the publications as fatty acid profile. Some correction factors were applied after checking methodology of individual studies. Only one study [11] had applied theoretical correction factors despite using a standard (following a recommended practice [38-40]); its data were taken as it was reported. In other studies that did not apply theoretical correction, corrections were applied following a common approach. We corrected the reported FAME profiles for the flame ionization detector (FID) response factor and methyl transformation [39]. Theoretical response correction factors (RCF) for FID to convert to weight percent methyl ester, and molecular weight correction factors (MWF) to convert to molar percent for some common fatty acids were used. The correction factors RCF and MWF used for conversion of FAME to fatty acid profile [39] are tabulated in supplementary table T1. The change in values were minute (clarified in supplementary data). All the experimental treatments given in various studies such as the fatty acid composition of (quiescent/post motile sperm/ after exposure to different dietary compositions/ aquaculture grown or wild-caught fish sperm/ sperm collected at different periods after hormonal stimulation/ fresh or cryopreserved sperm) were included in preliminary data collection. Wherever data was available as mean and standard deviation (mean±SD), the data was populated as ‘mean’, ‘mean+1SD’, and ‘mean-1SD’. A range covered by one standard deviation above the mean and one standard deviation below it includes about 68% of the observations [41], also used in quality control range calculation in pathological labs (example). Instead of relying on a single value (mean) from published literature, it also accounts for variability and gives some upper and lower limits near the central tendency of the data (see, “68-95-99.7 rule”). Thus, capturing data as close as possible to reality.

Additional studies that investigated specifically the phospholipid (PL) fractions in fish sperm, and fatty acid composition within different PL classes were reviewed too [12, 42-48]. Their data is presented separately. From all studies, CASA (computer-assisted semen analyses) parameters such as mean curvilinear velocity (VCL) within 0 to 20 seconds post-activation, and reported motility duration (in seconds) were collected to parameterize the “biomechanics” of fish sperm.

## 2.2. Characterization of dataset

Among all the selected articles, the data was extracted from 11 freshwater and four marine fish species. Studied freshwater fish species were: hilsa shad (Tenualosa ilisha) 1x [49], sterlet (Acipenser ruthenus) 1x [46], pikeperch (Sander lucioperca) 2x [50] including own data, common carp (Cyprinus carpio) 2x [26, 46], Eurasian perch (Perca fluviatilis) 2x [19, 51], rainbow trout (Oncorhynchus mykiss) 6x [11, 52-56], burbot (Lota lota) 3x [46, 57, 58], Arctic char (Salvelinus alpinus) 1x [59], ocellate river stingray (Potamotrygon motoro) 2x [60, 61], northern pike (Esox lucius) 2x [46] including own data and Nile tilapia (Oreochormis niloticus) 1x own data. The four studied marine fish were European sea bass (Dicentrarchus labrax) 2x [42, 43, 62], European eel (Anguilla anguilla) 1x [63], and Atlantic salmon (Salmo salar) 1x [64]. A detailed visualization in this regard can be found in supplementary Figure S7.

To clarify the influence of fish origin and, therefore a potential impact of natural or artificial feeding type on sperm quality, the selected articles were firstly screened for the origin of the fish (wild or hatchery reared), and the fatty acid composition data of artificial feeding (in case of hatchery reared fish) was collected. Among the cultured fish, the fatty acid composition of the feed was available for Anguilla anguilla, Oncorhynchus mykiss [52, 53, 55, 56], Lota lota [57], Perca fluviatilis [51], Dicentrarchus labrax [62], and Oreochormis niloticus (own data). The influence or change of fatty
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acid composition from feed to sperm was investigated only from those sources whose paired data
 fe (feed and sperm fatty acids composition) was available.

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Whereas, the artificial feed fatty acid composition of *Acipenser ruthenus* [61], *Cyprinus carpio* [26, 46], *Esox lucius* [46] including own data, *Lota lota* [58], *Oncorhynchus mykiss* [11, 54], *Perca fluviatilis* [65], *Potamotrygon motoro* [60, 61], *Salmo salar* [64], *Sander lucioperca* [50] including own data, *Salvelinus alpinus* [59] and *Tenualosa ilisha* [49] was not reported. For all freshwater fish species with unreported food data (*A. ruthenus*, *S. lucioperca*, *C. carpio*, *P. motoro*, *E. lucius*, and *S. alpinus*), we assumed the probable ARA: EPA and DHA: EPA stoichiometries of most common freshwater natural prey in temperate to sub-temperate freshwaters; as majority of those were naturally-raised broodstock in such climatic region. The data was selectively compiled from the literature [66-68], and from own data (Roy, K., Mraz, J., unpublished data). The food items included plankton consortia (≥200 µm mesh), zooplankton, macro-zoobenthos, aquatic insects, and freshwater minnows. *S. salar* and *T. ilisha* were excluded from such analyses due to the complexity of predicting their natural food base, as they were migratory (anadromous).

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**2.3. Formulation of hypotheses**

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Three independent hypotheses were used to frame the investigation:

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*Hypothesis 1:* The hypothesis 1 is inspired by Ahlgren and colleagues [69]. Being an aquatic animal, the total n-6: n-3 PUFA ratio of fish sperm is tightly conserved towards more preponderance of n-3 PUFA than n-6 PUFA. Mathematically, fish sperm n-6: n-3 PUFA ratio would be ≤1 irrespective of dietary perturbations.

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*Hypothesis 2:* The hypothesis 2 was inspired by Bell and colleagues [44], and Tocher [7], eicosanoids precursor ratio ARA: EPA in fish sperm is preferably inclined towards highly bioactive prostaglandins balance (high-bioactive PGE₂ > low-bioactive PGE₃). Mathematically, fish sperm ARA: EPA ratio would be ≥1 for high bioactive eicosanoid balance irrespective of dietary perturbations.

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*Hypothesis 3:* The hypothesis 3 was inspired by the current understanding of human sperm and multiple species comparisons [13, 14, 33, 70]. There is not much plasticity in sperm DHA to EPA balance. DHA: EPA stoichiometry must always favor DHA over EPA in fish sperm due to the key importance of DHA in sperm morphology, swimming, and membrane fusion reactions. Mathematically, the DHA: EPA ratio would be ≥1 for sperm irrespective of dietary perturbations.

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**2.4. Exploratory analyses on evolutionary/ genomic pre-disposition**

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In addition to investigating dietary influences on hypothesis 2 and 3, we performed an exploratory
analysis to find out if the evolutionary timeline is relatable with stoichiometry (ratio) of certain LC-
PUFA in fish sperm. For this purpose, we considered DHA: EPA ratio and ARA: EPA ratio in fish
sperm. Then we considered the evolutionary divergence era of fish species from Hughes and
colleagues [71], including the occurrence of whole genome duplication events in fish from Berthelot
and colleagues [72]. In wild fish, with swimming habitat across different temperatures, pH, salinity
(osmolality), and ecological niches (surface, pelagic, benthic; with different hydrostatic pressure), the
interquartile range of body DHA: EPA ratio is from ~1:1 to ~5:1 (median ~2:1); recalculated from
published dataset [73]. This analogy can be drawn for fish sperm too, as it must swim. We assumed a
minimum threshold DHA: EPA ratio of 1 as a lower threshold for efficient swimming functions
(membrane fluidity). On the other hand, a lower than 1 ARA: EPA ratio in sperm was assumed as an
inclination towards having less bioactive or reduced pro-inflammatory eicosanoid balance [7, 27]. The
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fatty acid ratios were arranged in descending order and the upper versus lower tails were
retrospectively evaluated using evolutionary knowledge. Era of divergence interpreted from the
graphical summarization provided by Hughes and colleagues [71].

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## 2.5. Data analysis framework and limitations

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All analysis and visualizations were done with a combination of GPT-4o Data Analyst and RStudio 2023.12.1 (Build 402) using packages 'ggplot2', 'corrplot', 'e1071', 'dunn.test', 'FSA'. Paired fatty acid profiles of feed and sperm were arranged species-wise, treatment-wise, and article-wise. First, a 2-tailed Pearson's correlation test was performed at alpha level 0.05, and a correlogram was generated using RStudio (library name: corrgram). A trend analysis using paired flows from feed to sperm was done using a violin-boxplot (for artificially fed fishes) or histogram with kernel density estimate and normal distribution fitting (for naturally fed fishes). A histogram of ARA: EPA and DHA: EPA ratios in the sperm of natural temperate freshwater fish species was overlayed on the range of those FA ratios expected in their natural food base to visually assess the skewness between the two. If a 2-tailed and significant Pearson's correlation coefficient is established, a bivariate linear regression analysis was performed with feed fatty acid profile (independent variable) and sperm fatty acid profile (dependent variable). For statistical tests of significant differences, the data distribution was first determined by the Shapiro-Wilk normality test. For normally distributed data, one-way ANOVA test was performed. For data not normally distributed, the Kruskal-Wallis rank sum test was performed. All statistical tests were interpreted at a set alpha level of 0.05. The scope of limitations and definitions of the analysis is declared in supplementary material.

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# 3. Distribution and drivers of fish sperm lc-pufa composition

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## 3.1. General observations

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By the term LC-PUFA in this review, we include only ARA, EPA, and DHA. Together they make from 1/3rd to half of fish sperm lipids (Fig S3-S5, Table 1). No dichotomy exists between freshwater and marine fish, or external and internal fertilizing fish in terms of LC-PUFA. The DPA (22:5n-3) can sometimes substitute DHA in fish sperm, as in the case of tilapia (own results). There were also cases where both 22:5n-6 and 22:5n-3 substituting DHA was apparent in blunt snouted mullet, annular seabream [48]. Fish species-wise sperm total n-6 PUFA (% of total FA), total n-3 PUFA (% of total FA), ARA (% of total FA), EPA (% of total FA), and DHA (% of total FA) can be found in supplementary Fig S1-S5. In our reviewed dataset, LC-PUFAs above DHA, such as 26:3 observed in human sperm [15], were not encountered.

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From reviewed data on fish sperm phospholipids (PL) in Table 1, phosphatidylcholine followed by phosphatidylethanolamine or sometimes sphingomyelin are dominant PL classes in fish sperm. Within each PL class, DHA was almost always the most predominant LC-PUFA followed by a competition between ARA and EPA for predominance (Table 1). DiPUFA molecular species mainly composed of LC-PUFAs dominate (65-72%) phosphatidylserine fraction of fish sperm membranes, as in sea bass, Atlantic or chinook salmon [12]. In the next section, we will establish its implication on sperm biomechanics.

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<table><caption>Table 1: Reported LC-PUFA share (% of total fatty acids) within phospholipid (PL), including PL classes distribution (% of total lipids) in fish sperm. Phosphatidylcholine (PC), Phosphatidylethanolamine (PE), Phosphatidylserine (PS), Phosphatidylinositol (PI), Sphingomyelin (SM), Lysophosphatidylcholine (LPC), Diphosphatidylglycerol (DPG), Plasmalogenic phosphatidylcholine (PPC), Plasmalogenic phosphatidylethanolamine (PPE), data not available (-).</caption><thead><tr><th>Fish species (habitat)</th><th>Phospholipid (%)</th><th>20:4n-6 (%)</th><th>20:5n-3 (%)</th><th>22:5n-3 (%)</th><th>22:5n-6 (%)</th><th>22:6n-3 (%)</th><th>Reference</th></tr></thead><tbody><tr><td rowspan="5">Dicentrarchus labrax (marine)</td><td>Total</td><td>0.2</td><td>9.3</td><td>1.9</td><td>-</td><td>37.3</td><td>[42]</td></tr><tr><td></td><td>0.2</td><td>9.3</td><td>2.0</td><td>-</td><td>37</td><td>[43]</td></tr><tr><td>PC (-)</td><td>1.7-4.0*</td><td>6.0-8.2*</td><td>0.6-1.3*</td><td>0.4-0.9*</td><td>19.7-24.8*</td><td rowspan="4">[44]</td></tr><tr><td>PE (-)</td><td>2.3-5.2*</td><td>4.5-7.7*</td><td>1.1-2.8*</td><td>0.8-1.3*</td><td>45.7-48.2</td></tr><tr><td>PS (-)</td><td>2.5-5.1*</td><td>1.9-4.3*</td><td>3.7-6.1*</td><td>1.1-2.0*</td><td>68.2-74.3</td></tr><tr><td>PI (-)</td><td>21.3-31.7*</td><td>5.0-17.7*</td><td>0-0.5*</td><td>0-0.4*</td><td>2.8-13.1*</td></tr><tr><td rowspan="5">Cyprinus carpio (freshwater)</td><td>PC (14.3%)</td><td>-</td><td>-</td><td>-</td><td>-</td><td>-</td><td rowspan="5">[46]</td></tr><tr><td>PE (24.7%)</td><td>-</td><td>-</td><td>-</td><td>-</td><td>-</td></tr><tr><td>PS (9.0%)</td><td>-</td><td>-</td><td>-</td><td>-</td><td>-</td></tr><tr><td>PI (8.0%)</td><td>-</td><td>-</td><td>-</td><td>-</td><td>-</td></tr><tr><td>SM (7.1%)</td><td>-</td><td>-</td><td>-</td><td>-</td><td>-</td></tr><tr><td rowspan="5">Esox lucius (freshwater)</td><td>PC (6.5%)</td><td>-</td><td>-</td><td>-</td><td>-</td><td>-</td><td rowspan="5"></td></tr><tr><td>PE (18.7%)</td><td>-</td><td>-</td><td>-</td><td>-</td><td>-</td></tr><tr><td>PS (7%)</td><td>-</td><td>-</td><td>-</td><td>-</td><td>-</td></tr><tr><td>PI (3.5%)</td><td>-</td><td>-</td><td>-</td><td>-</td><td>-</td></tr><tr><td>SM (5.5%)</td><td>-</td><td>-</td><td>-</td><td>-</td><td>-</td></tr><tr><td rowspan="5">Lota lota (freshwater)</td><td>PC (12%)</td><td>-</td><td>-</td><td>-</td><td>-</td><td>-</td><td rowspan="5"></td></tr><tr><td>PE (19.4%)</td><td>-</td><td>-</td><td>-</td><td>-</td><td>-</td></tr><tr><td>PS (8%)</td><td>-</td><td>-</td><td>-</td><td>-</td><td>-</td></tr><tr><td>PI (4.3%)</td><td>-</td><td>-</td><td>-</td><td>-</td><td>-</td></tr><tr><td>SM (2.8%)</td><td>-</td><td>-</td><td>-</td><td>-</td><td>-</td></tr><tr><td rowspan="3">Potamotrygon motoro (freshwater)</td><td>PC (-)</td><td>-</td><td>-</td><td>-</td><td>-</td><td>-</td><td rowspan="3">[45]</td></tr><tr><td>PE (-)</td><td>-</td><td>-</td><td>-</td><td>-</td><td>-</td></tr><tr><td>PS (-)</td><td>-</td><td>-</td><td>-</td><td>-</td><td>-</td></tr></tbody></table>
<table><tbody><tr><td></td><td>PI (2.8%)</td><td>-</td><td>-</td><td>-</td><td>-</td><td>-</td><td></td></tr><tr><td></td><td>SM (13%)</td><td>-</td><td>-</td><td>-</td><td>-</td><td>-</td><td></td></tr><tr><td rowspan="6">Oncorhynchus mykiss (freshwater)</td><td>PC (46.5-54%)</td><td>2.4-5.0*</td><td>4.4-10.8*</td><td>0.2-0.5*</td><td>0.2-0.6*</td><td>10.9-12.8</td><td rowspan="6">[47]</td></tr><tr><td>PE (2.5-33%)</td><td>5.2-12.9*</td><td>9.5-18.7*</td><td>0.2-0.5*</td><td>0.3-0.7*</td><td>15-17.6</td></tr><tr><td>PS (9.5-10.3%)</td><td>3-7.1*</td><td>3.5-2.7*</td><td>0.9-1.8*</td><td>0.4-1.4*</td><td>23.4-25.9</td></tr><tr><td>PI (2.5-4.5%)</td><td>7.3-13.4*</td><td>6.4-14.9*</td><td>0.2-0.7</td><td>0.2-0.6*</td><td>6.8-9.3</td></tr><tr><td>SM (1-2.67%)</td><td>-</td><td>-</td><td>-</td><td>-</td><td>-</td></tr><tr><td>LPC (2.5-3.5%)</td><td>-</td><td>-</td><td>-</td><td>-</td><td>-</td></tr><tr><td></td><td>DPC (0.7-1.5%)</td><td>-</td><td>-</td><td>-</td><td>-</td><td>-</td><td></td></tr><tr><td rowspan="6">Scophthalmus maeoticus (marine)</td><td>PC (42.3%)</td><td>1.5-2.1</td><td>0.4-1.2</td><td>2.5-2.9</td><td>0.9-2.9</td><td>6-7.1</td><td rowspan="6">[48]<sup>#</sup></td></tr><tr><td>PE (17.3%)</td><td>3.2-4.7</td><td>1.1-1.5</td><td>2.4-2.9</td><td>2.0-3.8</td><td>9.2-21.6</td></tr><tr><td>PS + PI (6.4%)</td><td>-</td><td>-</td><td>-</td><td>-</td><td>-</td></tr><tr><td>SM (6.8%)</td><td>-</td><td>-</td><td>-</td><td>-</td><td>-</td></tr><tr><td>PPC (12.4%)</td><td>-</td><td>-</td><td>-</td><td>-</td><td>-</td></tr><tr><td>PPE (14.8%)</td><td>-</td><td>-</td><td>-</td><td>-</td><td>-</td></tr><tr><td rowspan="6">Diplodus annularis (marine)</td><td>PC (42.2%)</td><td>4.3-5.7</td><td>9.7-14.2</td><td>0.7</td><td>1.0-2.2</td><td>0.9</td><td></td></tr><tr><td>PE (20.2%)</td><td>2.4-4.4</td><td>30.4-37.4</td><td>2.1-2.2</td><td>2.4-2.6</td><td>0.8</td><td></td></tr><tr><td>PS + PI (3.8%)</td><td>-</td><td>-</td><td>-</td><td>-</td><td>-</td><td></td></tr><tr><td>SM (14.9%)</td><td>-</td><td>-</td><td>-</td><td>-</td><td>-</td><td></td></tr><tr><td>PPC (10.5%)</td><td>-</td><td>-</td><td>-</td><td>-</td><td>-</td><td></td></tr><tr><td>PPE (8.4%)</td><td>-</td><td>-</td><td>-</td><td>-</td><td>-</td><td></td></tr><tr><td rowspan="6">Mullus burbatus ponticus (marine)</td><td>PC (25.5%)</td><td>8.7-9.1</td><td>7.4-7.8</td><td>1.7</td><td>3.1-6.3</td><td>2.3</td><td></td></tr><tr><td>PE (34.3%)</td><td>6.7-8.2</td><td>5.9-6.3</td><td>2.6-4.1</td><td>1.2-2.5</td><td>3.1</td><td></td></tr><tr><td>PS + PI (9.1%)</td><td>-</td><td>-</td><td>-</td><td>-</td><td>-</td><td></td></tr><tr><td>SM (8.6%)</td><td>-</td><td>-</td><td>-</td><td>-</td><td>-</td><td></td></tr><tr><td>PPC (6.6%)</td><td>-</td><td>-</td><td>-</td><td>-</td><td>-</td><td></td></tr><tr><td>PPE (15.9%)</td><td>-</td><td>-</td><td>-</td><td>-</td><td>-</td><td></td></tr><tr><td rowspan="2">Neogobius melanostomus (marine)</td><td>PC (52.9%)</td><td>0.7-2.2</td><td>1.5-8.3</td><td>3.0-6.1</td><td>1.0</td><td>7.2-8.7</td><td></td></tr><tr><td>PE (22.6%)</td><td>2.1-2.6</td><td>4.5</td><td>2.1-3.7</td><td>1.2-6.7</td><td>9.4-10.9</td><td></td></tr></tbody></table>
<table><tbody><tr><td></td><td>PS + PI (9.1%)</td><td>-</td><td>-</td><td>-</td><td>-</td><td>-</td></tr><tr><td></td><td>SM (9.1%)</td><td>-</td><td>-</td><td>-</td><td>-</td><td>-</td></tr><tr><td></td><td>PPC (5.1%)</td><td>-</td><td>-</td><td>-</td><td>-</td><td>-</td></tr><tr><td></td><td>PPE (5.5%)</td><td>-</td><td>-</td><td>-</td><td>-</td><td>-</td></tr><tr><td rowspan="5">Symphodus tinca (marine)</td><td>PC (53.9%)</td><td>2.0-3.5</td><td>1.0-11.7</td><td>1.2-2.3</td><td>1.0-1.4</td><td>4.4-5.8</td></tr><tr><td>PE (20.5%)</td><td>3.5-4.9</td><td>1.4-6.8</td><td>2.1-4.4</td><td>2.6-4.3</td><td>7.5-14.2</td></tr><tr><td>PS + PI (5.0%)</td><td>-</td><td>-</td><td>-</td><td>-</td><td>-</td></tr><tr><td>SM (9.3%)</td><td>-</td><td>-</td><td>-</td><td>-</td><td>-</td></tr><tr><td>PPC (5.7%)</td><td>-</td><td>-</td><td>-</td><td>-</td><td>-</td></tr><tr><td rowspan="5">Oreochromis mossambicus (freshwater)</td><td>PPE (5.6%)</td><td>-</td><td>-</td><td>-</td><td>-</td><td>-</td></tr><tr><td>PC (32.8%)</td><td>6.3-13.1</td><td>0.4-1.3</td><td>1.3-1.7</td><td>0.8-2.5</td><td>1.8-2.3</td></tr><tr><td>PE (18.6%)</td><td>7.1-22.9</td><td>0.7</td><td>1.0-2.9</td><td>2.5-6.2</td><td>2.8-4.3</td></tr><tr><td>PS (8.0%)</td><td>-</td><td>-</td><td>-</td><td>-</td><td>-</td></tr><tr><td>PI (4.7%)</td><td>-</td><td>-</td><td>-</td><td>-</td><td>-</td></tr><tr><td rowspan="5">Centrarchus macropterus (freshwater)</td><td>SM (6.7%)</td><td>-</td><td>-</td><td>-</td><td>-</td><td>-</td></tr><tr><td>PPC (11.3%)</td><td>-</td><td>-</td><td>-</td><td>-</td><td>-</td></tr><tr><td>PPE (16.1%)</td><td>-</td><td>-</td><td>-</td><td>-</td><td>-</td></tr><tr><td>PC (23.2%)</td><td>9.9-11.1</td><td>3.5-5.4</td><td>1.8-2.1</td><td>3.9-4.0</td><td>2.2-7.6</td></tr><tr><td>PE (24.1%)</td><td>13.1-17.3</td><td>1.9-6.4</td><td>5.7</td><td>1.6-2.1</td><td>14.8</td></tr><tr><td rowspan="5">Ictiobus cyprinellus (freshwater)</td><td>PS (11.8%)</td><td>-</td><td>-</td><td>-</td><td>-</td><td>-</td></tr><tr><td>PI (8.0%)</td><td>-</td><td>-</td><td>-</td><td>-</td><td>-</td></tr><tr><td>SM (18.0%)</td><td>-</td><td>-</td><td>-</td><td>-</td><td>-</td></tr><tr><td>PPC (6.3%)</td><td>-</td><td>-</td><td>-</td><td>-</td><td>-</td></tr><tr><td>PPE (11.6%)</td><td>-</td><td>-</td><td>-</td><td>-</td><td>-</td></tr><tr><td rowspan="5">Ictiobus cyprinellus (freshwater)</td><td>PC (21.9%)</td><td>11.1-13.2</td><td>0.6</td><td>0.5-0.9</td><td>2.3-2.7</td><td>4.6-4.9</td></tr><tr><td>PE (18.7%)</td><td>14.8-15.9</td><td>0.3</td><td>0.5</td><td>4.5-5.2</td><td>9.2-10.0</td></tr><tr><td>PS (8.8%)</td><td>-</td><td>-</td><td>-</td><td>-</td><td>-</td></tr><tr><td>PI (6.1%)</td><td>-</td><td>-</td><td>-</td><td>-</td><td>-</td></tr><tr><td>SM (18.6%)</td><td>-</td><td>-</td><td>-</td><td>-</td><td>-</td></tr></tbody></table>
<table><tbody><tr><td></td><td>PPC (15.0%)</td><td>-</td><td>-</td><td>-</td><td>-</td><td>-</td></tr><tr><td></td><td>PPE (9.2%)</td><td>-</td><td>-</td><td>-</td><td>-</td><td>-</td></tr><tr><td rowspan="7">Ctenopharyngodon idella (freshwater)</td><td>PC (34.1%)</td><td>17.1</td><td>8.4</td><td>0.6</td><td>-</td><td>3.2</td></tr><tr><td>PE (25.8%)</td><td>25.4</td><td>5.9</td><td>-</td><td>1.4</td><td>8.7</td></tr><tr><td>PS (9.4%)</td><td>-</td><td>-</td><td>-</td><td>-</td><td>-</td></tr><tr><td>PI (0%)</td><td>-</td><td>-</td><td>-</td><td>-</td><td>-</td></tr><tr><td>SM (20.9%)</td><td>-</td><td>-</td><td>-</td><td>-</td><td>-</td></tr><tr><td>PPC (-)</td><td>-</td><td>-</td><td>-</td><td>-</td><td>-</td></tr><tr><td>PPE (5.4%)</td><td>-</td><td>-</td><td>-</td><td>-</td><td>-</td></tr><tr><td rowspan="7">Cyprinus carpio (freshwater)</td><td>PC (30.4%)</td><td>13.5-28.9</td><td>-</td><td>-</td><td>1.3-9.8</td><td>1.4</td></tr><tr><td>PE (22.3%)</td><td>6.7-40.6</td><td>-</td><td>1.6-9.8</td><td>-</td><td>-</td></tr><tr><td>PS (5.4%)</td><td>-</td><td>-</td><td>-</td><td>-</td><td>-</td></tr><tr><td>PI (4.5%)</td><td>-</td><td>-</td><td>-</td><td>-</td><td>-</td></tr><tr><td>SM (10%)</td><td>-</td><td>-</td><td>-</td><td>-</td><td>-</td></tr><tr><td>PPC (5.8%)</td><td>-</td><td>-</td><td>-</td><td>-</td><td>-</td></tr><tr><td>PPE (9.6%)</td><td>-</td><td>-</td><td>-</td><td>-</td><td>-</td></tr><tr><td rowspan="3">Dicentrarchus labrax (marine)</td><td>PC (-)</td><td>-</td><td>-</td><td>-</td><td>-</td><td>1.7</td></tr><tr><td>PE (-)</td><td>-</td><td>-</td><td>-</td><td>-</td><td>10.9</td></tr><tr><td>PS (-)</td><td>-</td><td>-</td><td>-</td><td>-</td><td>50.2</td></tr><tr><td rowspan="3">Salmo salar (marine)</td><td>PC (-)</td><td>-</td><td>0.3</td><td>-</td><td>-</td><td>1.3</td></tr><tr><td>PE (-)</td><td>-</td><td>0.5</td><td>-</td><td>-</td><td>3.3</td></tr><tr><td>PS (-)</td><td>-</td><td>1.8</td><td>-</td><td>-</td><td>24.1</td></tr><tr><td rowspan="3">Onchorynchus tsawytscha (marine)</td><td>PC (-)</td><td>-</td><td>1.2</td><td>1.0</td><td>-</td><td>1.3</td></tr><tr><td>PE (-)</td><td>-</td><td>0.5</td><td>3.4</td><td>-</td><td>4.7</td></tr><tr><td>PS (-)</td><td>-</td><td>0.8</td><td>0.6</td><td>-</td><td>35.4</td></tr></tbody></table>

*Statistically significantly (p≤0.01) impacted by choice of food source [44, 47].

#Fatty acid data out of range (suspicious) compared to other literature [48].

##Data are of DiPUFA molecular species, not single molecule [12].
## 3.2. Influence of LC-PUFA and total n6: n-3 PUFA ratio on fish sperm biomechanics

Total n-6: n-3 PUFA ratio has no significant correlation with sperm biomechanics, but LC-PUFAs do. All LC-PUFAs are positively correlated with VCL, except EPA. The DHA is only LC-PUFA having a unanimous positive and statistically significant correlation to both VCL and motility duration. Results suggest DHA's positive influence on sperm biomechanics (discussed later), while both ARA and EPA involved in eicosanoids biology influence a reduced motility duration. Results can be found in Table 2.

Present correlations affirm eicosanoids balance interact with motility duration of fish sperm, with a small positive role in VCL (Table 2). Similar observations in human sperm showed sperm motility was related to the balance between the content of 19-hydroxy PGE and 19-hydroxy PGF but not to their separate concentrations [74]. The balance is further visualized in fish sperm using a power regression (Fig 1).

The eicosanoids balance of fish sperm is a black-box since the time of Bell and colleagues [44]. Not much is known in relation to fish sperm biomechanics, neither the right prostaglandin balance of fertile versus infertile males nor prostaglandin/eicosanoid receptors present in fish sperm. There is a legacy of understanding in human sperm along those lines (e.g., [74-76]). Infertile men (n = 82) had significantly higher ARA: DHA and ARA: EPA ratios that were both negatively correlated with total sperm count, sperm motility, and sperm morphology [15]. Our correlation analysis shows sperm LC-PUFA's interaction with fish sperm biomechanics, especially the ARA: EPA ratio and DHA (Table 2).

Using a 'spermatozoan spin-labeling' technique and without any CASA measurements or fertilization assays, it was argued that dietary lipids can alter the general fatty acids composition, but not fluidity, of trout sperm plasma membrane [47]. But such results are contradicted by recent literature that employ CASA or fertilization assays. For example, European eel sperm showed highest velocity and motility percentage (even at 30 seconds post activation) under dietary regime with highest LC-PUFA (ARA+ EPA+ DHA) clearly demonstrating enhanced biomechanics [77]. In internal fertilizing guppy, the importance of dietary LC-PUFA for share of paternity was observed [78] or motility duration [79]. In zebrafish, the addition of phospholipids in the diet (especially phosphatidylethanolamine) caused higher total and progressive motility and higher velocities [80]. The fertilization, eyed egg stage, and hatching rates were lower from male fish fed n-3 PUFA deficient diet [56]. In the next section, we will establish the dietary connection with sperm LC-PUFA composition.

<table><caption>**Table 2:** Pearson's 2-tailed correlation between selected PUFA parameters and sperm curvilinear velocity (VCL) or motility duration (MD). Non-significant *p* > 0.05 (ns).</caption><thead><tr><th>Variable 1</th><th>Variable 2</th><th>Correlation coefficient</th><th>p-value</th></tr></thead><tbody><tr><td rowspan="2">Total n6: n-3 PUFA ratio</td><td>VCL</td><td>-0.139</td><td>ns</td></tr><tr><td>MD</td><td>-0.259</td><td>ns (p=0.05)</td></tr><tr><td rowspan="2">20:4n-6 (ARA)</td><td>VCL</td><td>0.403</td><td>p&lt;0.001</td></tr><tr><td>MD</td><td>-0.666</td><td>p&lt;0.001</td></tr><tr><td rowspan="2">20:5n-3 (EPA)</td><td>VCL</td><td>0.281</td><td>ns</td></tr><tr><td>MD</td><td>-0.604</td><td>p&lt;0.001</td></tr><tr><td rowspan="2">22:6n-3 (DHA)</td><td>VCL</td><td>0.275</td><td>p&lt;0.01</td></tr><tr><td>MD</td><td>0.515</td><td>p&lt;0.001</td></tr><tr><td rowspan="2">ARA: EPA (eicosanoid actions)</td><td>VCL</td><td>0.296</td><td>p&lt;0.01</td></tr><tr><td>MD</td><td>-0.625</td><td>P&lt;0.001</td></tr></tbody></table>
<figure><img src="image_4.png" /><figcaption>Fig 1: Relationship of sperm eicosanoid actions (ARA: EPA ratio) and motility duration of fish sperm. Power regression (R² 0.651, p<0.001): Motility duration (sec) = 56.209 × (ARA: EPA)⁻⁰.⁷⁶⁵</figcaption></figure>

**3.3. Influence of dietary fatty acid profile on sperm fatty acid profile**

There are clear experimental evidence that choice of food source can significantly alter (p≤0.01) LC-PUFA composition in sea bass [44] or rainbow trout [47] sperm membrane phospholipids. Below is a further re-affirmation of such events based on reviewed data.

**3.3.1. Hypothesis 1**

Positive correlations were found between feed and sperm total n-6: n-3 PUFA ratio (r≈0.6, p<0.05), also total n-6: n-3 PUFA ratio in feed with sperm ARA: EPA ratio (r≈0.5, p<0.05). The correlogram can be found in supplementary material (Fig S6). In our paired dataset (feed and sperm), the total n-6: n-3 PUFA ratio ranged from 0.14 to 10 in feed, while total n-6: n-3 PUFA ratio in sperm (0.06-1.07) was significantly lower (p<0.01; Fig 2A). It shows signs of homeostatic control in sperm, with regards to total n-6: n-3 PUFA ratio. On the contrary, the ARA: EPA ratio in sperm (0.1-3.79) was significantly higher (p<0.01) than ARA: EPA ratio in feed (0.05 to 2.30) (Fig 2B). It shows signs of allostasis (defined as corrective measures) by sperm, with regards to ARA: EPA ratio to maintain a healthy or tolerable eicosanoids balance.

The hypothesis-1 holds true. Fish sperm tend to maintain lower (≤1 unit) total n-6 PUFA per unit of total n-3 PUFA (Fig 2A), but simultaneously prefer to maintain higher (>1 unit) ARA per unit of EPA (Fig 2B). Thus, both homeostasis and allostasis mechanisms co-exist, depending on specific physiological or biomechanical needs (further discussed).
<figure><img src="image_5.png" /><figcaption>**Fig 2:** Paired feed to sperm fatty acid ratio changes in artificially fed fish. (A) total n-6: n-3 PUFA decrease from feed to sperm. (B) ARA: EPA increases from feed to sperm. Red lines connect individual data points from feed to sperm. Downward lines indicate homeostatic control while upward lines indicate allostasis or corrective measures, perhaps happening during spermatogenesis.</figcaption></figure>

3.3.2. *Hypothesis 2*

The dietary ARA: EPA ratio is not correlated (*p*>0.05) with sperm ARA: EPA ratio (Fig S6). A case study on some temperate freshwater fish species in our dataset that grew on natural food suggests that the common ARA: EPA ratio in sperm is ≥1 (sperm ARA: EPA= 1.42±0.83; Fig 3). The natural food base, comprising of plankton consortia (≥200 µm mesh), zooplankton, benthic macro-zoobenthos, insects, smaller prey fish or minnows, can have ARA: EPA ratio from 0.05 to 0.72. The ARA: EPA ratio in wild fish sperm is higher than natural food (Fig 4). Same trend as artificially fed fish (Fig 2B). The hypothesis-2 holds true as well.

No relationship could be established between dietary and sperm ARA (Adj R² 0.02, p=0.1489), but a weak relationship exists between share of EPA in feed and sperm fatty acids (Adj R² 0.1254,
p<0.001). The relationship predicts per unit increase in dietary EPA, there is a 0.57 unit or 57% increase in sperm EPA (Fig 4). In an experiment with salmon, the fate of dietary EPA being deposited was close to 50% of what was fed, in diets that contained only EPA or EPA+DHA above minimum requirements. Rest was lost by β-oxidation and further bioconversion [81]. Whether the same bioenergetic partitioning of EPA happens during spermatogenesis too is presently unknown, but the prediction hints a close alignment.

<figure><img src="image_6.png" /><figcaption>Fig 3: Histogram of natural freshwater fish sperm ARA: EPA ratio, with kernel density estimate (KDE; green curve) and normal distribution fit (black curve). Solid red box on X-axis indicates ARA: EPA ratio range in natural food base (0.05-0.72). Dashed black box on X-axis indicates ARA: EPA ratio range in wild fish sperm (1.42±0.83). KDE is a non-parametric method to estimate the probability density function of a random variable.</figcaption></figure>

<figure><img src="image_7.png" /><figcaption>Fig 4: Relationship between EPA share in dietary fatty acids and EPA share in sperm fatty acids. Sperm EPA= 5.28 + 0.57 * Feed EPA (Adj. R² 0.125, p<0.01). Dark to lighter dots imply high to low FA quantity in feed.</figcaption></figure>
3.3.3. *Hypothesis 3*

The dietary DHA: EPA ratio is correlated (r≈0.6, p<0.05) with sperm DHA: EPA ratio (Fig S6). A weak but statistically significant relationship exist between dietary and sperm DHA: EPA ratio (Adj R² 0.2963, p<0.001). For every 1 unit increase in dietary DHA: EPA, we recorded a 1.67 unit increase in sperm DHA: EPA (Fig 5A). In artificially fed fish, sperm DHA: EPA ratio is mostly greater than feed DHA: EPA ratio (Fig 6B). But there are cases, shown by almost straight red lines (Fig 6B), the sperm or feed DHA: EPA ratio mimic each other. In wild fish, for example our case study on some temperate freshwater fish species grazed on natural food (DHA: EPA ratio range 0.1-1.6), the sperm DHA: EPA ratio is 1.97±1.33. Meaning sperm DHA: EPA ratio can be commonly >1, with possibility of values <1 too (Fig 6A). Thus, hypothesis-3 holds true, with some genetic limitations. There are fish species that cannot sufficiently endogenously fortify DHA during spermatogenesis (next section).

A weak relationship exists between share of DHA in feed and sperm fatty acids (Adj R² 0.127, p<0.001). The relationship predicts per unit increase in dietary DHA, there is a 0.71 unit or 71% increase in sperm DHA (Fig 5B). In an experiment with salmon, the fate of dietary DHA being deposited was almost 100% of what was fed. The reason being DHA being too important or complex for catabolism [81]. Such research is not present in fish sperm, but the prediction hints at a close alignment.

<figure><img src="image_8.png" /></figure>

<figure><img src="image_9.png" /></figure>
**Fig 5:** (A) Relationship between dietary and fish sperm DHA: EPA ratio, Sperm DHA: EPA ratio = 0.30 + 1.67 * Feed DHA: EPA ratio (Adj. R² 0.29, p<0.01) (B) Relationship between DHA share in dietary fatty acids and DHA share in sperm fatty acids. Sperm DHA = 14.05 + 0.71 * Feed DHA (Adj. R² 0.13, p<0.01). Dark to lighter dots imply high to low FA quantity in feed.

<figure><img src="image_10.png" /></figure>

**Fig 6:** (A) Histogram of natural freshwater fish sperm ARA: EPA ratio, with kernel density estimate (KDE; blue curve) and normal distribution fit (black curve). Solid red box on X-axis indicates ARA: EPA ratio range in natural food (0.1-1.6). Dashed black box on X-axis indicates ARA: EPA ratio range in wild fish sperm (1.97±1.33). KDE is a non-parametric method to estimate the probability density function of a random variable. (B) Paired feed to sperm DHA: EPA ratio changes in artificially fed fish.

It is worth mentioning that the weak relationships in Fig 4, 5A, and 5B are derived from a limited dataset, with different diets and across different fish species. The relationship although weak cannot be overlooked, as fish sperm phospholipids data reviewed in Table 2 has shown significant effect of
diets altering fatty acids composition. Future controlled experiments with graded dietary treatments of EPA, DHA, and DHA: EPA and their deposition in sperm need to be conducted, to ascertain the true correlation. The inter-relationships in Fig 4, 5A, and 5B are quite weak, and it may account for other governing factors that are not explained. Among such factors could be the evolutionary and genetic pre-disposition, which will be presented below.

**3.4. Evolutionary connection to LC-PUFA stoichiometries in fish sperm**

**3.4.1. DHA-to-EPA stoichiometry**

Fig 7 shows three species, *Lota lota*, *Esox lucius*, and *Potamotrygon motoro*, having sperm DHA: EPA ratio below 1 (threshold). *Those below* threshold represent some uniquely diverged fish lineages. The ocellate river stingray (*P. motoro*) is an elasmobranchii but uniquely displaced to freshwaters before the third-round whole genome duplication event (3R-WGD). Burbot (*L. lota*) is a unique descendant of marine cod lineage into freshwaters. The Northern pike (*E. lucius*) is a uniquely diverged sister group from freshwater salmonids. They were uniquely diverged during the fourth-round whole genome duplication event (4R-WGD). The DHA fortification seems limited or inefficient in these species (Fig 7), neither DPA substituted for DHA in their sperm (see data).

<figure><img src="image_11.png" /><figcaption>**Fig 7: Sperm DHA: EPA ratio of different fish species in our dataset.** The red line is a demarcation of sperm DHA: EPA ratio greater than or less than 1. Red arrow (↔) indicates uniquely diverged fish lineages *during 3R- or 4R-WGD events*.</figcaption></figure>

**3.4.2. ARA-to-EPA stoichiometry**

In Table 3, fish species are arranged in descending order of their sperm ARA: EPA ratio (median) and matched with their era of evolutionary divergence. The fish genomes that diverged in the Devonian, Cretaceous, or Jurassic eras seem to have sperm ARA: EPA ratio ≥1. While fish genomes that
diverged in the Paleozoic or Triassic era seem to have sperm ARA: EPA ratio ≤1. There is one exception of *D. labrax* (European seabass), a marine Perciformes genome diverged in the Cretaceous era, having sperm ARA: EPA ratio <1; unlike freshwater Perciformes genome or others in Cretaceous era (Table 3).

<table><caption>**Table 3: Median sperm ARA: EPA ratio of different fish species and their evolutionary timeline.** Era of divergence interpreted from the graphical summarization in Hughes and colleagues [71]. The red line is a demarcation of sperm ARA: EPA ratio above or below 1 (rounded-off).</caption><thead><tr><td>Fish Species</td><td>Sperm ARA: EPA</td><td>Order</td><td>Era of divergence</td></tr></thead><tbody><tr><td>Acipenser ruthenus</td><td>2.570</td><td>Acipenseriformes</td><td>Devonian</td></tr><tr><td>Oreochromis niloticus</td><td>2.030</td><td>Cichliformes</td><td>Cretaceous</td></tr><tr><td>Potamotrygon motoro</td><td>1.930</td><td>Myliobatiformes</td><td>Devonian</td></tr><tr><td>Lota lota</td><td>1.710</td><td>Gadiformes</td><td>Cretaceous</td></tr><tr><td>Sander lucioperca</td><td>1.435</td><td>Perciformes</td><td>Cretaceous</td></tr><tr><td>Cyprinus carpio</td><td>1.190</td><td>Cyprinifomes</td><td>Jurassic</td></tr><tr><td>Perca fluviatilis</td><td>0.970</td><td>Perciformes</td><td>Cretaceous</td></tr><tr><td>Esox lucius</td><td>0.930</td><td>Esociformes</td><td>Paleozoic</td></tr><tr><td>Anguilla anguilla</td><td>0.470</td><td>Anguilliformes</td><td>Triassic</td></tr><tr><td>Salvelinus alpinus</td><td>0.370</td><td>Salmoniformes</td><td>Paleozoic</td></tr><tr><td>Oncorhynchus mykiss</td><td>0.290</td><td>Salmoniformes</td><td>Paleozoic</td></tr><tr><td>Salmo salar</td><td>0.260</td><td>Salmoniformes</td><td>Paleozoic</td></tr><tr><td>Tenualosa ilisha</td><td>0.130</td><td>Clupeiformes</td><td>Triassic</td></tr><tr><td>Dicentrarchus labrax</td><td>0.020</td><td>Perciformes</td><td>Cretaceous</td></tr></tbody></table>

## 4. Basis of fish sperm lc-pufa composition

### 4.1. Biomechanics perspective

When it comes to sperm PUFA composition, the total n-6: n-3 PUFA ratio is less than 1, be it aquatic [82] or terrestrial animals, including humans [83], irrespective of dietary fatty acid supply. Similar to our observations in fish, the total n-6: n-3 PUFA ratio in boar (*Sus scrofa*) sperm was consistently below 1 (0.14-0.23) despite a wide variability in dietary total n-6: n-3 PUFA ratio (0.05-29.6) across four different diets [84]. Total n-6: n-3 PUFA ratio in human spermatozoa and sperm quality parameters such as motility, viability, morphology, and total spermatozoa were significantly inversely correlated (r≈ -0.31 to -0.58, *p*<0.01), clearly suggesting sperm performances being improved by a lower n-6: n-3 PUFA ratio. Fatty acid composition in sperm membrane of normozoospermic human male subjects had total n-6: n-3 PUFA ratio lower than 1 (0.77±0.05, *p*<0.05), compared to oligozoospermic (1.17±0.14, *p*>0.05), asthenozoospermic (0.9±0.06, *p*>0.05), and oligoasthenozoospermic human male subjects (1.41±0.2, *p*>0.05) which had sperm total n-6: n-3 PUFA ratio equal to or higher than 1 [85]. Our present analysis suggests that this may be true for fish as well. In rainbow trout, fed with a diet deficient in total n-3 fatty acids, lower sperm motility was observed [56]. Some earlier discussions by Dzyuba and colleagues postulated that fish sperm phospholipid bilayer containing more n-6 PUFA than n-3 PUFA to be softer and more deformable (≈higher flagellar beating in three-dimensional space) [60], but we observed no correlation between total n-6: n-3 PUFA ratio and biomechanics of sperm (Table 1).

Changes in the phospholipid class composition during sperm maturation have not been investigated in fish [12], but from the reviewed data it seems PC, PE, and SM (Table 2) are the dominant
phospholipid class in mature spermatozoa of fish. As previously described, the phospholipids from all sperm samples were very unsaturated with almost all molecular species from PE, PS, and PI containing at least one polyunsaturated acyl chain [12]. The same could be corroborated by our reviewed data across species, as the sum of total PUFA in sperm, irrespective of n-3 or n-6 series (Fig S1-S2), is ≥50% of all fatty acids, with 1/3rd or up to half of all PUFAs being the sum of ARA, EPA and DHA (Fig S3-S5). We have considered these three as the main LC-PUFA (ARA, EPA, DHA) in sperm. Among the three, DHA is the only LC-PUFA that showed a unanimous positive correlation with both VCL and motility duration simultaneously, whereas either EPA or ARA did not. Across the spectrum DHA also seems to be present in high proportions, likely due to its combined/ synergistic impact on both physiology and biomechanics. First, although DHA-specific lipid mediators are not described in fish sperm, DHA is traditionally believed to be 'not a substrate' of eicosanoids synthesis in fish sperm [44]. Whereas recent research suggests otherwise that resolvin D1 (RvD1), produced by DHA, exerts anti-inflammatory and pro-resolving activities in sperm [86]. DHA-derived specialized pro-resolving mediators such as resolvins and protectins are recently identified molecules that can orchestrate the timely resolution of inflammation [87, 88]. DHA and DHA-derived mediators have even been described to be an 'analgesic' [89], so its high presence in sperm is worth investigating beyond just structural significance. Second, the structural significance of DHA is huge for sperm phospholipid bilayer membranes. The best description in this regard is given by Bell and colleagues [12]. In the bilayer, DHA showed a high tendency to link with itself (DHA×DHA) and other LC-PUFA (DHA×EPA, DHA×ARA, DHA×DPA) giving 'DiPUFA' molecular speciation in sperm membranes of seabass, Atlantic or chinook salmon. [12]. Once esterified into phospholipids, DHA has been demonstrated to significantly alter many basic properties of membranes including acyl chain order and "fluidity", phase behavior, elastic compressibility, permeability, fusion, flip-flop and protein activity [90]. DHA's interaction with other membrane lipids, particularly cholesterol [90], may play a prominent role in modulating sperm membrane fluidity and "fusogenic properties" (discussed below). The fish sperm, besides having phospholipids, have a fair share of cholesterol too, with cholesterol-to-phospholipid ratio of 0.3 [47]. Under such circumstances, a DHA dominated phospholipid bi-layer membrane gives the most optimal biomechanical properties to sperm to swim and fuse with egg, with n-3 series DPA being the closest substitute in some cases/ species (discussed later).

The relationships between membrane composition (i.e., fatty acid unsaturation and acyl chain length, cholesterol, phospholipids, and proteins) and the physical parameter often referred to as "membrane fluidity" have been historically discussed [91-93]. Many reports now demonstrate that DHA-containing bilayers behave differently from bilayers containing the other major membrane PUFA, such as ARA [90, 94]. We postulate ARA's (20:4n-6) role in fish sperm is mostly limited to inflammatory eicosanoids synthesis. Its 22-carbon chain length elongated and desaturated product (i.e., 22:5n-6) is known not to be as efficient as 22:5n-3 or 22:6n-3 in biomembrane functions [95, 96]. Among features, in particular, DHA dominated membrane bi-layers reportedly [90, 94]: (a) increases the area/headgroup (e.g., lateral surface area for sperm swimming); (b) increases permeability to water and other solutes (e.g., enable osmoregulation, ion-channel function of sperm); (c) increases acyl chain flexibility (e.g., sperm flagellar beating and waveform function), and; (d) increases packing free volume, drives $H_{II}$ phase, induces negative curvature strain and may enhance formation of lateral domains (e.g., all relevant while fusing with egg).

## 4.2. Dietary perspective

It is now well established that the apparent digestibility of LC-PUFA (ARA, EPA, DHA) is generally highest of all practical fatty acids, increasing with increased unsaturation, and possibly aided by
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selective uptake of these essential fatty acids. The digestion, emulsification, absorption and transport of dietary lipids in fish is well described by Turchini and colleagues [97]. The lipid digestion starts with lingual and/or gastric lipases, partitioning lipid droplets into smaller vesicles that are easier to digest once entering the intestinal system. After leaving the stomach, or the anterior part of the intestine in stomach-less fish, lipid is usually solubilized by bile salts and pancreatic lipases (e.g., pancreatic triacylglycerol lipase, bile-stimulated triacylglycerol lipase, and phospholipase A₂) and other lipases secreted by enterocytes to further reduce droplet size and increase lipolytic efficiency. Following primary digestion, the partially digested lipids, acylglycerols (DAG, 1,3- and 2-MAG), lyso-phospholipids (lyso-PL), free fatty acids (FFA), and cholesterol will then reconstitute into smaller disc-shaped mixed micelles. Once delivered to the surface of enterocytes, they are absorbed across the enterocyte membrane, by membrane-associated proteins, and transported to subcellular compartments (endoplasmic reticulum) or cytosol by fatty acid transport proteins (FATP) [97]. Fish seem to express a variety of FATP in the intestine. Furthermore, due to the gene duplications that have taken place in fish, more variants of FATP are found in fish (genes *fatp 1-6*, including their α or β variants) [97]. After absorption into enterocytes, the products (lyso-PL, FFA, acylglycerols, cholesterol) go on their way to target tissues such as testes, via lipoprotein synthesis and exocytosis to the blood as mature chylomicrons containing high-, low- or very low-density lipoproteins (HDL, LDL, VLDL). The n-3 LC-PUFA (EPA, DHA) are mostly embedded on HDL, while n-6 LC-PUFA (ARA) is embedded on LDL or VLDL [97]. The preponderance of main intestinal and blood lipoproteins in fish differs by evolution too. For example, in primitive fishes like class Chondrichthyes, the major blood lipoprotein appears VLDL-like [98] and relatively high levels of VLDL/LDL are also seen in primitive ray-finned fish, like eels [99]. Higher ray finned fish, and especially teleost, have HDL as the prime lipoprotein [98, 100, 101]. Whether the differences hypothesized in sperm ARA: EPA ratio by evolutionary period (Table 3) could be due to dichotomy in LC-PUFA export mechanisms or eicosanoid preference, merits investigation.

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*De novo* synthesis of PL is largely dependent on the amount and type of dietary lipid, with PL and TAG having different contributions to endogenous PL re-synthesis [102]. It is believed that the principal precursor of PL synthesis is dietary PL, with lyso-PL and FFA taken up by enterocytes and re-acylated to reform PL [97], in testes for example. After exporting the main products, FFA, lyso-PL and acylglycerols, to target sites such as testicular cells, the triacylglycerol (TAG) and PL re-synthesis begins in endoplasmic reticulum. The most common PL classes in fish sperm membrane (PC, PE, SM) is reconstructed as follows (Fig 8), based on biosynthetic pathways for the major phospholipid classes described [97]. Interestingly, PC can be synthesized from multiple routes (possibilities) while PE offers multiple possibilities to synthesize other PL classes in sperm. This peculiar distribution can be compared to having a population with the majority universal donors with O blood group (the PE), as well as universal recipients with AB blood group (the PC) offering the largest flexibility. It is presently unknown whether such distribution of PL classes in fish sperm is intentional to allow maximum flexibility during spermatogenesis process. In Atlantic salmon, an increase in total testis content of fatty acids and PL, and a shift to higher PL composition was observed in maturing males, concomitant with increases in mRNA levels for genes involved in FA uptake and synthesis, and production of LC-PUFA and PL [103]. Testis fatty acid composition of gilthead seabream fed fish oil or blended vegetable oil diets showed significant differences. Vegetable oils decreased/ halved share of LC-PUFA (ARA, EPA, DHA) within total PUFA in testis (which was somewhat maintained constant due to homeostasis) [104].
<figure><img src="image_12.png" /><figcaption>**Fig 8: Biosynthetic pathways for the major phospholipid (PL) classes in fish testes or during spermatogenesis.** Numbers indicate enzymes: (1) CDP-DAG synthases, (2) PA-phosphatases, (3) PI synthase, (4) PS synthase, (5) CDP-choline: DAG phosphotransferase, (6) CDP-ethanolamine: DAG phosphotransferase, (7) PE methyltransferase, (8) PS decarboxylase, (9) SM synthase, (10) PE N-methyltransferase, and (11) PS synthases via base exchange. The green boxes constitute the majority PL classes in fish sperm (Table 2). The map is re-drawn from Turchini and colleagues [97].</figcaption></figure>

The ARA: EPA ratio in sperm could, in theory, be influenced by diet, as it has been discussed previously by Bell and colleagues [44] that improper ARA: EPA ratio in hatchery fish feed may have implications on broodstock gamete quality and unknown repercussions on larval vitality. For example, in goldfish testis, it was shown that n-6 LC-PUFA (ARA) stimulates testosterone production, whereas n-3 LC-PUFA (EPA) may function as inhibitory regulators of steroid production [105]. Our analysis suggests that ARA concentration, as well as ARA: EPA ratio in sperm may be quite resilient to dietary manipulation. There are some allostasis/ corrective measures to achieve the right n-6: n-3 PUFA ratio and ARA: EPA balance in fish sperm, independent of dietary perturbations. Rather, sperm EPA and DHA could be influenced directly by dietary levels. This is even true at the fish level, as fillet EPA+DHA reserves are synchronous with the richness of EPA+DHA in feed [1, 106]. However, from the dietary supply, EPA can be more prone to β-oxidation and further bioconversion, whereas DHA is almost non-catabolized in fish and is preferentially deposited and retained [81]. The same was corroborated in our analysis.

DHA is a very long chain n-3 PUFA, which is perhaps the most important ‘structural’ fatty acid for gametes [107], and more important than EPA. DHA’s central role has been established in human and other animal spermatozoa by several authors [13, 14, 33, 70]. While DHA in human spermatozoa could be positively correlated (r= 0.26-0.67, p<0.01) with motility, viability, morphology, and total spermatozoa count, such correlation could not be found for EPA [85]. Our analysis suggests DHA is stoichiometrically dominating over EPA in fish sperm and in most fish species. DHA is more abundant than EPA in sperm, even when the food base has the opposite composition (DHA < EPA). Systematic reviews of randomized controlled clinical trials show that DHA supplementation treats male infertility in human subjects by improving fertility markers in semen [108, 109]. Beirão and colleagues [110] reported that the sperm quality of *Solea senegalensis* improved through an enriched
DHA diet, specifically the sperm curvilinear velocity and share of progressive sperm. Once it is a very long chain (≥22) PUFA such as DHA, it is likely to have a direct effect on sperm membrane fluidity, as shown in human sperm [14, 33]. In a study by Bell and colleagues [12], it appeared that docosapentaenoic acid (22:5n-3, DPA) can substitute functionally for DHA within sperm phospholipid classes of two salmonids. In fact, large amounts of DPA were found in sperm from rabbit (*Oryctolagus cuniculus*), boar (*S. scrofa*), rat (*Rattus rattus*), stallion (*Equus ferus*), and rooster (*Gallus domesticus*), together with lower amounts of DHA [12]. The sperm of Nile tilapia, blunt snouted mullet, annular seabream in our dataset showed such peculiarity of a high DPA share, with lower share of DHA. It is interesting that a simple elimination of one double bond from DHA producing DPA (but omega-6; 22:5n-6) results in the loss of several behavioral features in animals as concluded by Stillwell and Wassall [90], whereas, multiple species of fish or animal substitute DHA by DPA (but omega-3; 22:5n-3). The biomechanical and physiological function of sperm membrane may remain uncompromised by such substitution. The n-3 DPA can form and substitute for some D-series resolvins, otherwise produced by DHA [88].

Sperm ARA: EPA ratio is an index of good sperm quality in human [33]. The ARA-to-EPA stoichiometry in cellular membranes determines eicosanoid actions [7, 27], such as in fish sperm [44]. Our analysis suggests that the stoichiometry of ARA to EPA in fish sperm is not completely malleable by dietary perturbations; in fact, it may be physiologically regulated. The ARA produces pro-inflammatory eicosanoids that are biologically more active (potent), such as 2-series prostaglandins (PGE₂). The EPA produces reduced pro-inflammatory or anti-inflammatory eicosanoids that are biologically less active (potent), such as 3-series prostaglandins (PGE₃; biologically less active) [7]. Our analysis showed that ARA is selectively preserved among n-6 PUFAs in fish sperm, but not all fish species have ARA > EPA in sperm. Although eicosanoids and prostaglandins have not been studied in fish sperm in detail, during motility, the ARA: EPA ratio (high activity: low activity eicosanoids) in fish sperm may change significantly, but not DHA: EPA ratio (both low bioactive eicosanoid pre-cursors) [19]. The results indicate there could be a deliberate ARA: EPA balancing in fish sperm, independent of diet and related to species-specific eicosanoids biology or tolerance/detection limits of eicosanoids action (inflammatory, anti-inflammatory responses, chemotaxis, etc.) [27] on prostaglandin receptors on sperm membrane [76], presently unknown in fish.

### 4.3. Evolutionary and physiological perspective

As proved in the rabbit or stallion testis/ epididymis [111, 112], fish testes could be an active site of fatty acid metabolism. Especially *de-novo* LC-PUFA (ARA, EPA, DHA) biosynthesis by desaturation and elongation, as active as in other PL-rich tissue such as brain [65, 113]. The presence of enzymes (Δ4, Δ5, and Δ6 desaturases, along with elongase-2, elongase-5) necessary for ARA, EPA, and DHA synthesis in fish testes, primary spermatocytes/ spermatogonia (diploid cell) have not been widely studied. However, a recent transcriptomic profiling of fish spermatozoa (haploid from diploid cell) hinted at their existence [114]. During spermatogenesis in eel, the EPA, ARA and DHA were reported to be fortified in testes relative to other fatty acids, although it was hypothesized as transported from liver, and not synthesized on-site [115, 116]. In Atlantic salmon testis, an increased expression of genes for elongation/desaturation to DHA was observed in sexually maturing males relative to immature fish. Besides, there was concomitant increases in phosphatidic acid, a backbone of PL (Fig 8), in maturing males compared to immature fish [103]. This coincidence must be towards spermatogenesis, as DHA is a major LC-PUFA and PL is a major lipid class in fish sperm. It is hence suggested that fish testes are a major site of *de-novo* LC-PUFA biosynthesis.
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It seems that the fish sperm itself has preserved both lipid catabolism and anabolism functions [117], much like human spermatozoa [118], albeit primitive spermatozoan design or motility duration limited to minutes in fish [16, 34]. Fish sperm has enzymatic capacity for glycolysis, triglyceride, and phospholipid catabolism, as well as triglyceride synthesis [22]. Fatty acid synthase (FAS) activity responsible for initiating *de-novo* fatty acids synthesis is present. The FAS activity is comparable to that of phospholipase, pyruvate kinase, and glucose-6-phosphate dehydrogenase activities in fish sperm [22]. Transcriptomic study in haploid fish spermatozoa recently [114] revealed most enriched genes in fish sperm could be related to fatty acid biosynthesis, namely elongases *elovl1*, *elovl5*, *elovl6* (but no desaturases, *fads*, or *scd* reported). Compared to haploid germ cells, fish sperm could have several-fold upregulated mRNAs pertaining to eicosanoids, namely arachidonate lipoxygenase synthesis (*alox15b*); leukotrienes, thromboxane, or prostaglandin too, mentioned in supplementary data of Castro-Arnau and colleagues [114]. This evidence indicates that haploid fish spermatozoa may share partly the capabilities of diploid somatic cells, which can adjust the fatty acid composition of its membrane as or when necessary, like epithelioma papillosum cells (EPC) or liver cells in fish [119, 120]. Recently, eicosanoid actions in Eurasian perch sperm, as it ascended from basal metabolism (pre-activation quiescent state) to active metabolism (post-activation exhaustion), was also hypothesized [19]. Our results of feed to sperm changes in ARA: EPA and DHA: EPA stoichiometries corroborate the presence of such bio-machineries. An absence of desaturases in the transcriptome of spermatozoa of the marine carnivorous fish [114] could be related to marine habitat, as they thrive on an EPA+DHA rich diet with no real need for endogenous bioconversion [3, 4]. A similar transcriptomic study on spermatozoa of a freshwater fish species is needed in the future, to determine if probe *elovls* and *fads*, *scd* are present. As freshwaters are DHA deficient, there is a general need for its endogenous bioconversion to reach physiologically relevant levels [67, 69]. However, our results show, not all freshwater species may engage in allostasis (corrections) of DHA in sperm, relative to food base. Some species, such as northern pike, could simply reflect in sperm what it eats in ponds/ lakes [121].

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It may be related to the loss of certain genetic repertoire at cellular levels to endogenously biosynthesize or do bioconversion of DHA [2, 3]. The 3R WGD (third round whole genome duplication) event occurred around 320-350 mya, leading to the emergence of teleost [122], from marine and cartilaginous ancestors. Before 3R WGD event, there was a similar divergence from marine cartilaginous fishes who had both *fads1* and *fads2* [3] to freshwater cartilaginous fishes such as riverine sting rays (e.g., *Potamotrygon* sp.) that may have partly retained *fads1* or *fads2* [72, 123]. In our analysis, the early-diverged freshwater elasmobranchii *P. motoro* (ocellate river stingray), which evolved before the 3R WGD event, shows a weakened DHA biosynthesis capacity or DHA: EPA ratio in sperm. The 4R WGD event (fourth-round whole genome duplication), that happened around 100 mya [122], could be related to *Salmo salar*, *Oncorhynchus mykiss*, *Lota lota*, and *Esox lucius*. After 4R WGD, fishes either retained (e.g., rainbow trout) or completely lost (e.g., northern pike, burbot) DHA synthesis capacity [2, 3, 124]. After 4R WGD, Northern pike or burbot became a uniquely diverged genome from a sister group of freshwater Salmoniformes or marine Gadiformes; living in freshwaters [71, 72]. The loss of efficient DHA biosynthesis machinery in Northern pike have been discussed before [125]. In these species, absence of Δ5 desaturase [126], absence of Δ4 desaturase forcing partial DHA synthesis via less efficient Sprecher pathway [127], presence of monofunctional Δ5 *fads2* forcing partial DHA synthesis only from C20 precursors, or loss of *elovl2* functions (inability to elongate C20 or C22 substrates for DHA biosynthesis) have weakened DHA synthesis capacity [2, 3, 124]. Irrespective of a loss in desaturation capacity, a single-step elongated product of EPA, 20:5n-3 (i.e., DPA, 22:5n-3) have the potential to substitute DHA within fish sperm phospholipid [12], as observed in some fish species in this review (tilapia, blunt snouted mullet,
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annular seabream). However, a loss of *elovl2* in such fish lineages would prevent such substitution from happening too. Figure 9 presents a general LC-PUFA biosynthesis pathway, which may be active in fish testes or sperm.

<figure><img src="image_13.png" /></figure>

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<caption>Fig 9: Biosynthetic pathways for the major LC-PUFA (ARA, EPA, DHA or DPA) in fish testes or during spermatogenesis. Major sperm LC-PUFA are highlighted in red box. Above arrows, the enzymes and genes (in parenthesis) are mentioned, based on a general understanding [97, 113].</caption>

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In wild fish with swimming habitat across different gradients of temperature, pH, salinity/ osmolality, and ecological niches or hydrostatic pressure, the interquartile range (IR) of whole-body DHA: EPA ratio is from ~1:1 to ~5:1 (median ~2:1); recalculated from published dataset [73]. This analogy can be drawn for swimming fish sperm in external or internal environment too. Our observed range of sperm DHA: EPA ratio was identical to IR of whole-body DHA: EPA by Gladyshev and colleagues [73]. There were several species having DHA: EPA stoichiometry in sperm below the median of 2 or even worse <1. An analysis is yet to be done on how missing DHA in sperm impacts swimming behavior [34] in those species relative to others. Our analysis shows the DHA: EPA stoichiometry in fish sperm may have an evolutionary/ genetic pre-disposition to engage or not engage in allostasis or corrective measures from food to sperm. Molecular modeling studies have suggested that DHA favored conformations (angle-iron or helix) which give phospholipids containing DHA unusual packing properties. Or, DHA dominated phospholipid fractions give unstable lipid bilayers with fusogenic properties [12]. The former aspect (packing properties) could be important for sperm flagellar waveform [34], while the latter (fusogenic properties) may be important for promoting fusion between the plasma membrane of fish eggs and sperm, as discussed by Bell and colleagues [12]. DHA has the smallest volume change with the temperature of any fatty acid, thus important in preserving a thermal adaptation of sperm biomembrane across temperatures [12].

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In fish sperm, it has been hypothesized that ARA-derived PGE₂ serve more important functions than EPA-derived PGE₃ [44], and linked with acrosome reactions [75]. In male fish, such prostaglandins are linked with chemotaxis for opposite sexes [128]; same may be speculated for sperm seeking an egg. Our analysis suggests, in addition to influences of feed/ diet, Paleozoic and Triassic era diverged genomes of fish, may be potentially inclined towards lower amounts of highly bioactive eicosanoids
in sperm (i.e., ARA-derived PGE₂ < EPA-derived PGE₃). Whereas, the argument by Bell and colleagues that sperm may prefer more of highly bioactive and pro-inflammatory eicosanoids [44] may hold true for fish genomes diverged in Devonian, Jurassic or Cretaceous era. For example, in the Triassic era, diverged fish genome such as European eel [63, 115], diets with high ARA induced medium milt volumes and high sperm motility, but diets with higher EPA induced 'remarkable' volumes of milt with high sperm motility. It is to be noted that like *fads* and *elovl* gene repertoire (discussed above), the cyclooxygenase (*cox*) gene sequences too show a complicated history of duplications and losses [129]. The *cox* produces prostaglandins in animals. In our reviewed data, we observed that some fish sperm function at higher ARA: EPA ratio, while some function at lower ARA: EPA ratio, showing a potential dichotomy in choice of eicosanoids or prostaglandin synthesis pathways. It may also mean a competition between rate-limiting *cox-1* and *cox-2* enzymes for a common substrate (ARA or EPA; [7]) in a species-specific manner, in fish sperm, which is currently an unexplored territory. However, both isoforms (*cox-1*, *cox-2*) can be expressed constitutively in the testes [88]. In simplistic terms, *cox-1* is the enzyme responsible for basal, constitutive prostaglandin synthesis, whereas *cox-2* is important in various inflammatory and "induced" (e.g., induced breeding) settings [27]. Havird and colleagues [129] hinted at a functional diversification of *cox-2a* and *cox-2b* genes before cyprinids genome diverged, with a possible loss of *cox-2a* in some derived teleosts during that time. The previous era before cypinids genome emerged (around Jurassic era) was Triassic era and some derived teleost genome during this era could have been clupeids and eel-like fishes [71]. Some fish genomes diverged during paleozoic era [71] had also documented possible loss in *cox-1a* gene in some species of the order Salmoniformes [129]. Not much is known in this field, see discussions [129], and our exploratory analyses justifies further scientific investigation with spermatozoa as a model. Recent research has demonstrated an altered behavior of fish sperm under exposure to ovarian fluid. For example, guiding or promoting the spermatozoon to find its way to the egg [31, 32], enhanced sperm motility, prolonged sperm longevity [130], or activation from a prolonged hibernation [131]. This is an indication that eicosanoids or prostaglandin receptors must exist in fish sperm too, as in human or *C. elegans* enabling sperm rheotaxis/ chemotaxis (see introduction). In fact, eicosanoids could be the main drive stimuli, besides ATP, in fish sperm that must swim in external aquatic environment, without immediate direct access to ovarian fluid always (except internal-fertilizing fish). Current research on fish sperm rheotaxis or biomechanics (exposed to ovarian fluid; [31, 32, 130, 131]) discuss theories limited to salinity, osmotic pressure/ osmolality, pH, membrane ion-channel functions, ion, energy, proteins, amino acids and sugar uptake, etc. But an alternating mechanism where ARA-derived eicosanoids cause inflammation, and EPA/DPA/DHA-derived specialized pro-resolving mediators (SPMs; [88]) work to resolve it, cannot be overlooked. All n-3 LC-PUFA in fish sperm (EPA, DHA) is involved in SPM, including n-3 DPA which can substitute DHA in production of D-series resolvins (e.g., RvDn-3DPA [88]). Hence, lipid mediators and eicosanoid biology of fish sperm (Fig 10) is suggested as a new area of research [65].
<figure><img src="image_14.png" /><figcaption>**Fig 10: Hypothesized lipid-mediators in fish sperm membrane modulating inflammation and its resolution during motility.** The map is based on Harwood [88], Oliver and colleagues [132].</figcaption></figure>

## 5. Conclusion

This review shows that LC-PUFAs are not merely structural components of fish sperm, but biomechanical, physiological cornerstones too. For enabling a smooth function or behavior of sperm, a fish ejaculating in an external water environment and not in ovarian fluid, must have some auto-guidance capabilities modulated by eicosanoids balance. A lower ARA: EPA ratio enables searching longer (motility duration) perhaps, as a higher preponderance of inflammatory eicosanoids or ARA presence may induce swelling/ inflammation that is not very comfortable to move. Yet some fish sperm function at higher ARA: EPA ratio, while some function at lower ARA: EPA ratio, showing a dichotomy in choice of eicosanoids or prostaglandin synthesis pathways. In classic fish sperm literature, a lot of emphasis has been on biophysics or osmolality-induced swelling of sperm, due to water intake or malfunctioning of membrane ion channels or depleted energy/ substrates, resulting in cessation of motility. But the immunological aspects of fish sperm motility such as eicosanoids modulating inflammation or anti-inflammation are not explored.

Negative association of ARA with sperm motility duration but not VCL could be related to an example of a swollen finger which is movable, but poses some resistance to move, caused by some local inflammation by higher ARA-oxidized eicosanoids. Conversely, higher DHA (or at times substituted by DPA) may enable faster searching and fertilizing, as associated positively with both VCL and motility duration. Additionally, the dietary influences show highest conservation of DHA in sperm over EPA or ARA, being the only LC-PUFA that allow highest membrane packaging/ re-packaging/ fusion capabilities, least change to biophysical stress (thermal-pressure conditions), and
752 not usually subjected to catabolism or inflammatory eicosanoid production. The fact that diets can
753 influence them significantly, and some fish genomes (lineages) cannot undertake sufficiently
754 corrective measures during spermatogenesis, needs careful consideration. Especially in hatchery-bred
755 fish for aquaculture or released to natural waters, undesirable man-made selection (trade-off with
756 natural selection) or downstream consequences to progeny may go unchecked/ overlooked due to
757 usually high *libido* and sperm count in male fish, compared to female fish where research is more
758 concentrated.

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