Differential distribution of eicosanoids and polyunsaturated fatty acids in the Penaeus monodon male reproductive tract and their effects on total sperm counts

All experiments were approved by the Institutional Animal Care and Use Committee of the National Center for Genetic Engineering and Biotechnology, Thailand (Approval Code BT-Animal 13/2560). This permit covered the purchase wild-caught shrimp, shrimp transportation, shrimp rearing experiment, and shrimp dissection. No permit was required for the collection site access as the wild-caught broodstock collection from the Andaman Sea was conducted by local fishermen and purchased through a local shrimp farm. All experiments were performed in accordance with Animal Research: Reporting of In Vivo Experiments (ARRIVE) and conformed with international and national legal and ethical requirements, including the U.K. Animals (Scientific Procedures) Act, 1986 and associated guidelines, EU Directive 2010/63/EU for animal experiments, and the National Research Council’s Guide for the Care and Use of Laboratory Animals.

Wild-caught male shrimp were captured from the Andaman Sea, Thailand (salinity level at approximately 31 ppm) (N = 10). Eleven-month-old domesticated male P. monodon, which had been raised in earthen ponds and fed with commercial feed pellets, were acquired from the Shrimp Genetic Improvement Center (SGIC), Surat Thani, Thailand (N = 10). Average body weights of wild-caught and domesticated shrimp were 86.9 ± 9.0 and 66.8 ± 7.6 g, respectively. Shrimp testes and vas deferens were dissected and flash frozen in liquid N₂ for the quantification of eicosanoids and PUFAs using UHPLC-HRMS/MS. Spermatophores were collected and used for total sperm counts.

To determine changes in eicosanoid and PUFA levels in shrimp fed with different diets, eleven-month-old, domesticated males from the SGIC were fed with either polychaetes or feed pellets for four weeks (N = 8 each). Fatty acid profiles in polychaetes and feed pellets (N = 4 per feed) were analyzed using gas chromatography coupled with flame ionization detector (GC-FID) by the Nutrition Service at Central Lab Co., Ltd. (Thailand) (www.centrallabthai.com↗). Shrimp testes and vas deferens were dissected and flash frozen in liquid N₂. Spermatophores were collected and used to determine total sperm counts and percentage of sperm abnormality.

Spermatophores were individually homogenized in a calcium-free sea water solution. After debris sedimentation, sperms were counted using a hemocytometer under a light microscope [19]. Abnormal sperms were defined as sperms with a misshaped head or tail as well as sperms with no head or tail [20]. Total sperm counts and abnormal sperm counts were determined from both spermatophores of each shrimp using average counts of four aliquots from each spermatophore homogenate. The percentage of abnormal sperm were then calculated based the percentage of abnormal sperm from the number of total live sperm.

Eicosanoid standards were purchased from Cayman Chemicals (Michigan, USA). Standard compounds include prostaglandin D₂ (PGD₂), prostaglandin E₁ (PGE₁), PGE₂, PGF_2α, 15d-PGJ₂, (±)5-hydroxy-6E,8Z,11Z,14Z-eicosatetraenoic acid ((±)5-HETE), (±)8-hydroxy-5Z,9E,11Z,14Z-eicosatetraenoic acid ((±)8-HETE), (±)9-hydroxy-5Z,7E,11Z,14Z-eicosatetraenoic acid ((±)9-HETE), (±)11-hydroxy-5Z,8Z,12E,14Z-eicosatetraenoic acid ((±)11-HETE), 12(R)-hydroxy-5Z,8Z,10E,14Z-eicosatetraenoic acid (12(R)-HETE), (±)5-hydroxy-6E,8Z,11Z,14Z,17Z-eicosapentaenoic acid ((±)5-HEPE), (±)8-hydroxy-5Z,9E,11Z,14Z,17Z-eicosapentaenoic acid ((±)8-HEPE), (±)9-hydroxy-5Z,7E,11Z,14Z,17Z-eicosapentaenoic acid ((±)9-HEPE), (±)12-hydroxy-5Z,8Z,10E,14Z,17Z-eicosapentaenoic acid ((±)12-HEPE), (±)15-hydroxy-5Z,8Z,11Z,13E,17Z-eicosapentaenoic acid ((±)15-HEPE), (±)18-hydroxy-5Z,8Z,11Z,14Z,16E-eicosapentaenoic acid ((±)18-HEPE), ARA, DHA, and EPA. Deuterated compounds, namely PGE₂-d₄, 5(S)-HETE-d₈, 12(S)-HETE-d₈, and EPA-d₅, were used as internal standards to determine percent recovery during chemical extraction and during UHPLC-HRMS/MS analysis. All solvents and chemicals used in this study were HPLC grade or higher. Glacial acetic acid, acetonitrile, methanol, and ethanol were purchased from Merck (Darmstadt, Germany). Formic acid and cyclohexane were purchased from Fisher Scientific (Loughborough, UK). Hexane was purchased from J.T. Baker (New Jersey, USA). Ethyl acetate was purchased from Mallinckrodt Baker (New Jersey, USA). Isopropanol was purchased from RCI labscan (Bangkok, Thailand). Butylated hydroxytoluene (BHT) and Hank’s Balanced Salt Solution (HBSS) were purchased from Sigma-Aldrich (Missouri, USA). Ethylenediaminetetraacetic acid (EDTA) was purchased from Fluka (Steinheim, Switzerland). Water was purified by Barnstead GenPure Pro (Thermo Fisher Scientific, Massachusetts, USA).

Shrimp testes and vas deferens were individually homogenized in liquid N₂ and diluted in HBSS to adjust tissue concentration to 0.1 g/mL (wet weight). Organ homogenates were divided into 500 μL aliquots and adjusted to pH 4.0 using 5 μL of glacial acetic acid. Ten microliters of 10% BHT in HPLC-grade ethanol (w/v) were added as an antioxidant. Internal standards, including PGE₂-d₄, 5(S)-HETE-d₈, and EPA-d₅, were added to determine the percent recovery in each sample. An optimal extraction method was selected for each organ based on the recovery yields of the internal standards (S1 Table).

Five hundred microliters of testis homogenates were subjected to ethyl acetate extraction at a 1:1 ratio (v/v) of tissue homogenate to ethyl acetate. Extraction mixtures were shaken in the dark for 15 min at 290 rpm and spun down at 8,000 rpm (8,228 ×g) for 10 min at 20°C. The organic phase (upper phase) was collected, and the extraction process was repeated one more time. The extracts were evaporated to dryness and dissolved with 200 μL of 100% HPLC-grade ethanol for UHPLC-HRMS/MS analysis.

Five hundred microliters of vas deferens homogenates were subjected to methanol-chloroform extraction using the procedure modified from Folch extraction method [21]. Tissue homogenates were sequentially mixed with 3.75 mL of methanol, 6.25 mL of chloroform, and 3.12 mL of water. Samples were mixed rigorously for 1 min after each solvent was added. The mixture was shaken for 15 min at 290 rpm at room temperature and spun down at 8,000 rpm at 20°C for 10 min. The organic phase (lower phase) was collected in a clean tube. The extraction was repeated by adding 3.75 mL of chloroform to the remaining aqueous phase. The mixture was vortexed for 1 min, shaken for 15 min at 290 rpm, and then spun down at 8,000 rpm at 20°C for 10 min. The organic phase was collected, pooled, dried, and dissolved with 200 μL of 100% HPLC-grade ethanol for UHPLC-HRMS/MS analysis.

Chromatographic separation was performed on a Dionex UltiMate 3000RS UHPLC system (Thermo Fisher Scientific) with an Acclaim^TM RSLC 120 C18 column (2.1×150 mm, 2.2 μm; Thermo Fisher Scientific) under gradient conditions using mobile phase A (0.01% (v/v) acetic acid in water) and B (0.01% (v/v) acetic acid in acetonitrile) as previously described [22]. The linear gradient went from 30% B to 100% B within 17 min, followed by holding 100% B for 2 min. The elution gradient was returned to the starting condition of 30% B within 0.5 min and kept constant for 3.5 min before starting the next injection. UHPLC conditions included setting auto-sampler temperature at 10°C, column temperature at 40°C, injection volume at 5 μL, and flow rate at 300 μL/min for a total run time of 23 min.

Mass spectrometry analyses were performed on an Orbitrap Fusion™ Tribrid™ Mass Spectrometer (Thermo Scientific), equipped with electrospray ionization (ESI) source, and operated in negative ion mode. The mass spectrometer was controlled by the Xcalibur software (version 4.4.16.14) and calibrated using the ESI negative ion calibration solution (Pierce® LTQ velos ESI negative ion calibration) according to the manufacturer’s protocol. Conditions for the mass spectrometer were set with the ESI voltage at 2,500 V in negative mode. Nitrogen was used as the sheath gas at 40 psi and as the auxiliary gas at 12 psi. Ultra-pure helium was used as the collision gas with the ion transfer tube temperature at 333°C. The vaporizer temperature was 317°C. Fragment ions of PUFA and eicosanoid standards were detected by the Orbitrap analyzer operated under target mass resolution of 120,000 with an automatic gain control (AGC) setting of 5×10⁴ and a maximum ion injection time of 250 ms. The time-scheduled parallel reaction monitoring (PRM) method was used for data acquisition. Analytical characteristics of PUFA and eicosanoid standards used to identify and quantify the compounds in P. monodon tissues are provided in S2 Table. Both limit of detection (LOD) and limit of quantification (LOQ) were calculated based on the standard deviation (SD) of the response as well as the slope [23, 24]. LOD=3.3×SD(slopeoftheregressionline)LOQ=10×SD(slopeoftheregressionline) SD represents standard deviation of a blank sample with very low concentration (0.24–7.81 nM) of the measurand.

Extracted-ion chromatograms (XIC) and mass spectra of eicosanoids and PUFAs obtained from the UHPLC-HRMS/MS analysis were processed and interpreted using Quan Browser (4.3.73.11), Xcalibur software (version 4.3.16.14). The area-under-the-curve (AUC) ratio of each metabolite was calculated by dividing the AUC of chromatographic peak of each respective metabolite with the AUC of the internal standard (12(S)-HETE-d₈). A pivot table of metabolite AUC ratios was constructed using Pandas (version 1.1.3, http://pandas.pydata.org↗), Python package [25, 26]. A heat map illustrating the AUC ratios of the metabolites was generated using Matplotlib (version 3.3.2, https://matplotlib.org/↗) and Seaborn (version 0.11.0, https://seaborn.pydata.org/↗), Python package [27, 28]. AUC ratios were converted to amounts of metabolites (ng/g tissue) using standard equations shown in S2 Table.

Significant differences between the means of independent samples from the two sets of samples were assessed using the t-test with the threshold for significance set at P < 0.05 (*, † and #) or P < 0.01 (**, †† and ##).

Wild-caught male P. monodon broodstocks were captured from the Andaman Sea, Thailand (Fig 1A). Shrimp body weight and body length were recorded prior to dissection to obtain testes, vas deferens, and spermatophores (Fig 1B–1D). Similarly, domesticated males were obtained from SGIC, a biosecure facility located in Surat Thani Province, Thailand (Fig 1E). Their testes, vas deferens, and spermatophores were also collected (Fig 1F–1H). It should be noted that all shrimp spermatophores were intact without melanization. Data analysis revealed that wild-caught shrimp had larger body weight (Fig 1I), longer body length (Fig 1J), and higher spermatophore weight (Fig 1K) than those of domesticated shrimp. Additionally, the total sperm counts of wild-caught shrimp were also higher than those of domesticated shrimp (Fig 1L).

To determine eicosanoid and PUFA profiles in P. monodon male reproductive organs, shrimp testes and vas deferens were subjected to ethyl acetate extraction and methanol-chloroform extraction, respectively. The organ extracts were then analyzed using UHPLC-HRMS/MS as depicted in Fig 2. The identity of each metabolite was verified based on retention time, precursor ion, proposed fragment ion, and m/z distribution (S3 Table). Testes and vas deferens of wild-caught and domesticated shrimp contained a combined number of 10 eicosanoids, including three prostaglandins (PGE₂, PGF_2α, and 15d-PGJ₂), three HETEs ((±)8-, (±)11-, and 12(R)-HETEs), and four HEPEs ((±)8-, (±)12-, (±)15-, and (±)18-HEPEs) (Fig 3A–3J). Additionally, all three PUFAs, namely ARA, DHA, and EPA, were detected in all organ samples (Fig 3K–3M).

Heat map analysis was used to compare relative levels of eicosanoids and PUFAs based on the AUC ratio obtained from the UHPLC-HRMS/MS analysis (Fig 4). Testes of wild-caught shrimp contained seven eicosanoids, including PGE₂, PGF_2α, 15d-PGJ₂, (±)8-HETE, 12(R)-HETE, (±)8-HEPE, and (±)12-HEPE (Fig 4A, upper panel). Among these, PGF_2α, (±)8-HETE, and (±)8-HEPE were present with high intensities in the heat map, suggesting that these eicosanoids may play crucial roles in spermatogenesis. Additionally, all three PUFAs were present in shrimp testes, in which ARA, DHA, and EPA were detected at low, medium, and high intensities relative to one another, respectively.

UHPLC-HRMS/MS analysis revealed that eight eicosanoids and three PUFAs were detected in vas deferens of wild-caught shrimp. In addition to the seven eicosanoids previously identified in testes, (±)18-HEPE was present in vas deferens with low intensities in the heat map (Fig 4A, lower panel). In contrast, (±)8-HETE and (±)8-HEPE were present with high intensities in vas deferens. Relative levels of ARA, EPA, and DHA in vas deferens were also similar to those in testes of wild-caught shrimp.

Heat map analysis of eicosanoids and PUFAs in testes and vas deferens of domesticated shrimp revealed different patterns from those in wild-caught shrimp. Three PUFAs and ten eicosanoids were detected in both testes and vas deferens of domesticated shrimp. The two additional eicosanoids identified only in domesticated shrimp were (±)11-HETE and (±)15-HEPE, which were detected at low intensities in both testes and vas deferens. When relative levels of eicosanoids were examined in testes of domesticated shrimp, it was observed that all ten eicosanoids were present at relatively low intensities in the heat map, which was different from the pattern observed in testes of wild-caught shrimp. On the other hand, the heat map of vas deferens of domesticated shrimp displayed similar metabolic profiles to those of wild-caught shrimp, in which (±)8-HETE and (±)8-HEPE were major products of this pathway. Moreover, EPA was consistently the most abundant metabolite in testes and vas deferens of both wild-caught and domesticated shrimp, which illustrates the importance of EPA in the P. monodon sperm maturation process.

To follow metabolic changes that occurred during the sperm maturation process, levels of eicosanoids and PUFAs in shrimp testes were compared with those in vas deferens. In both wild-caught and domesticated shrimp, testes contained higher levels of PGE₂ (Fig 5A), but lower levels of 15d-PGJ₂ (Fig 5C), (±)8-HETE (Fig 5D), and (±)12-HEPE (Fig 5H) than vas deferens. On the other hand, levels of the remaining eicosanoids and PUFAs varied, depending on the shrimp source. In wild-caught shrimp, (±)18-HEPE (Fig 5I) was below the detection limit in testes but was detected at 4.18 ± 1.76 ng/g tissue in vas deferens. On the other hand, levels of PGF_2α (Fig 5B), (±)11-HETE (Fig 5E), 12(R)-HETE (Fig 5F), (±)8-HEPE (Fig 5G), ARA (Fig 5J), DHA (Fig 5K), and EPA (Fig 5L) were comparable between testes and vas deferens of wild-caught shrimp.

In domesticated shrimp, testes contained higher levels of PGF_2α (Fig 5B), but lower levels of 12(R)-HETE (Fig 5F), (±)8-HEPE (Fig 5G), ARA (Fig 5J), DHA (Fig 5K), and EPA (Fig 5L) than vas deferens. Interestingly, (±)11-HETE was detected only in vas deferens of domesticated shrimp (Fig 5E). As (±)11-HETE was below the limit of detection in vas deferens of wild-caught shrimp, it is likely that this metabolite is not essential for the sperm maturation process in P. monodon.

As wild-caught shrimp produced higher total sperm counts than domesticated shrimp, levels of eicosanoids and PUFAs in testes of wild-caught shrimp were compared to those in domesticated shrimp to determine correlations between these metabolites and total sperm counts. Testes of wild-caught shrimp contained higher levels of (±)8-HEPE (Fig 5G), but lower levels of (±)12-HEPE (Fig 5H), (±)18-HEPE (Fig 5I), and EPA (Fig 5L) than domesticated shrimp. On the other hand, levels of PGE₂ (Fig 5A), PGF_2α (Fig 5B), 15d-PGJ₂ (Fig 5C), (±)8-HETE (Fig 5D), 12(R)-HETE (Fig 5F), ARA (Fig 5J), and DHA (Fig 5K) in the testes of wild-caught and domesticated shrimp were comparable. Lastly, (±)11-HETE (Fig 5E) was not detected in testes in both wild-caught and domesticated shrimp, suggesting that this metabolite was not involved in shrimp spermatogenesis.

In vas deferens, wild-caught shrimp contained higher levels of PGE₂ (Fig 5A) and PGF_2α (Fig 5B), but lower levels of 15d-PGJ₂ (Fig 5C), (±)8-HETE (Fig 5D), (±)11-HETE (Fig 5E), 12(R)-HETE (Fig 5F), (±)12-HEPE (Fig 5H), ARA (Fig 5J), DHA (Fig 5K), and EPA (Fig 5L) than domesticated shrimp. Based on these data, it was deduced that high levels of (±)8-HEPE in testes and high levels of PGE₂ and PGF_2α in vas deferens are associated with high sperm counts. On the other hand, high levels of (±)12-HEPE, (±)18-HEPE, and EPA in testes and high levels of 15d-PGJ₂, (±)8-HETE, (±)11-HETE, 12(R)-HETE, (±)12-HEPE, and PUFAs in vas deferens are correlated with low sperm counts in P. monodon. Although (±)15-HEPE was identified in both the testes and vas deferens of domesticated shrimp as shown in the XIC (Fig 3I) and the heat map (Fig 4B), this metabolite was detected at the level above the limit of detection in only 2 out of 10 shrimp samples (S4 File). Therefore, (±)15-HEPE was excluded from the quantitative analysis.

In hatcheries, domesticated males are typically fed with live Perinereis nuntia polychaetes instead of commercial feed pellets to increase total sperm counts. To test the effects of shrimp feed on PUFA and eicosanoid profiles in male reproductive tract, eleven-month-old, domesticated males from the same genetic background were fed with either polychaetes or feed pellets for four weeks. Polychaetes and feed pellets were analyzed using GC-FID, revealing that polychaetes contained higher levels of total saturated fatty acids, monounsaturated fatty acids, and polyunsaturated fatty acids, including ARA, EPA, and DHA, than feed pellets (Table 1). Shrimp fed with polychaetes also had higher sperm counts (Fig 6A), but comparable percentage of sperm abnormality to those of pellet-fed shrimp (Fig 6B).

To determine whether eicosanoid and PUFA profiles in the male reproductive tract were affected by shrimp diet, testes and vas deferens of polychaete- and pellet-fed shrimp were analyzed using UHPLC-HRMS/MS. First, levels of eicosanoids and PUFAs were compared between testes and vas deferens of shrimp in each feed group to determine metabolic changes during spermatogenesis and sperm maturation process, respectively. Data analysis revealed that the majority of the metabolites, including 15d-PGJ₂ (Fig 7C), (±)8-HETE (Fig 7D), 12(R)-HETE (Fig 7F), (±)12-HEPE (Fig 7G), (±)18-HEPE (Fig 7H), ARA (Fig 7I), DHA (Fig 7J), and EPA (Fig 7K), were detected at higher levels in vas deferens than testes of shrimp in both feed groups, suggesting that these metabolites were more essential in the sperm maturation process than spermatogenesis. Meanwhile, levels of PGF_2α were comparable between testes and vas deferens of shrimp in both feed groups (Fig 7B). The two eicosanoids with distinct metabolic patterns according to feed types were PGE₂ and (±)11-HETE (Fig 7A and 7E). More specifically, levels of PGE₂ were comparable between testes and vas deferens of polychaete-fed shrimp (Fig 7A). In pellet-fed shrimp, however, PGE₂ was detected at similar levels in testes but became undetectable in vas deferens, suggesting that the use of feed pellets reduced the levels of PGE₂ in this organ. In contrast, (±)11-HETE was absent in most tested samples except in vas deferens of polychaete-fed shrimp (Fig 7E). These data suggest that although changes in shrimp diet did not alter relative levels of most PUFAs and eicosanoids in shrimp testes and vas deferens, the distribution of certain ARA-derived eicosanoids, namely PGE₂ and (±)11-HETE, in vas deferens was affected by shrimp feed. Lastly, (±)8-HEPE and (±)15-HEPE were excluded from the analysis as they were quantifiable in less than 50% of samples.

Results from other studies as well as data from our own analysis (Fig 6) revealed that polychaete-fed shrimp had higher total sperm counts than pellet-fed shrimp [18, 20] However, the effects of shrimp diets on levels of PUFAs and eicosanoids in shrimp testes and vas deferens have yet to be investigated. In this study, levels of eicosanoids and PUFAs in testes were compared between polychaete- and pellet-fed shrimp to assess the impact of shrimp feed. Testes of polychaete-fed shrimp contained higher levels of (±)8-HETE (Fig 7D), but lower levels of 15d-PGJ₂ (Fig 7C), (±)12-HEPE (Fig 7G), (±)18-HEPE (Fig 7H), ARA (Fig 7I), DHA (Fig 7J), and EPA (Fig 7K) than those in pellet-fed shrimp. On the other hand, levels of PGE₂ (Fig 7A), PGF_2α (Fig 7B), and 12(R)-HETE (Fig 7F) were comparable in testes of polychaete- and pellet-fed shrimp. Lastly, (±)11-HETE (Fig 7E) was not detected in testes of shrimp from both feed groups, indicating that this compound was not involved in shrimp spermatogenesis.

A similar analysis was performed to compare levels of eicosanoids and PUFAs in vas deferens between polychaete- and pellet-fed shrimp. The UHPLC-HRMS/MS analysis revealed that PGE₂ (Fig 7A) and (±)11-HETE (Fig 7E) were present only in vas deferens of polychaete-fed shrimp. As levels of these metabolites were below the limit of detection in vas deferens of pellet-fed shrimp, it is possible that the lack of these eicosanoids might be correlated with low sperm counts. On the other hand, levels of (±)12-HEPE (Fig 7G) were higher in vas deferens of pellet-fed shrimp than in those of polychaete-fed shrimp, suggesting a negative correlation between high levels of (±)12-HEPE and total sperm counts. Meanwhile, levels of PGF_2α (Fig 7B), 15d-PGJ₂ (Fig 7C), (±)8-HETE (Fig 7D), 12(R)-HETE (Fig 7F), (±)18-HEPE (Fig 7H), ARA (Fig 7I), DHA (Fig 7J), and EPA (Fig 7K) were comparable in vas deferens of polychaete- and pellet-fed shrimp, indicating that the difference in shrimp feed did not affect the production of these eicosanoids in vas deferens.

Spermatophore quality of decapod crustaceans can be evaluated using several parameters, including melanization, spermatophore weight, sperm number, sperm viability, sperm abnormality, and spermatophore absence rates [35, 38]. The loss of spermatophore quality can be attributed to stress, poor nutrient, and the length of time spent in captivity for wild-caught shrimp [19, 39]. In this study, all spermatophores were present and no melanization was observed in all collected samples. Wild-caught shrimp had higher spermatophore weights and higher total sperm counts than domesticated shrimp, suggesting that the spermatophore quality of wild-caught shrimp was higher than those of domesticated shrimp in this study. Our data were supported by Rodríguez et al. (2007), in which the wild-caught Pacific white shrimp Litopenaeus vannamei produced higher total sperm counts than the domesticated counterparts [32]. However, other studies reported that spermatophore weights and total sperm counts of wild-caught and domesticated shrimp were comparable [18, 31]. The discrepancy between these studies may stem from the difference in shrimp size. A positive correlation between shrimp size and total sperm count has previously been reported in a different study in L. vannamei [40]. Upon closer examination of our data and the data from Rodríguez et al. (2007), it was confirmed that wild-caught males with higher body weights also had higher total sperm counts than domesticated males in both studies [32], whereas wild-caught and domesticated males with similar body weights also contained comparable total sperm counts [18, 31]. As a result, shrimp body weight should also be taken into consideration during the comparison of total sperm counts between shrimp from different sources.

One of the contributing factors that affect sperm quality is the amounts of PUFAs in crustacean diets [41]. Supplementation of fish oil enriched in n-3 and n-6 PUFAs has been shown to increase levels of ARA, EPA, and DHA in P. monodon testis and enhance the number of spermatozoa in male broodstocks [41]. In fact, spermatophore quality can be used to determine the efficiency of crustacean maturation diets [18, 42, 43]. In this study, the consumption of polychaetes, which contained higher levels of n-3 and n-6 PUFAs than feed pellets, did not result in higher levels of ARA, EPA, and DHA in shrimp testis and vas deferens than those of pellet-fed shrimp. Moreover, a negative correlation between levels of dietary PUFAs and levels of PUFAs in testis and vas deferens was observed, suggesting that aside from the dietary intake, other factors also influenced levels of PUFAs in crustacean male reproductive organs.

In the oriental river prawn Macrobrachium nipponense, a positive correlation between high levels of n-6 PUFAs in the testis and crustacean spermatogenesis has been reported [44]. Levels of EPA and DHA in the testis increased as shrimp progressed from early to mid and late stages of gonad development [44]. Nevertheless, this observation might be species-specific as there was no correlation between levels of EPA and DHA in the testis and spermatogenesis or mating activities in M. rosenbergii [45]. On the other hand, high levels of ARA have typically been correlated with low sperm counts and poor sperm motility in mammals [46]. However, the effects of high levels of ARA in male reproductive organ have never been reported in crustaceans. In this study, the analysis of wild-caught and domesticated shrimp revealed a negative correlation between total sperm counts and high levels of EPA in testes as well as high levels of ARA, EPA, and DHA in vas deferens. These data were supported by the analysis of polychaete- and pellet-fed shrimp, in which higher levels of EPA were observed in testes of pellet-fed shrimp than those of polychaete-fed shrimp.

As PUFAs are known precursors of eicosanoids, the increased levels of PUFAs in shrimp testis and vas deferens could potentially result in higher production of eicosanoids in these organs. In this study, the UHPLC-HRMS/MS analysis revealed that ten eicosanoids and three PUFAs were found in P. monodon testes and vas deferens. These included PGE₂, PGF_2α, (±)8-HETE, (±)11-HETE, 12(R)-HETE, (±)8-HEPE, (±)12-HEPE, and (±)18-HEPE, all of which had previously been identified in crustaceans [8, 9, 11–13, 15, 47–49]. Additionally, to the best of our knowledge, this is also the first identification of 15d-PGJ₂ and (±)15-HEPE in crustaceans. The roles of 15d-PGJ₂ in male reproductive maturation has been firmly established in mammals [4, 50]. High levels of 15d-PGJ₂ in the testis and vas deferens were associated with impaired spermatogenesis in pigs and male infertility in humans, respectively [4, 50]. In the testis, 15d-PGJ₂ acted through the reactive oxygen species (ROS) pathway, which prevented the differentiation of human testicular peritubular cells [4]. This resulted in the loss of contractility of the peritubular cells of the testis, which led to impaired spermatogenesis. On the other hand, high levels of 15d-PGJ₂ in vas deferens activated the PPARγ pathway, which regulated luminal electrolytes in the reproductive ducts that affected sperm functions and viability [50]. As high levels of 15d-PGJ₂ were detected in vas deferens of P. monodon, we propose that excess levels of 15d-PGJ₂ might impair sperm function and viability in shrimp vas deferens, which subsequently result in low sperm counts in penaeid shrimp.

Although the roles of 15d-PGJ₂ in spermatogenesis are well-established in mammals, the function of 15-HEPE in testis and vas deferens has not been reported in any organism. Nevertheless, the inhibition of 15-lipoxygenase, which converts EPA to 15-HEPE, can improve sperm motility and acrosome reaction rates as well as reduce the oxidative stress via ROS pathway [51]. Therefore, the identification of 15-HEPE in testis and vas deferens of domesticated shrimp might also indicate that the ROS pathway may be activated in domesticated shrimp.

In this study, the heat map analysis of relative abundance of PUFAs and eicosanoids in shrimp reproductive tract revealed that (±)8-HEPE and (±)8-HETE were the two most abundant eicosanoids in shrimp testes and vas deferens. In fact, high levels of (±)8-HETE and (±)8-HEPE were reported in E. pacifica [52] and high levels of (±)8-HEPE were also detected in the hepatopancreas of P. monodon [49], suggesting that these hydroxy fatty acids were major metabolites and ubiquitously expressed in crustaceans.

To assess the roles of eicosanoids in shrimp male reproductive organs, two sets of shrimp samples were selected for analysis. Shrimp from different sources, namely wild-caught and domesticated shrimp, were used as representatives of shrimp with high and low total sperm counts, respectively. The effects of shrimp diets on total sperm counts were also examined as the use of polychaetes as live feed for male brooders has been shown to produce higher spermatophore weights and higher total sperm counts than the use of feed pellets [18, 20]. The results from this study are summarized in Fig 8. The comparative analysis of levels of eicosanoids and PUFAs in testes and vas deferens revealed that levels of 15d-PGJ₂, (±)8-HETE, and (±)12-HEPE in shrimp testes were lower than those in vas deferens in all shrimp samples (Fig 8A), suggesting that these eicosanoids may be essential for the sperm maturation process.

Eicosanoid and PUFA profiles were also compared for shrimp from different sources (wild-caught vs. domesticated shrimp; Fig 8B) and for shrimp fed with different diets (polychaete- and pellet-fed shrimp; Fig 8C). In both sets of samples, high levels of (±)12-HEPE, (±)18-HEPE, and EPA in testes as well as high levels of (±)12-HEPE in vas deferens were negatively correlated with total sperm counts (Fig 8B and 8C). In contrast, high levels of PGE₂ in vas deferens were positively correlated with high sperm counts in shrimp from both sets of samples. In humans, addition of PGE₂ and PGF_2α at low physiological levels to spermatozoa has been shown to improve sperm function [6]. Furthermore, transcriptomic analyses in crab gonads also provided supporting evidence regarding the positive effects of eicosanoid biosynthesis pathway in crustacean male reproductive maturation. This led to the identification of prostaglandin E synthase 2 and prostaglandin F synthase as candidates for the regulators of growth, sexual differentiation, and reproduction in the testis of the orange mud crab Scylla olivacea [53]. Similarly, prostaglandin E synthase and prostaglandin E2 receptor were also identified as potential regulators of gonadal development in P. trituberculatus [54]. These data were also supported by a study in mammals, in which cyclooxygenase-2 and prostaglandin synthase enzymes that regulate the conversion of ARA to PGE₂ could serve as a local modulator of testicular activity in Leydig and Sertoli cells [55]. Therefore, we propose that eicosanoids also serve as modulators for testicular development and sperm maturation process in P. monodon. Our results not only expand the coverage of eicosanoid biosynthesis pathway in crustaceans, but also suggest that the roles of eicosanoids in spermatogenesis are conserved between crustaceans and mammals. Furthermore, the correlations between total sperm counts and high levels of eicosanoids in shrimp testis and vas deferens also suggest an alternative approach to improve total sperm counts by increasing the prostaglandin biosynthesis while suppressing the production of HEPEs in the male reproductive tract of penaeid shrimp.