Revista Cienfica, FCV-LUZ / Vol. XXXV Recibido: 08/02/2025 Aceptado:15/04/2025 Publicado: 13/05/2025 hps://doi.org/10.52973/rcfcv-e35636 UNIVERSIDAD DEL ZULIA Serbiluz Sistema de Servicios Bibliotecarios y de Información Biblioteca Digital Repositorio Académico 1 of 5 Assessment of semen quality in freshwater mussel Unio elongatulus eucirrus: analysis of sperm molity, morphometry, and pH Evaluación de la calidad seminal en el mejillón de agua dulce Unio elongatulus eucirrus: análisis de la molidad, morfometría espermáca y pH Mustafa Erkan Özgür Turgut Özal University, Faculty of Agriculture, Department of Fishery Engineering, 44280, Malatya, Türkiye *Corresponding Author: mustafa.ozgur@ozal.edu.tr ABSTRACT The freshwater mussel (Unio elongatulus eucirrus), nave to the Euphrates River basin in Türkiye, holds lile economic value; however, it plays an important ecological role in natural aquac ecosystems. Consequently, understanding its biology parcularly its reproducve traits, such as semen quality, sperm molity, and morphology is of scienfic interest. This study aimed to invesgate several reproducve parameters in 10 male specimens of this species, including sperm concentraon, seminal pH, molity characteriscs, and sperm morphometry. Sperm kinemacs were assessed using a computer-assisted sperm analysis (CASA) system. The results showed the following sperm velocity values: straight- line velocity (VSL: 40.75 ± 6.01 μm/s), curvilinear velocity (VCL: 103.00 ± 2.62 μm/s), and average path velocity (VAP: 54.24 ± 6.75 μm/s). Sperm morphometry, analyzed via scanning electron microscopy (SEM), revealed a head length of 3.90 ± 0.11 μm, head width of 1.70 ± 0.17 μm, and tail length of 37.64 ± 0.45 μm. Regarding the physicochemical parameters, the seminal pH was 6.25 ± 0.26, and sperm concentraon was 15.48 ± 0.53 × 10 9 cells/mL. These results indicate that the morphological and kinemac characteriscs of freshwater mussel (Unio elongatulus eucirrus) spermatozoa are quite like those observed in other mussel and fish species. However, it was determined that spermatozoa are easily acvated upon contact with water. Key words: Freshwater mussel; sperm cell characteriscs; Euphrates River Basin; Türkiye RESUMEN El mejillón de agua dulce (Unio elongatulus eucirrus), que habita en la cuenca del río Éufrates en Turquía, carece de importancia económica; no obstante, representa un valioso aporte ecológico en los ecosistemas acuácos naturales. Por esta razón, resulta de interés conocer su comportamiento, especialmente en lo relacionado con la reproducción, lo cual incluye la calidad seminal, en parcular la molidad y la morfología espermáca. El objevo de este estudio fue invesgar algunos parámetros reproducvos en 10 individuos machos de esta especie, tales como la concentración espermáca, el pH seminal, la cinemáca y la morfometría de los espermatozoides. La cinemáca espermáca fue determinada mediante un sistema de análisis asisdo por computadora (CASA) bajo microscopio. Los resultados obtenidos revelaron los siguientes valores: velocidad reclínea (VSL: 40,75 ± 6,01 μm/s), velocidad curvilínea (VCL: 103,00 ± 2,62 μm/s) y velocidad media de trayectoria (VAP: 54,24 ± 6,75 μm/s). La morfometría fue evaluada mediante microscopía electrónica de barrido (SEM), obteniéndose las siguientes dimensiones: longitud de la cabeza (3,90 ± 0,11 μm), ancho de la cabeza (1,70 ± 0,17 μm) y longitud de la cola (37,64 ± 0,45 μm). En cuanto a los parámetros fisicoquímicos, el pH seminal fue de 6,25 ± 0,26, mientras que la concentración espermáca alcanzó los 15,48 ± 0,53 x 10⁶ células/mL. Estos resultados indican que las caracteríscas morfológicas y cinemácas de los espermatozoides del mejillón de agua dulce (Unio elongatulus eucirrus) son bastante similares a las observadas en otras especies de mejillones y peces. No obstante, se determinó que los espermatozoides se acvan fácilmente al entrar en contacto con el agua. Palabras clave: Mejillón de agua dulce; caracteríscas de los espermatozoides; cuenca del río Éufrates; Turquía

Revista Cienfica, FCV-LUZ / Vol. XXXV UNIVERSIDAD DEL ZULIA Serbiluz Sistema de Servicios Bibliotecarios y de Información Biblioteca Digital Repositorio Académico INTRODUCTION The mussels have contributed to aquac ecosystem include that are biofiltraon, enviromental monitoring, habitat modificaon, nutrient cycling and food webs [1]. In addion, they are important components of the aquac ecosystem because they constute a large part of the benthic biomass, play major funcons in water filtraon, water clarity, play a role in biogeochemical cycles and sedimentaon, and support biodiversity [2]. Despite their important role in the ecosystem, mussels are rapidly declining worldwide, and 224 of the 511 freshwater mussel species (nearly half) are classified as Near Threatened on the 2015 IUCN Red List of Threatened Species [3]. Otherhand, Çetinkaya [4] reported that the freshwater mussels have lived totally 5 species such as Unio spp (2 species)., Sinanodonta sp., Legumina sp. and Pseudodontopsis sp. in Euphrates River in Türkiye. The low reproducve capacity is generally a liming factor on reproducve efficiency of aquac organisms in their environment [5]. There is a posive correlaon between the molity and velocity of sperm cells. Therefore, the decreasing in these parameters may reduce the reproducve efficiency [6]. Other hand, the molity and sperm cell molity descriptors which are indicator for sperm quality are determined by the computer assisted sperm analysis (CASA). Because, this instrument is the most objecve according to tradional invesgaon methods and facilitates rapid assessment [7]. Even, generally, the molity of sperm cells can live only few minutes, even less than 1 min aſter contaminate with water [8]. Due to the short duraon of molity, it is crucial that operaon and study be rapidly accomplished. It is why more invesgators started to use CASA systems and its usage has been growing up all over the world [9]. Some spermatological characteriscs have been described on bivalves such as Ruditapes philippinarum, Crassostrea gigas and Panopecten yessoensis [10], Truncilla truncate [11], Neotrigonia margaritacea [12], zebra mussel, Dreissena polymorpha [13 , 14]. But there is not any study about sperm cell molity descriptors and morphology of sperm cell in Unio elongatulus eucirrus in literature. The taxonomy of this species is that Phylum: Mollusca, Class: Bivalvia, Family: Unionidae, Genus: Unio, Species: elongatulus, Subspecies: eucirrus [15]. Thus, this research aimed to determine the external morphological characterisc and sperm molity descriptors of freshwater mussel species, Unio elongatulus eucirrus. MATERIAL AND METHODS Animal collecon and husbandry for sperm samples For sperm samples at the end of April 2024, the mature males of Unio elongatulus eucirrus were collected on the shores of Karakaya Dam Lake (38°29’26.0”N 38°21’23.7”E) on Upper Part of the Euphrates River in Malatya, Türkiye. The mussels kept in an aquarium (60 L) with tap water non-stop aerated (23 °C same like water temperature of lake). The body of each mussel was wiped dry to avoid any contaminaon with water before sperm samples collecon. Sperm samples were collected into Eppendorf tubes from 10 individuals’ mussel and kept on ice for the duraon of the experiment [14]. The kinemacs and concentraon of sperm cells The kinemacs of sperm cells in such as VSL: straight line velocity (μm/s), VCL: curvilinear velocity (μm/s), VAP: angular path velocity (μm/s), LIN, linearity (%), the rao of net distance moved to total path distance, BCF: beat cross frequency turning points of the sperm cell head (Hz), ALH: amplitude of lateral displacement of the sperm cell head (μm) and MAD: mean angular displacement ( o ), average change in direcon of the sperm head from frame to frame [8 , 16] were carried out by the computer assisted sperm analysis systems, BASA-Sperm Aqua which has Olympus CX31 microscope (Japan) with 200x magnificaon lens, Sony CCD camera (Japan) with 30 frames/ second. The sperm samples from 10 males were acvated with acvaon soluon (AS) at a rao 1:250 (Sperm: AS). Acvaon medium was aquarium water (23 0 C, pH 7.9). Sperm cell concentraon (sperm cell/mL) was determined from males by using a Neubauer hemocytometer (Germany) [17]. The sperm pH in tess was determined pH indicator papers (Merck Millipore, Germany). The morphology of sperm cells For scanning electron microscope (SEM) imaging, sperm samples were collected by pipee, concentrated by centrifugaon (Hech Universal 320R Refrigerated Centrifuge, Germany) and fixed for 3 h in freshly prepared 2.5% glutaraldehyde in 0.1 M phosphate buffer (pH 7.4) at 0–4 o C. Then the samples rinsed in the same buffer and immersed for 2 h in 1.0% osmium tetroxide in 0.1 M phosphate buffer (pH 7.4) at 20 ° C. Aſter rinsing in the buffer, the samples were transferred crical point drying apparatus. The dried samples were coated with gold palladium and imaged using a LEO EVO40 model scanning electron microscope (Oberkochen, Germany) at accelerang voltages of 20 kV [18]. The length and width of head and tail length were determined on 25 spermatozoa specimens from scanning electron micrographs. The photographs of sperm cells were also taken from 100 x lenses, immersion oil of light microscopy by staining Diff-Quick. In staining, the samples immersed in Diff- Quick A soluon for 10 sec, Diff-Quick I soluon for 5 seconds and Diff-Quick II soluon for 5 sec. Then slides were washed with water, dried air for 1 min. It is described spermatozoa dimensions from photographs by Corel Draw soſtware. Stascs analysis The SPSS 17 soſtware was used for stascal analysis. All parameters were expressed as mean ± standard deviaon (Mean±SD) and the stascally significant level was accepted as P < 0.05. The homogeneity of the data in the parameters was tested using Skewness and Kurtosis in descripve analysis. RESULTS AND DISCUSSION The kinemacs, concentraon, head length, head width and tail length of sperm cells and pH of sperm in Unio elongatulus eucirrus have shown in TABLE I. The pH of the sperm in the tescle is slightly acidic. 2 of 5

Semen quality in freshwater mussel / Mustafa Erkan Özgür UNIVERSIDAD DEL ZULIA Serbiluz Sistema de Servicios Bibliotecarios y de Información Biblioteca Digital Repositorio Académico TABLE I. Sperm parameters of Unio elongatulus eucirrus Sperm parameters (n=10) Mean ± S.D. Minimum Maximum Concentraon of sperm cell (x10 9 /mL) 15.48±0.53 14.80 16.10 pH of sperm 6.25±0.26 6.00 6.5 VSL (μm/s) 40.75±6.01 31.39 51.67 VCL (μm/s) 103.00±2.62 99.54 118.63 VAP (μm/s) 54.24±6.75 49.48 68.26 LIN (%) 30.22±6.30 22.20 44.73 BCF (Hz) 5.92±1.52 3.27 8.76 ALH (μm) 23.97±7.94 11.49 33.75 MAD ( o ) 0.04±0.02 0.01 0.09 Head length of sperm cell (μm) 3.90±0.11 3.66 3.99 Head width of sperm cell (μm) 1.70±0.17 1.35 1.92 Tail length of sperm cell (μm) 37.64±0.45 36.95 38.44 VSL, straight line velocity (μm/s), VCL, curvilinear velocity (μm/s), VAP, angular path velocity (μm/s), LIN, linearity (%), BCF, beat cross frequency (cross/second), ALH, amplitude of lateral displacement of the spermatozoa head (μm), MAD: mean angular displacement ( o ), average change in direcon of the sperm head from frame to frame. The morphological characterizaon of sperm cell in Unio elongatulus eucirrus has shown in FIG. 1. The head of sperm cells has formed to approximately elongate cylindrical or bulled in the shape which has length 3.90±0.11 μm and width 1.70 ± 0.17 μm. The flagellum (lenght 37.64±0.45 μm) was very long as 10 mes longer than head (TABLE I). FIGURE 1. Figure a, b and c show images of different magnificaons in scanning electron microscope (SEM) micrograph. Figure d shows image in 100x lens of light microscopy by staining Diff-Quick in freshwater mussel, Unio elongatulus eucirrus sperm cells The structure of both morphology and ultra-structure of sperm cell in fish has been used to establish phylogenec relaonships among species [19 , 20]. However, the idenfied character of head morphology in sperm cells can be used to both predict semen quality and an indicator for ferlity [21]. Assessment of sperm cell head morphology is also important in aquaculture for cryopreservaon and molity [22 , 23]. Spermatozoa are immole in the tess, however, the acvaon of sperm molity in the freshwater mussel, Unio elongatulus eucirrus is the same as fish and other aquac invertebrates (Video S1). According to the obtained parameters, sperm cells which generally move on a straight or slightly curved route aſter acvaon which found to be similar to and / or closely related to the results of some authors [6 , 10 , 24 , 25]. The formaon and structure of mussel sperm cell have been studied mostly in marine bivalves, but have been less in parcularly freshwater bivalves. Some of the scienfic studies on sperm characteriscs of freshwater mussels are as follows. For example, it was studied about concentraon and morphology of sperm cell two freshwater unionid mussel such as Ligumia subrostrata and Ligumia straminea. Researchers focused especially differences in seasonal. According to their results, largest sperm cell concentraon of L. subrostrata was 19.60 × 10 9 cells/mL in between end of September to start of November. Greatest sperm concentraon of L. straminea was 20.0 × 10 9 cells/mL on September. They reported that the sperm cells for both species were uniflagellated. Other hand, the dimensions of sperm cell in scanning electron microscopy (SEM), the mean head length and width (mid-spawning) of sperm cell were 3.38 ± 0.04 µm for L. subrostrata and 1.61 ± 0.01 µm and 3.37 ± 0.04 µm and 1.61 ± 0.01 µm for L. straminea [17]. At other study, it was researched about reproducve cycle, fecundity and growth of the freshwater mussel Unio tumidus (Bivalvia: Unionidae). Researchers reported the five main stages in spermatogenesis such as spermatogonia, spermatocytes, spermads, spermatozoa and spermad morulae. They determined the spherical shaped cell in all stages of spermatogenesis. The diameter of spermatogonia was 3.6– 4.8 μm, while the head diameter of spermatozoa was 2.1 μm and bullet-shaped [26]. Moreover, the sperm dimensions of some freshwater unionid mussels were the head of spermatozoa measured 1.5 × 4.0 μm cylindrical and the 35 μm long flagella for Anodonta grandis [27], 4.0 μm long and 1.5 μm wide with 20-24 μm long flagella for Diplodon chiliensis chiliensis [28]. The dimensions of head and tail of spermatozoa and the cell density obtained in this study are parallel to sperm dimensions of those freshwater mussels presented in the literatures [17 , 26 , 27 , 28]. Other hand, the researchers’ studies about spermatozoa morphology about four marine bivalve species from the Beibu Gulf of the South China Sea. According to their results these 3 of 5

Revista Cienfica, FCV-LUZ / Vol. XXXV UNIVERSIDAD DEL ZULIA Serbiluz Sistema de Servicios Bibliotecarios y de Información Biblioteca Digital Repositorio Académico bivalves had cone-shaped of sperm. The length-width of head and the length of tail in sperm of these bivalves were found for Paphia schnelliana (2.26-1.59 µm and 47.37 µm), Lutraria sieboldii (1.76-1.55 µm and 35.38 µm), Angona lamellaris (2.84-1.26 µm and 38.19 µm), and Paphia textzle (2.59-1.09 µm and 45.95 µm) [29]. A deep-sea bivalve, Calyptogena pacifica has bullet-shaped sperm head and measured about 5.8 μm in length and 1.8 μm in width of sperm head [30]. The dimensions of spermatozoa in Arcca islandica, a marine bivalve from the North Atlanc had the rod-shaped ‘head’ regions (= acrosomal complex plus nucleus) about 9-9.5 µm on head, about 60 µm of flagellum and tapered, rod-shaped, while Neotrapezium sublaevigatum, a marine bivalve from Indo-Pasific from had 9.5 µm of spermatozoa head and short, barrel shaped [31]. In the present study, there was differences in total sperm head width and length, flagella length and spermatozoa shape amongst other marine bivalves. The gamete quality and the dimensions or morphology of spermatozoa can be highly variable among invertebrates. This situaon has also been among populaons of the same species [29 , 30]. CONCLUSIONS Finally, it has been documented the presence of sperm char- acteriscs of Unio elongatulus eucirrus in this manuscript. Over- all, the results from the present study indicate there is compa- ble or similar with the semen characteriscs and the dimensions of spermatozoa in some freshwater mussels living in other hab- itats. Addionally, in the present study, there was development of a rapid and reliable technique to quanfy sperm molity us- ing computer assessment sperm analysis system. We have also hoped that this data may lead to understand to reproducon physiology of this species. Further studies are necessary to clarify the exact me, ions, temperatures and fer- lizaon capacies of sperm acvaon in this freshwater mussel species. ACKNOWLEDGEMENTS The Department of Fishery Engineering, Faculty of Agriculture in Malatya Turgut Özal University for providing laboratory equipment of the work described in this paper. Funding No funding was received for conducng this study. BIBLIOGRAPHICS REFERENCE [1] Vaughn CC. Ecosystem services provided by freshwater mussels. Hydrobiol. 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