Sperm handling in aquatic animals for artificial reproduction

Artificial reproduction involves collection and handling of gametes in a way that secures their quality and maximizes the fertilization outcome. In addition to initial sperm quality, numerous steps can affect the final result of fertilization, from the sperm collection process until gamete mixing (or co-incubation) when the spermatozoon enters or fuses with the oocyte. In this review, we summarize the whole process of sperm handling, from collection until fertilization for fish, penaeid shrimp, bivalve mollusks and marine mammals. To obtain sperm from captive animals, techniques vary widely across taxa, and include stripping by abdominal massage or testis surgical removal in fish, spermatophore collection in penaeid shrimps, gonadal scarification or temperature shock in bivalve mollusks, and voluntary collection via positive reinforcement in mammals. In most cases, special care is needed to avoid contamination by mucus, seawater, urine, or feces that can either activate sperm motility and/or decrease its quality. We also review techniques and extender solutions used for refrigerated storage of sperm across the aforementioned taxa. Finally, we give an overview of the different protocols for in vivo and in vitro fertilization including activation of sperm motility and methods for gamete co-incubation. The present study provides valuable information regarding breeder management either for animal production or species conservation.Agência financiadora CRB-anim ANR-11-INBS-003 Reproseed (FP7-KBBE-2009-3) MICIU (Juan de la Cierva-Incorporacion) IJCI-2017-34200 project ReproFl - MAR2020 Program 16-02-01-FMP-59 Fundacao para a Ciencia e Tecnologia (FCT, Portugal) through project FCT UID/Multi/04326/2019info:eu-repo/semantics/publishedVersio

Biological characteristics of sperm in European flat oyster (Ostrea edulis)

In brooding mollusc species that incubate larvae before their release in seawater, the biology of gametes remains poorly explored. The present study describes some biological characteristics of sperm of the European flat oyster, a native species that has been over-exploited in the past and is nowadays classified as an endangered species in Europe. Flat oysters were collected by divers in the Rade de Brest (Finistère, France), during its natural reproduction period. Gonadal pH is acidic (6.31 ± 0.10). Spermatozoa are clustered in spermatozeugmata, an acellular structure in which the sperm heads are embedded. After their transfer in seawater, spermatozeugmata have a mean diameter of 64 ± 3 μm and they release free spermatozoa for a mean duration of 21 ± 3 min. Immediately after their release, the mean percentage of motile spermatozoa was 48.5 ± 12.6%. At 10 min after dilution in seawater, movement of spermatozoa was no more observed. Biological characteristics of European flat oyster sperm are compared to those observed in the Pacific oyster, regarding the unique reproductive behaviour of the former species and the role of spermatozoa transfer played by spermatozeugmata. The present results aim to improve the knowledge of reproduction and natural recruitment processes, support conservation and restoration measures and favour the establishment of management protocols of gametes and larvae in this endangered species

Métabolisme énergétique du spermatozoïde d'huître creuse (Crassostrea gigas)

The present study aims at describing the energy metabolism of Pacific oyster, Crassostrea gigas, spermatozoon. The main objectives are: (1) describing the kinetic of cellular and biochemical characteristics of Pacific oyster spermatozoon during its movement phase, (2) identifying the metabolic pathways to produce energy and their substrates, (3) determining the involvement of energy metabolism in fertilization success. The results showed the long duration (ranging 24 to 72h) of the movement of Pacific oyster spermatozoon and the maintenance of a high intracellular ATP content until the end of the movement phase. The role of oxidative phosphorylation (OXPHOS) in ATP production was highlighted. Alternative ATP producing pathways might contribute to sustain energy production when OXPHOS is impaired, showing the high adaptation capacity of Pacific oyster spermatozoa. An increase of phosphoarginine content observed at the end of the movement phase suggests energy storage capacity. Endogenous substrates involved in ATP synthesis must be determined. Both the high regulation capacity of the energy metabolism of Pacific oyster spermatozoa and their ability to restore energy stores catabolized during movement likely contribute to the successful settlement observed in the wild. In conclusion, this work provided a fine description of energetic metabolism of Pacific oyster spermatozoon.Cette thèse présente une étude du métabolisme énergétique du spermatozoïde d’huître creuse, Crassostrea gigas. Les principaux objectifs sont (1) d’établir la dynamique des caractéristiques cellulaires et biochimiques du spermatozoïde de cette espèce durant la phase de mouvement, (2) d’identifier les voies de synthèse d’ATP et leurs substrats et (3) de déterminer l’implication du métabolisme énergétique dans le succès à la fécondation du spermatozoïde de cette espèce. Les résultats de ce travail montrent la longue durée du mouvement (de 24 à 72h) du spermatozoïde d’huître creuse et le maintien d’une concentration élevée en ATP intracellulaire, jusqu'à la fin du mouvement. Le rôle de la phosphorylation oxydative (OXPHOS) dans la synthèse d’ATP a été mis en évidence. Le spermatozoïde d’huître creuse montre une grande plasticité dans l’utilisation de ses voies de production d’énergie, assurant la synthèse d’ATP par d’autres voies qu’OXPHOS lorsque la mitochondrie est inhibée. L’accumulation de phosphoarginine en fin de mouvement montre une capacité de stockage de l’ATP synthétisé. La nature des substrats endogènes utilisés pour la synthèse d’ATP reste cependant à déterminer. La forte capacité de régulation du métabolisme énergétique du spermatozoïde d’huître creuse, associée à la possibilité de synthétiser de l’énergie au cours de sa longue phase de nage, contribuent probablement au succès du peuplement observé chez cette espèce. Ce travail apporte une première description du métabolisme énergétique du spermatozoïde d’huître creuse, améliorant la connaissance fine de cette cellule

Energy metabolism of Pacific oyster (Crassostrea gigas) spermatozoa

The present study aims at describing the energy metabolism of Pacific oyster, Crassostrea gigas, spermatozoon. The main objectives are: (1) describing the kinetic of cellular and biochemical characteristics of Pacific oyster spermatozoon during its movement phase, (2) identifying the metabolic pathways to produce energy and their substrates, (3) determining the involvement of energy metabolism in fertilization success. The results showed the long duration (ranging 24 to 72h) of the movement of Pacific oyster spermatozoon and the maintenance of a high intracellular ATP content until the end of the movement phase. The role of oxidative phosphorylation (OXPHOS) in ATP production was highlighted. Alternative ATP producing pathways might contribute to sustain energy production when OXPHOS is impaired, showing the high adaptation capacity of Pacific oyster spermatozoa. An increase of phosphoarginine content observed at the end of the movement phase suggests energy storage capacity. Endogenous substrates involved in ATP synthesis must be determined. Both the high regulation capacity of the energy metabolism of Pacific oyster spermatozoa and their ability to restore energy stores catabolized during movement likely contribute to the successful settlement observed in the wild. In conclusion, this work provided a fine description of energetic metabolism of Pacific oyster spermatozoon.Cette thèse présente une étude du métabolisme énergétique du spermatozoïde d’huître creuse, Crassostrea gigas. Les principaux objectifs sont (1) d’établir la dynamique des caractéristiques cellulaires et biochimiques du spermatozoïde de cette espèce durant la phase de mouvement, (2) d’identifier les voies de synthèse d’ATP et leurs substrats et (3) de déterminer l’implication du métabolisme énergétique dans le succès à la fécondation du spermatozoïde de cette espèce. Les résultats de ce travail montrent la longue durée du mouvement (de 24 à 72h) du spermatozoïde d’huître creuse et le maintien d’une concentration élevée en ATP intracellulaire, jusqu'à la fin du mouvement. Le rôle de la phosphorylation oxydative (OXPHOS) dans la synthèse d’ATP a été mis en évidence. Le spermatozoïde d’huître creuse montre une grande plasticité dans l’utilisation de ses voies de production d’énergie, assurant la synthèse d’ATP par d’autres voies qu’OXPHOS lorsque la mitochondrie est inhibée. L’accumulation de phosphoarginine en fin de mouvement montre une capacité de stockage de l’ATP synthétisé. La nature des substrats endogènes utilisés pour la synthèse d’ATP reste cependant à déterminer. La forte capacité de régulation du métabolisme énergétique du spermatozoïde d’huître creuse, associée à la possibilité de synthétiser de l’énergie au cours de sa longue phase de nage, contribuent probablement au succès du peuplement observé chez cette espèce. Ce travail apporte une première description du métabolisme énergétique du spermatozoïde d’huître creuse, améliorant la connaissance fine de cette cellule

Spermatozoa motility in bivalves: Signaling, flagellar beating behavior, and energetics

WOS:000476579700004Though bivalve mollusks are keystone species and major species groups in aquaculture production worldwide, gamete biology is still largely unknown. This review aims to provide a synthesis of current knowledge in the field of sperm biology, including spermatozoa motility, flagellar beating, and energy metabolism; and to illustrate cellular signaling controlling spermatozoa motility initiation in bivalves. Serotonin (5-HT) induces hyper-motility in spermatozoa via a 5-HT receptor, suggesting a serotoninergic system in the male reproductive tract that might regulate sperm physiology. Acidic pH and high concentration of K+ are inhibitory factors of spermatozoa motility in the testis. Motility is initiated at spawning by a Na+-dependent alkalization of intracellular pH mediated by a Na+/H+ exchanger. Increase of 5-HT in the testis and decrease of extracellular K+ when sperm is released in seawater induce hyperpolarization of spermatozoa membrane potential mediated by K+ efflux and associated with an increase in intracellular Ca2+ via opening of voltage-dependent Ca2+ channels under alkaline conditions. These events activate dynein ATPases and Ca2+/calmodulin-dependent proteins resulting in flagellar beating. It may be possible that 5-HT is also involved in intracellular CAMP rise controlling cAMP-dependent protein kinase phosphorylation in the flagellum. Once motility is triggered, flagellum beats in asymmetric wave pattern leading to circular trajectories of spermatozoa. Three different flagellar wave characteristics are reported, including "full", "twitching", and "declining" propagation of wave, which are described and illustrated in the present review. Mitochondrial respiration, ATP content, and metabolic pathways producing ATP in bivalve spermatozoa are discussed. Energy metabolism of Pacific oyster spermatozoa differs from previously studied marine species since oxidative phosphorylation synthetizes a stable level of ATP throughout 24-h motility period and the end of movement is not explained by a low intracellular ATP content, revealing different strategy to improve oocyte fertilization success. Finally, our review highlights physiological mechanisms that require further researches and points out some advantages of bivalve spermatozoa to extend knowledge on mechanisms of motility

ATP content and viability of spermatozoa drive variability of fertilization success in the Pacific oyster (Crassostrea gigas)

WOS:000408034700015International audienceFertilization of the oocyte is a critical process of sexual reproduction depending among other factors on spermatozoa functionality. Mitochondria participate in many crucial processes for spermatozoa motility and fertilizing ability. The Pacific oyster, Crassostrea gigas, is characterized by a high inter-individual variability of its reproductive success which has been shown to be partially due to the variability of gamete quality. The present study explored spermatozoa characteristics such as: i) relationships among mitochondrial functionality (ATP content, mitochondrial membrane potential (MMP), reactive oxygen species production); sperm viability (percentages of live, dying, and dead); and motility; and ii) their involvement in inter-oyster variability of fertilizing ability demonstrated using simple and multiple regressions. Our results showed that ATP content depends on both MMP and viability of spermatozoa. Using multiple regressions, 61% of the variability of the trochophore-larval yield was explained by a model combining the ATP content and the percentage of dying spermatozoa (P\textless0.001). Our results reveal that capacity of spermatozoa to maintain a high level of ATP via OXPHOS partly explains the inter-individual variability of fertilization success in the Pacific oyster. Sperm ATP content and viability assays will provide valuable tools for assessing sperm quality of this species in aquaculture production, cryopreservation, and bioassays

The toxic dinoflagellate Alexandrium minutum affects oyster gamete health and fertilization potential

Dinoflagellates from the globally distributed genus Alexandrium are known to produce both paralytic shellfish toxins (PST) and uncharacterized bioactive extracellular compounds (BEC) with allelopathic, ichthyotoxic, hemolytic and cytotoxic activities. In France, blooms of Alexandrium minutum appear generally during the spawning period of most bivalves. These blooms could therefore alter gametes and/or larval development of bivalves, causing severe issues for ecologically and economically important species, such as the Pacific oyster Crassostrea (=Magallana) gigas. The aim of this work was to test the effects of three strains of A. minutum producing either only PST, only BEC, or both PST and BEC upon oyster gametes, and potential consequences on fertilization success. Oocytes and spermatozoa were exposed in vitro for 2 hours to a range of environmentally realistic A. minutum concentrations (10 to 2.5 × 104 cells mL-1). Following exposure, gamete viability and reactive oxygen species (ROS) production were assessed by flow cytometry, spermatozoa motility and fertilization capacities of both spermatozoa and oocytes were analysed by microscopy. Viability and fertilization capacity of spermatozoa and oocytes were drastically reduced following exposure to 2.5 × 104 cells mL-1 of A. minutum. The BEC-producing strain was the most potent strain decreasing spermatozoa motility, increasing ROS production of oocytes, and decreasing fertilization, from the concentration of 2.5 × 103 cells mL-1. This study highlights the significant cellular toxicity of the BEC produced by A. minutum on oyster gametes. Physical contact between gametes and motile thecate A. minutum cells may also contribute to alter oyster gamete integrity. These results suggest that oyster gametes exposure to A. minutum blooms could affect oyster fertility and reproduction success

Application of Flow Cytometry to Assess Deepwater Horizon Oil Toxicity on the Eastern Oyster Crassostrea virginica Spermatozoa

International audienceThe Deepwater Horizon (DWH) oil spill in 2010 resulted in the release of millions of liters of oil into the Gulf of Mexico. Dispersants such as Corexit 9500A were used to disperse oil both at the well-head and at the surface. Polycyclic aromatic hydrocarbons and dispersants have been shown to deleteriously affect early life stages of organisms including oysters. This study examined the impacts of chemically enhanced water-accommodated fractions [CEWAF; 1.29–26.14 µg/l tPAH50 (a sum of 50 different polycyclic aromatic hydrocarbons)], high-energy water-accommodated fractions (HEWAF; 16.53–248.89 µg/l tPAH50), and dispersants (0.625–10 mg/l) on the cellular functions (viability, mitochondrial membrane potential (MMP), reactive oxygen species production (ROS), and acrosomal integrity) and resulting fertilization success of eastern oyster Crassostrea virginica spermatozoa. While viability of spermatozoa was not affected by CEWAF and HEWAF at concentrations tested, dispersant exposure caused significant decrease in viability at the highest concentration tested. Fertilization success as well as MMP and ROS production were significantly decreased upon exposure to CEWAF, HEWAF, and dispersants. Also, although not affected by HEWAF exposure, acrosomal integrity decreased upon exposure to CEWAF and dispersants at concentrations tested. The results of this study suggest that impaired fertilization and reduced viability observed after exposure to DWH oil spill contaminants may result, at least partially, from alterations of cellular functions of spermatozoa and contribute to negative effects on oyster populations, and thus the ecology and economy of the Gulf of Mexico

Involvement of Mitochondrial Activity and OXPHOS in ATP Synthesis During the Motility Phase of Spermatozoa in the Pacific Oyster, Crassostrea gigas

In the Pacific oyster, spermatozoa are characterized by a remarkably long movement phase (i.e., over 24 h) sustained by a capacity to maintain intracellular ATP level. To gain information on oxidative phosphorylation (OXPHOS) functionality during the motility phase of Pacific oyster spermatozoa, we studied: i) changes in spermatozoa mitochondrial activity (i.e., mitochondrial membrane potential, MMP) and intracellular ATP content in relation to motion parameters and ii) the involvement of OXPHOS for spermatozoa movement using CCCP. The percentage of motile spermatozoa decreased over a 24 h movement period. MMP increased steadily during the first 9 h of the movement phase and was subsequently maintained at a constant level. Conversely, spermatozoa ATP content decreased steadily during the first 9 h post activation and was maintained at this level during the following hours of the movement phase. When OXPHOS was decoupled by CCCP, the movement of spermatozoa was maintained 2 h and totally stopped after 4 h of incubation, whereas spermatozoa were still motile in the control after 4 h. Our results suggest that ATP sustaining flagellar movement of spermatozoa may partially originate from glycolysis or from mobilization of stored ATP or potential phosphagens during the first 2 h of movement as OXPHOS was decoupled by CCCP. However, OXPHOS is required to sustain the long motility phase of Pacific oyster spermatozoa. In addition, spermatozoa may hydrolyse intracellular ATP content during the early part of the movement phase, stimulating mitochondrial activity. This stimulation seems to be involved in sustaining a high ATP level until the end of the motility phase