Catherine Labbé

Egg and sperm quality in fish.

Fish egg quality can be defined as the ability of the egg to be fertilized and subsequently develop into a normal embryo. Similarly, sperm quality can be defined as its ability to successfully fertilize an egg and subsequently allow the development of a normal embryo. In the wild or under aquaculture conditions, the quality of fish gametes can be highly variable and is under the influence of a significant number of external factors or broodstock management practices. For these reasons, the topic of gamete quality has received increasing attention. Despite the significant efforts made towards a better understanding of the factors involved in the control of gamete quality, the picture is far from being complete and the control of gamete quality remains an issue in the aquaculture industry. Some of the factors responsible for the observed variability of gamete quality remain largely unknown or poorly understood. In addition very little is known about the cellular and molecular mechanisms involved in the control of egg and sperm quality. In the present review, the molecular and cellular characteristics of fish gametes are presented with a special interest for the mechanisms that could participate in the regulation of gamete quality. Then, after defining egg and sperm quality, and how can it can be accurately estimated or predicted, we provide an overview of the main factors that can impact gamete quality in teleosts.

Characterization of Sperm Plasma Membrane Properties after Cholesterol Modification: Consequences for Cryopreservation of Rainbow Trout Spermatozoa

Abstract During cryopreservation, the cell plasma membrane faces severe perils, including lipid phase separation, solute effects, and osmotic stresses associated with ice crystallization. How the initial biophysical properties of the plasma membrane can be modulated before cryopreservation in order to influence cellular resistance to the freeze-thaw stress is addressed in this study. Rainbow trout (Oncorhynchus mykiss) spermatozoa were chosen because the lack of an acrosome in this species suppresses potential interactions of cryopreservation with capacitation. Methyl-beta cyclodextrin-induced modulation of membrane cholesterol revealed the presence of a significant cholesterol exchangeable pool in the trout sperm plasma membrane, as membrane cholesterol content could be halved or doubled with respect to the basic composition of the cell without impairing fresh sperm motility and fertilizing ability. Biophysical properties of the sperm plasma membrane were affected by cholesterol changes: membrane resistance to a hypo-osmotic stress increased linearly with membrane cholesterol whereas membrane fluidity, assessed with DPH (1,6-diphenyl-1,3,5-hexatriene) and with several spin-labeled analogues of membrane lipids, decreased. Phosphatidyl serine translocation between the bilayers was slowed at high cholesterol content. The increased cohesion of fresh trout sperm plasma membrane as cholesterol increased did not improve the fertilizing ability of frozen-thawed sperm whereas the lowest cholesterol contents impaired this parameter of sperm quality. Our study demonstrated that cholesterol induced a stabilization of the plasma membrane in rainbow trout spermatozoa, but this stabilization before cryopreservation brought no improvement to the poor freezability of this cell.

Thermal acclimation and dietary lipids alter the composition, but not fluidity, of trout sperm plasma membrane

The effect of a long-term adaptation of rainbow trout to 8 and 18°C combined with a corn oil-or a fish oil-supplemented diet on the characteristics of the spermatozoan plasma membrane was investigated. The experiment lasted up to 22 mon during which spermatozoa were collected from the mature males. Spermatozoan plasma membranes were isolated by nitrogen cavitation, and the cholesterol content, phospholipid composition and fatty acid pattern were investigated. Membrane viscosity was assessed on whole cells by electron spin resonance using spin-labeled phospholipids. Neither diet nor rearing temperature influenced the cholesterol content of the plasma membrane nor the phospholipid class distribution. The rearing temperature of the broodstock only slightly affected the phospholipid fatty acids. A minor decrease in 18∶0 and increase in monounsaturated fatty acids was observed for the cold-adapted fish. These modifications were not sufficient to affect membrane fluidity, and we conclude that trout spermatozoa do not display any homeoviscous adaptations in these conditions. On the contrary, the dietary fatty acid intake greatly modified the fatty acid profile of plasma membrane phospholipids. The fish oil-fed trout displayed a much higher n−3/n−6 fatty acid ratio than did the corn oil-fed ones, but the 22∶6n−3 levels remained unchanged. Modifications in plasma membrane composition by the diet were obtained although neither of the two diets was deficient in essential fatty acids. The enrichment in n−3 fatty acids, however, did not affect plasma membrane fluidity which was unchanged by the diets.

Influence of rainbow trout thermal acclimation on sperm cryopreservation: relation to change in the lipid composition of the plasma membrane

The ability of rainbow trout (Oncorhynchus mykiss) spermatozoa to withstand cryopreservation was tested after the fish and sperm were exposed to various temperature conditions. In the first experiment, trout were acclimated at either 8 °C or 18 °C during gametogenesis, and transferred to 13 °C at the beginning of the spawning season. Following this 13 °C transfer, the fertilizing ability of frozen-thawed sperm from the fish reared at 18 °C was found to be twice as better (60–80% fertilization rate) than that of the fish reared at 8 °C (20–30% fertilization rate) up to 42 days after the transfer. A low membrane cholesterol/phospholipid ratio was strongly correlated (P < 0.002) with the better freezing resistance of the corresponding sperm. The relevance of this parameter in predicting sperm ability to survive cryopreservation is discussed. In another experiment, fish with the same thermal background during gametogenesis were transferred to 13 °C or to 5 °C before the beginning of the spawning season. The same fertilization rates after freeze-thawing were observed between the two groups (30–35%). Weekly or bi-weekly stripping frequencies did not alter the fertilization rates. Thus, no damage can be attributed to a long in vivo storage of sperm at a relatively high temperature. In an in vitro experiment, the storage temperature (0 °C to 25 °C) of sperm before freezing was shown to have little influence on the fertilizing results after thawing although membrane fluidity was higher in sperm stored at 24 °C compared with those stored at 4 °C. It is concluded from this study that the quality of sperm cryopreservation can be improved by appropriate broodstock handling. A high rearing temperature during gametogenesis followed by transfer to colder water improves sperm resistance during cryopreservation. The stripping frequency of the fish does not influence sperm quality at the temperature tested. Storing sperm in vitro below 5 °C is recommended, although higher temperatures are not markedly deleterious.

Cryobanking of aquatic species

This review is focused on the applications of genome cryobanking of aquatic species including freshwater and marine fish, as well as invertebrates. It also reviews the latest advances in cryobanking of model species, widely used by the scientific community worldwide, because of their applications in several fields. The state of the art of cryopreservation of different cellular types (sperm, oocytes, embryos, somatic cells and primordial germ cells or early spermatogonia) is discussed focusing on the advantages and disadvantages of each procedure according to different applications. A special review on the need of standardization of protocols has also been carried out. In summary, this comprehensive review provides information on the practical details of applications of genome cryobanking in a range of aquatic species worldwide, including the cryobanks established in Europe, USA, Brazil, Australia and New Zealand, the species and type of cells that constitute these banks and the utilization of the samples preserved. Statement of relevance This review compiles the last advances on germplasm cryobanking of freshwater and marine fish species and invertebrates, with high value for commercial aquaculture or conservation. It is reviewed the most promising cryopreservation protocols for different cell types, embryos and larvae that could be applied in programs for genetic improvement, broodstock management or conservation of stocks to guarantee culture production.

Characteristics and freezing tolerance of brown trout spermatozoa according to rearing water salinity

Abstract In most salmonid species, sperm cryopreservation induces a drastic decrease in percentage of motile spermatozoa after thawing, and this is at odds with the high motility observed on cryopreserved sperm of most marine species studied. This study was carried out to investigate whether water salinity could modify sperm fitness to cryopreservation. During the 9 months preceding sexual maturation, 30 brown trout Salmo trutta f. fario males were reared in seawater, while 30 other males from the same family were reared in freshwater. Sperm was collected once and analyzed. Sperm motility was lower and more variable in the seawater group (10% to 90%; mean 55%±29S.D.) than in the freshwater one (60% to 95%; mean 89%±7S.D.). Resistance of spermatozoa plasma membrane to osmotic shock was assessed by calculating the time before 50% spermatozoa became permeable to propidium iodide upon exposure to deionized water. Fifty percent permeable cells occurred 2 min earlier in sperm from seawater fish than in sperm from freshwater fish. The two groups displayed the same sperm fatty-acid profile and the same sperm cholesterol/phospholipid ratio (0.526±0.092S.D. in seawater fish vs. 0.523±0.076S.D. in freshwater fish). Cryopreservation induced the same decrease in sperm ATP content in the two groups and fertilization rates were not different, whether fish were reared in seawater or in freshwater (27%±12 and 32%±12, respectively). It is concluded that water salinity is not a determinant of sperm fitness to cryopreservation, and that salinity is not a direct modulator of the cholesterol/phospholipid ratio.

Liposomes as an alternative to egg yolk in stallion freezing extender.

Egg yolk is normally used as a protective agent to freeze semen of equine and other species. However, addition of egg yolk in extenders is not without disadvantages and the demand to find cryoprotective alternatives is strong. The objective of this study was to test the cryoprotective capacities of liposomes composed of egg yolk phospholipids. Two experiments were conducted: 1) the first to determine the optimal composition and concentration of liposomes to preserve post-thaw motility and membrane integrity of spermatozoa; 2) the second to assess in vivo the cryoprotective capacities of these liposomes. In Experiment 2, post-thaw motility and membrane integrity of spermatozoa were also analyzed. Experiment 1 demonstrated that liposomes composed of phospholipids E80 (commercial lecithins from egg yolk composed mainly of phosphatidylcholine and phosphatidylethanolamine) and of Hanks salts-glucose-lactose solution (E80-liposomes) were the most efficient in preserving post-thaw motility. The optimal concentration was 4 % (v/v). In Experiment 2, fertility rate after artificial insemination of semen frozen with E80-liposomes was 55 % (22/40) compared with 68 % (27/40) with the control extender containing egg yolk (EY) (p = 0.23). Post-thaw motility parameters were higher with EY than with E80-liposomes (p < 0.0001). For post-thaw membrane integrity no difference was observed between the two extenders (p = 0.08). Liposomes composed of egg yolk phospholipids appeared to be a promising alternative to replace egg yolk in semen freezing extenders in equine species.

Changes in motility, ATP content, morphology and fertilisation capacity during the movement phase of tetraploid Pacific oyster (Crassostrea gigas) sperm

Changes in sperm features during the movement phase are especially interesting to study in external fertilization species whose sperm duration movement is long because this implies a significant adaptation of moving cells to the external medium. This study describes the changes in tetraploid Pacific oyster sperm characteristics in relation to time post activation. Sperm individually collected on three tetraploid males were activated in seawater. Their features were analysed over a 24h period and compared to a sperm pool collected on three diploid males as a reference. The percentage of motile spermatozoa, the intracellular ATP content, and the fine structure of spermatozoa were studied in relation to time post activation. Furthermore, the fertilisation capacity of sperm individually collected on five diploid males was assessed after 1 and 24h post activation. A forward progressive movement was maintained for at least a 20h duration. Compared to diploid males, the percentage of motile spermatozoa was lower in tetraploid males. The intracellular ATP concentration was higher in spermatozoa from tetraploid males than in spermatozoa from diploid males. A decrease in ATP content was observed in the first 6h post activation and severe alterations were observed in sperm morphology after 24h. Then, a lower fertilisation capacity of sperm from diploid males was observed at the end of the movement phase. The cessation of Pacific oyster sperm motility was unlikely caused by ATP consumption as ATP concentration was still high at the end of sperm movement but rather caused by drastic changes in sperm morphology. Compared to sperm collected on diploid males, the lower quality of sperm from tetraploid males was emphasized by a shorter movement duration and deeper morphological alterations at the end of the movement phase.

Cryopreservation of goldfish fins and optimization for field scale cryobanking

When gametes and embryos are not available, cryobanking of somatic tissues is one possibility to keep a genetic record of fish valuables in a context of biodiversity conservation and animal breeding management. Cryopreservation of whole fin pieces would be more advantageous than the commonly used cryopreservation of cells after fin culture, as it would allow extensive sampling without immediate need for laboratory facilities. The objective of this work was to assess the cryopreservation ability of fin pieces from goldfish (Carassius auratus) and to test whether a laboratory procedure could be adapted to field conditions. Caudal fin explants were cryopreserved in culture medium with 125mM sucrose and 10% Me(2)SO. After 14days of culture, the frozen-thawed explants showed the same cell growth rate and grew the same somatic cell number as the fresh ones. Cells proliferated inside and around the explants as shown by BrdU labeling. Neither the size of the fin pieces nor the freezer type, -70 degrees C upright or -20 degrees C chest, influenced the outcome of cryopreservation. Fin pieces were stored 4days at 4 degrees C in dry conditions prior to cryopreservation without alteration of the fin explant culture success. This study demonstrated that field collecting of goldfish fin pieces is possible as whole fin pieces can be stored in standard fridge or be shipped at subzero temperature before they are frozen into a plain -20 degrees C chest freezer. After incorporation in cryobanks in liquid nitrogen, thawed fin pieces reliably produce somatic cells in cell culture conditions.

Optimization of somatic cell injection in the perspective of nuclear transfer in goldfish.

BackgroundNuclear transfer has the potential to become one strategy for fish genetic resources management, by allowing fish reconstruction from cryopreserved somatic cells. Survival rates after nuclear transfer are still low however. The part played by unsuitable handling conditions is often questioned, but the different steps in the procedure are difficult to address separately. In this work led on goldfish (Carassius auratus), the step of somatic cells injection was explored. Non-enucleated metaphase II oocytes were used as a template to explore the toxicity of the injection medium, to estimate the best location where the cell should be injected, and to assess the delay necessary between cell injection and oocyte activation.ResultsTrout coelomic fluid was the most suitable medium to maintain freshly spawned oocytes at the metaphase II stage during oocyte manipulation. Oocytes were then injected with several media to test their toxicity on embryo development after fertilization. Trout coelomic fluid was the least toxic medium after injection, and the smallest injected volume (10 pL) allowed the same hatching rates as the non injected controls (84.8% ± 23). In somatic cell transfer experiments using non enucleated metaphase II oocytes as recipient, cell plasma membrane was ruptured within one minute after injection. Cell injection at the top of the animal pole in the oocyte allowed higher development rates than cell injection deeper within the oocyte (respectively 59% and 23% at mid-blastula stage). Embryo development rates were also higher when oocyte activation was delayed for 30 min after cell injection than when activation was induced without delay (respectively 72% and 48% at mid-blastula stage).ConclusionsThe best ability of goldfish oocytes to sustain embryo development was obtained when the carrier medium was trout coelomic fluid, when the cell was injected close to the animal pole, and when oocyte activation was induced 30 min after somatic cell injection. Although the experiments were not designed to produce characterized clones, application of these parameters to somatic cell nuclear transfer experiments in enucleated metaphase II oocytes is expected to improve the quality of the reconstructed embryos.