Post-thawed motility and fertility from Atlantic salmon (Salmo salar L.) sperm frozen with four cryodiluents in straws or pellets

The cryopreservation of salmonid sperm is a complex process involving the interplay of many factors. Although cryopreservation protocols can be evaluated through a range of responses at various stages in the process, the number of progeny is the ultimate indicator of success. We compared reproductive success from freezing Atlantic salmon (Salmo salar L.) sperm using the eight combinations of (1) the penetrating cryoprotectants, 10% dimethyl sulfoxide (DMSO) or methanol (MeOH); (2) the nonpenetrating cryoprotectants glucose (0.3 M) or sucrose (0.6 M), and freezing in 0.1 mL pellets or 0.25 mL straws. All cryodiluents were supplemented with 10% (v/v) of hen's egg yolk. Response variables were the percentage and degree of motility of thawed and activated sperm using computer assisted sperm analysis (CASA), and rates of eyed embryos, hatch and egg sac larvae. Growth rates of alevins were assessed to two months post hatch. Atlantic salmon milt cryopreserved in straws had higher spermatozoa motility and fertilization success than milt cryopreserved in pellets (P < 0.05). Type of sugar tested did not significantly affect the response variables. In the MeOH treatment, thawed spermatozoa achieved higher speed and a higher fertilization rate evaluated at the eyed embryo stage than spermatozoa subjected to the DMSO treatment. Higher mortality rate (especially before hatching) of MeOH offspring than DMSO offspring led to equal numbers of progeny for the two treatments from the swimming stage to the end of the study. Moreover, during feeding fish from the MeOH group produced significantly lower weight larvae than the DMSO and control groups. Even so, the weight of the MeOH group was satisfactory. Length and the condition factors did not differ significantly among the larvae groups. Significant positive correlations were found between fertilization success (measured in number of eyed eggs) and both motility (rs = 0.81), and velocity (rs = 0.49). Freezing in straws gave betters results than freezing in pellets for cryopreservation of salmon milt; whereas type of sugar tested (glucose vs sucrose) did not have significant effects. Penetrating cryoprotectants DMSO and MeOH differed in their effect on post-thawed sperm velocity, fertilization rate and mortality rate of progeny, suggesting the need for further research on the influence of these cryoprotectants on frozen sperm and and post-fertilization devopmental processes.     

Temporal and spatial patterns of sea lice levels on sea trout in western Scotland in relation to fish farm production cycles

The relationship between aquaculture and infestations of sea lice on wild sea trout (Salmo trutta) populations is controversial. Although some authors have concluded that there is a link between aquaculture and lice burdens on wild fish, others have questioned this interpretation. Lice levels have been shown to be generally higher on Atlantic salmon farms during the second years of two-year production cycles. Here we investigate whether this pattern relates to lice burdens on wild fish across broad temporal and spatial axes. Within Loch Shieldaig across five successive farm cycles from 2000 to 2009, the percentage of sea trout with lice, and those above a critical level, were significantly higher in the second year of a two-year production cycle. These patterns were mirrored in 2002–2003 across the Scottish west coast. The results suggest a link between Atlantic salmon farms and sea lice burdens on sea trout in the west of Scotland.     

Temporal variations in the diet of brown trout (Salmo trutta L.) and rainbow trout (S. gairdneri R.) in Rutland Water

The diet of brown trout (Salmo trutta L.) and rainbow trout (S. gairdneri Richardson) in Rutland Water were compared during the first two fishing seasons (April–October 1977 and 1978).Fortnightly samples of approximately forty stomachs were obtained from boat and bank, rod-and-line caught trout giving a total of 1046 stomachs over the two seasons.During 1977 seasonal changes in the diet were divided into two phases; the first being a period of abundant drowned terrestrial food until June. This was followed by a period of more stable water level from July onwards when chironomid larvae and pupae were consistently the most important food items and the diversity of food also increased.In 1978 the proportion of chironomid pupae and larvae declined and they were replaced in the diet by Gammarus and Asellus.     

Preliminary studies on cryopreservation of snakehead (Channa striata) embryos

This paper reports the findings of the ongoing studies on cryopreservation of the snakehead, Channa striata embryos. The specific objective of this study was to collect data on the sensitivity of C. striata embryo hatching rate to low temperatures at two different developmental stages in the presence of four different cryoprotectants. Embryos at morula and heartbeat stages were selected and incubated in 1 M dimethyl sulfoxide (Me₂SO), 1 M ethylene glycol (EG), 1 M methanol (MeOH) and 0.1 M sucrose solutions at different temperatures for a period of time. Embryos were kept at 24 °C (control), 15 °C, 4 °C and −2 °C for 5 min, 1 h and 3 h. Following these treatments, the embryos were then transferred into a 24 °C water bath until hatch to evaluate the hatching rate. The results showed that there was a significant decrease of hatching rate in both developmental stages following exposure to 4 °C and −2 °C at 1 h and 3 h exposure in each treatment. Heartbeat stage was more tolerant against chilling at −2 °C for 3 h exposure in Me₂SO followed by MeOH, sucrose and EG. Further studies will be conducted to find the best method to preserve embryos for long term storage.     

Comparative cryopreservation study of trochophore larvae from two species of bivalves: Pacific oyster (Crassostrea gigas) and Blue mussel (Mytilus galloprovincialis)

Oysters and mussels are among the most farmed species in aquaculture industry around the world. The aim of this study was to test the toxicity of cryoprotective agents to trochophore larvae from two different species of bivalves and develop an improved cryopreservation protocol to ensure greater efficiency in the development of cryopreserved trochophores (14 h old oyster larvae and 20 h old mussel larvae) to normal D-larvae for future developments of hatchery spat production. The cryopreservation protocol producing the best results for oyster trochophores (60.0 ± 6.7% normal D-larvae) was obtained by holding at 0 °C for 5 min then cooling at 1 °C min⁻¹ to −10 °C and holding for 5 min before cooling at 0.5 °C to −35 °C, holding 5 min and then plunging into liquid nitrogen (LN), using 10% ethylene glycol. For mussel experiments, no significant differences were found when cooling at 0.5 °C min⁻¹ or at 1 °C min⁻¹ for CPA combinations with 10% ethylene glycol and at 0.5 °C min⁻¹. Using these combinations, around half of trochophores were able to develop to normal D-larvae post-thawing (48.9 ± 7.6% normal D-larvae).     

Viability of fastidious Phytophthora following different cryopreservation treatments

Living stock cultures with constant phenotypes and genotypes are required for a wide range of research and industrial applications; however, long-term, stable preservation of fastidious Phytophthora strains has been challenging. In this study, we systematically evaluated different cryopreservation treatments to identify and clarify freezing, thawing, and other conditions appropriate for long-term maintenance. Optimal preservation conditions were largely strain-specific, with robust strains remaining fully viable and the fastidious yielding lower recovery under all test conditions. Nevertheless, several procedures were shown to be generally applicable for effective cryopreservation of most Phytophthora organisms. Fastidious strains retained higher viability following the −1 °C min⁻¹ freezing protocol (Mr Frosty's) than either of two widely used programmed freezing procedures. Revival was higher when frozen mycelium plugs were thawed at 37 °C for 2 min or 25 °C for 5 min, while lower viability was apparent for fastidious strains thawed at 55 °C for 1.5 min. Among 15 cryoprotective solutions assessed, 5 % dimethyl sulfoxide produced the highest viability for all fastidious strains. The effect of prefreeze and postfreeze treatments on revival was mild, if any, and strain-dependent. This study has generated reliable, practical, long-term preservation solutions applicable to a majority of Phytophthora species. It also has revealed a need for in-depth physiological and morphological investigations to further enhance the preservation methods for fastidious strains.     

Investigating sperm cryopreservation in a model tunicate, Ciona intestinalis sp. A

In cryopreservation procedures, the capacity to protect the cells from freezing and thawing processes is sensitive to the choice of the cryoprotective agent (CPA) and to its optimal concentration. The advancement of research on Tunicate model species has raised interest in liquid nitrogen cryopreservation for the storage and distribution of genetic resources. Ciona intestinalis (Linnè, 1767) consists of a complex of cryptic taxa that are central to several areas of investigation, from comparative genomics to invasive biology. Here we investigated how five CPAs, three chilling rates and two freezing rates influence semen cryopreservation in C. intestinalis sp. A. By using larval morphology and motility as endpoints, we estimated that long term semen storage requires 10% dimethyl sulfoxide as a protective agent, −1 °C/min chilling rate (18 °C to 5 °C) and −13 °C/min freezing rate (5 °C to −80 °C), followed by immersion in liquid nitrogen.     

Effects of six trace metals on calcium fluxes in brown trout (Salmo trutta L.) in soft water

Summary Calcium fluxes were measured simultaneously in brown trout fry maintained in an artificial soft water medium of [Ca] 20 mol·l^-1 and pH 5.6, and exposed to each of six trace metals (Al, Cu, Fe, Ni, Pb, and Zn). The trace metal concentrations represented typical and maximum levels found in acid waters experiencing declining fishery status. In the absence of trace metals, evidence is presented which suggests that ca. 91% of Ca taken up from the external medium was by extraintestinal active transport. Calcium efflux was stimulated by both concentrations of Al, Cu, Fe, and Pb. Efflux was also stimulated by [Ni] 170 nmol·l^-1 and [Zn] 3000 nmol·l^-1. In some cases, response to increased efflux was stimulation of influx. Lack of stimulation of influx resulted in negative net Ca fluxes. Net Ca losses were recorded at both concentrations of Al, Pb, and Ni, lower concentrations only of Fe, and higher concentrations only of Cu and Zn.Abbreviations J _in influx - J _net net flux - J _out efflux Henceforward in this paper, chemical elements are referred to by their chemical symbols rather than by full names     

Cryobanking as tool for conservation of biodiversity: effect of brown trout sperm cryopreservation on the male genetic potential

Sperm cryobanking could be a good alternative to breeding in captivity in order to preserve genetic diversity. Sperm from two well-characterized brown trout populations originating from two river basins in the Northwest of Spain (Esla and Duerna), both threatened by extinction, was cryopreserved. In order to determine whether a sperm cryobank is the best option for preserving genetic profiles, cell viability, chromatin fragmentation, fertility and genetic variability of the offspring obtained with fresh and frozen sperm, were analyzed. Sperm viability was not reduced by freezing (87.0 ± 3.32% to 77.9 ± 3.59% and 77.6 ± 6.53% to 76.6 ± 2.61% in fresh and frozen sperm from Esla and Duerna, respectively). The percentage of fragmented DNA increased after freezing in spermatozoa from Esla males (from 4.7 ± 0.23% to 6.0 ± 0.28%), but not those from Duerna males.After freezing/thawing, the percentage of eyed embryos drops from 66.8 ± 6.77% to 16.1 ± 3.46% and from 50 ± 8.97% to 11.5 ± 2.50% in the Esla and Duerna basins, respectively. This reduction indicates that many spermatozoa have lost their ability to contribute to embryo development and this loss is not related to either spermatozoa viability or the DNA integrity. Genotypic determination by microsatellite analysis showed that frozen/thawed sperm provided offspring with a similar genetic profile to unfrozen milt, demonstrating the accuracy of the cryopreservation procedure.Taking into account the prolificacy of this species, even a low rate of success of fry after cryopreservation, could provide enough individuals to recover stable populations without altering the genetic profiles of the preserved strains. Therefore, cryopreservation is considered a safe, simple and cheap technology for gene banking in the analyzed brown trout populations.     

Effects of cryoprotectant agents and freezing protocol on motility of black-lip pearl oyster (Pinctada margaritifera L.) spermatozoa

Gamete cryopreservation techniques have been applied to several bivalve mollusc species. However, research activity in this area has primarily focused on cryopreserving gametes from edible oysters (Ostreiidae). Few studies have examined the effect of cryoprotectants and freezing protocols in the preservation of spermatozoa from cultured pearl oysters (Pteriidae). Pearl oyster producers are increasingly looking towards the development of improved family lines and, as a consequence, the ability to cryopreserve gametes would bring about significant benefits to the cultured pearl industry. In response to this need, we evaluated the effect of three cryoprotectant additives (CPA) on motility of spermatozoa from the black-lip pearl oyster, Pinctada margaritifera. These additives have previously been used to cryopreserve gametes of other bivalve species. The following CPA mixtures were evaluated: (1) 0.45M trehalose and 0, 0.64, 1.02 and 1.53 M dimethyl sulfoxide (Me(2)SO); (2) 0.2M glucose and 2M Me(2)SO and (3) 1.31 M propylene glycol (PG). The effects of four different freezing protocols on motility of P. margaritifera spermatozoa were also evaluated (slow, medium, medium-rapid and rapid cooling). This study showed that total motility was best retained when spermatozoa were cryopreserved in 0.45 M trehalose and 0, 0.64, 1.02 or 1.53 M Me(2)SO and frozen using slow to medium-rapid cooling rates (2.1-5.2 degrees Cmin(-1)). Rapid freezing through direct plunging of spermatozoa into liquid nitrogen resulted in the lowest overall retention of motility regardless of the CPA additive; however, CPA mixture also influenced retention of motility, with 0.2M glucose in 2M Me(2)SO and 1.31 M PG retaining the lowest levels of motility for the CPAs evaluated.     

Dormancy of Medicago marina (L.) seed

Medicago marina (L.) is a Mediterranean species whose seeds show strong dormancy that prevents germination. We used an integrated approach of physiological analyses and proteomics to investigate the mechanisms that control M. marina dormancy/germination and that underlie stress tolerance. First, we evaluated the effects on dormancy breaking of the following treatments: mechanical scarification, freezing at −20 °C, storage for 4 months and heating at 100 °C for 1 h. Mechanical scarification and freezing were the most effective treatments in overcoming dormancy. The role of abscisic acid (ABA) in M. marina dormancy was studied by ELISA immuno-enzymatic assay. The ABA content of germinated and non-germinated mature (control) and treated seeds was determined. The level of ABA was higher in treated seeds than in control seeds; the most significant increase occurred in the heated seeds. A comparison of the ABA level in the germinated, control and treated seeds suggests that different mechanisms modulate ABA content in response to different stresses, and that a specific ABA-signalling pathway regulates germination. Proteomic analysis revealed 46 proteins differentially expressed between treated and untreated seeds; 14 of these proteins were subsequently identified by mass spectrometry. Several of the proteins identified are important factors in the stress response, and are involved in such diverse functions as lipid metabolism, protein folding and chromatin protection. Lastly, an analysis of the phosphoproteome maps showed that the function of many proteins in seeds subjected to temperature treatment is modulated through post-translational modifications.     

Reproductive ecology and growth of a population of brown trout (Salmo trutta L.) in an aquifer-fed stream of Old Castile (Spain)

In the River Lobos-Ucero and its tributary the River Avión-Milanos (Duero basin, Old Castile, Central Spain), two limestone streams fed by aquifers, the population of brown trout, as compared with the populations of other European streams, shows a high growth rate, high condition coefficients, short life-span and early age at first maturity. Gonad cycle was also studied. Size distributions of unshed eggs exhibit a dynamic activity with a bi-modal distribution from June onwards, spawning occurred in the last days of November. Fecundity (F) can be predicted from trout length (L, mm) according to the equation: F= –646.47+5.6167 · L. Numbers and standing crop of trout range from 18 to 3903 ind. ha^–1 and 3.6 to 452.9 Kg ha^–1, reaching higher values in the sites close to the aquifers. Egg production had values of 22.4 and 18.0 eggs m^–2 in the Rivers Ucero and Avión-Milanos respectively. Some factors suggested as regulators of these demographical characteristics are discussed in the light of recent literature.     

Essential oil variability in Thymbra capitata (L.) Cav. growing wild in Southern Apulia (Italy)

Essential oils of Thymbra capitata (Thymus capitatus) collected from Southern Apulia (Italy) were analysed using gas chromatography and gas chromatography–mass spectrometry techniques, to check for chemical variability. The study showed that among the 75 components of the oils the most recurrent ones were thymol and carvacrol, which always constituted more than 50% of the oils, as well as γ-terpinene, borneol and p-cymene. Cluster analysis led to the identification of three chemotypes: thymol, carvacrol and thymol/carvacrol; this was presumably a crossbreed between the other two chemotypes. Principal component analysis showed the direct correlation among myrcene, α-terpinene and γ-terpinene; anti-correlation between thymol and carvacrol, and the inverse correlation between linalool and myrcene. Moreover, lower thymol concentrations were accompanied by an increase in myrcene, α-terpinene and γ-terpinene.     

The reproductive response of the sea urchins Paracentrotus lividus (G.) and Psammechinus miliaris (L.) to a hyperproteinated macrophytic diet

The sea urchins Paracentrotus lividus and Psammechinus miliaris are submitted to the same environmental conditions in the Bay of Brest. The relationship between seasonal changes in food source quality and their gonad production was investigated in reproducing experimentally these conditions. In a first stage two macroalgae (Palmaria palmata and Laminaria digitata) were tested. P. miliaris showed a stronger preference for P. palmata and over a year-long experiment both urchins progressively preferred P. palmata. Seasonal variations in the chemical composition of P. palmaria were observed in the Bay of Brest: total carbohydrates were important and the relative maximum (about 50%) was reached between February and August; the lipid level was low and had a relative maximum of about 1% in June and August. Total protein in P. palmaria was high compared to other seaweeds: the maximum value (25%) was observed in June, which was probably due to the maintenance of nitrogen nutrient in the bay.In the second stage of the study, seasonal changes in biochemical components of ingestion and absorption of the two sea urchins were followed in the laboratory using a monospecific diet of P. palmaria. The patterns of total carbohydrates and lipid absorption were very similar for both sea urchin species. Carbohydrates were absorbed strongly and uniformly, year round. Lipid absorption mimicked the lipid nutrient pattern in the food source. Only changes in protein absorption varied slightly between the two urchin species. Protein absorption was maximal for both species in February and June, but the quantity of absorbed protein was significantly higher in P. miliaris than in P. lividus during February. This increase was concomitant with protein storage in the sea urchin gonads, which peaked in February for P. miliaris and in June for P. lividus. P. lividus had a higher gonad production efficiency, based on gonad yield. The comparison between in situ data and the experimental results suggests that an algal diet more nitrogenous than the in situ algal food source would benefit the herbivorous P. lividus, rather than the more omnivorous species P. miliaris. Although P. milaris has been described as a species with large gonad production potential, P. lividus appears to be a more suitable species for echiniculture conditions.