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1.
Semen cryopreservation in the Salmonidae and in the Northern pike   总被引：1，自引：0，他引：1  
The present paper summarizes the data on a semen cryopreservation method for the Salmonidae (Oncorhynchus mykiss, Salmo trutta f. lacustris, Salvelinus fontinalis, Salvelinus alpinus, Salmo trutta f. fario, Hucho hucho, Coregonus lavaretus, Thymallus thymallus) and for the Northern pike (Esox lucius) published during recent years. It describes (1) methods used for the determination of sperm viability; (2) the protective efficiency of substances specifically for protection of internal and external parts of cells and the process of extender development; (3) the freezing, thawing and fertilization conditions; and (4) the tolerable deviations from the freezing protocol for more easy application. Finally, biomarkers are reported that predict the suitability of semen for cryopreservation and the quality of frozen–thawed semen.  相似文献   

2.
The present study describes a uniform method for cryopreservation of semen of Salmonidae (Oncorhynchus mykiss (Walbaum), Salmo trutta f. fario L., Salmo trutta f. lacustris L., Coregonus sp.). It presents a new type of extender and experiments demonstrating that warming of frozen/thawed semen to 20°C prior to fertilization significantly increases the fertilization rate. Freezing is performed in straws in the vapour of liquid nitrogen and for insemination a diluent technique is used. The consistency of the method was tested by repeating the experiments with different batches of semen and eggs. The following fertilization rates (% of control) were obtained: Oncorhynchus mykiss: 89.6 ± 16.0% (mean ± standard deviation, n= 25, n of control = 20, sperm/egg ratio of 1.6 ± 0.2 × 106 spermatozoa/ egg). Salmo trutta f. fario: 93.8 ± 6.4% (n= 12,9.9 ± 1.2 × 106spermatozoa/egg), Coregonus sp.: 92.8 ± 2.4% (n= 6, 0.5 × 106 spermatozoa/egg), Salmo trutta f. lacustris: 85.0 ± 8.4% (n= 12, 4.8 ± 1.4 × 106 spermatozoa/egg).  相似文献   

3.
For salmonid semen, the cryoprotective action of 10% methanol was compared with a 5% dimethyl sulphoxide (DMSO), 1% glycerol mixture, until now one of the most effective cryoprotectants. In Oncorhynchus mykiss (Walbaum), Salmo trutta L. f. fario, Salmo trutta L. f. lacustris and Salvelinus alpinus (L.), semen cryopreserved with both cryoprotectants yielded post-thaw fertilization rates of 90-100% of control with untreated semen at sperm-to-egg ratios of 1.8 × 106-2.4 × 106 spermatozoa per egg. However, at sperm-to-egg ratios of 0.9 × 106-1.2 × 106 spermatozoa per egg, semen cryopreserved with methanol had significantly higher fertilization rates than semen frozen with the DMSO/glycerol mixture. In other studies we obtained similar data for Coregonus sp., Salvelinus fontinalis (Mitchill), Thymallus thymallus (L.) and Hucho hucho (L.), proving that methanol is the most effective and generally applicable cryoprotectant for semen of the studied salmonid species. To facilitate the insemination of large egg batches we investigated the suitability of 1.2 ml and 5 ml straws for deep freezing of semen of Oncorhynchus mykiss, Salmo trutta f. fario, Salmo trutta f. lacustris and Salvelinus alpinus. With 1.2 ml straws the fertilization rates were similar to 0.5 ml straws when using lower freezing and higher thawing temperatures. The 5 ml straws resulted in a fertilization success of only about 40% of fresh semen control.  相似文献   

4.
Since 2016, annually occurring species-specific die-offs of brown trout (Salmo trutta fario) occurred in the Thur River, situated in the Eastern part of Switzerland. These events lead to drastically reduced population densities in the impacted river regions. Clinical signs in brown trout and mortality were restricted to few weeks in August/September. To characterize the syndrome and to find possible causes, from end of March to November 2018, one-year-old brown trout (Salmo trutta fario) and rainbow trout (Oncorhynchus mykiss) were exposed to water from Thur River, fish were sampled regularly and screened for infectious agents, including viral metagenomics, and pathology was described. Starting approximately four months post-exposure, brown trout showed severe lymphohistiocytic pancarditis and necrotizing and haemorrhagic hepatitis. These lesions were recorded until the end of the experiment in November. Rainbow trout were not affected at any point in time. No infectious agents could be identified so far as cause of disease, especially no viral aetiology. Even if pathogenesis and pathology point in the direction of an infectious agent, a causative relationship could not be confirmed and aetiology remains unclear.  相似文献   

5.
The effects of extender composition and freezing rate on motility and fertility of frozen‐thawed Arctic char, Salvelinus alpinus, spermatozoa were investigated. Three freezing rates, two semen diluents and three cryoprotectants were tested. Semen frozen in 0.3 mol L?1 glucose diluent with 10% methanol as a cryoprotectant or in a diluent described by Lahnsteiner with 10%N,N‐dimethylacetamide (DMA) resulted in the highest sperm motility. Fertility was the highest for semen frozen in a glucose–methanol extender but was not significantly different than that for semen frozen in Lahnsteiner's diluent with 10% DMA. Dimethyl sulphoxide (DMSO) at 10% was a relatively ineffective cryoprotectant with either semen diluent. Semen frozen at 6 cm above the surface of liquid nitrogen resulted in a higher post‐thaw sperm motility and fertility than semen frozen at 5 cm. The addition of 7% fresh egg yolk to glucose diluent containing methanol or DMSO did not improve the fertility of frozen‐thawed spermatozoa. However, the addition of 7% fresh egg yolk to glucose–DMA extender significantly improved the fertilization percentages of frozen‐thawed spermatozoa. In conclusion, dilution of semen 1:3 in 0.3 mol L?1 glucose with 10% methanol and freezing 6 cm above the surface of liquid nitrogen (freezing rate of 40±8°C min?1, mean±SD from ?5 to ?55°C) is a promising protocol for cryopreservation of Arctic char semen.  相似文献   

6.
Collection of fish milt by stripping risks the danger of milt contamination by urine. This may seriously influence milt characteristics and quality, including usefulness for cryopreservation. Urine contamination of milt may be avoided by using a catheter for sperm collection. The objectives of this study were to provide basic characteristics of milt collected with a catheter, to test the usefulness of this milt for cryopreservation, and to correlate characteristics of fresh and cryopreserved semen with sperm fertility rates. Milt from 25 rainbow trout Oncorhynchus mykiss (Walbaum) males were used. All samples were cryopreserved using the pellet method within 1 h of collection, using 0.6 m sucrose and 10% dimethyl sulphoxide (DMSO) as an extender. Catheterization resulted in semen of very good motility (> 90% motile spermatozoa) and high fertilization rates after cryopreservation (mean fertilization rate 81.8 ± 13.3% of control, at a sperm/egg ratio of 2.4 ± 0.3 × 106). Osmolality of seminal plasma and concentrations of sodium, potassium and magnesium ions had low variability, which suggests that they are important for creating a stable environment for sperm storage in the sperm duct. Higher variability of certain seminal plasma characteristics, such as protein concentration and antiproteinase activity, suggests that these characteristics are related to individual semen features of particular males. A strong correlation of seminal plasma zinc concentration with protein concentration may reflect an importance of zinc in semen biology. Cryopreservation caused a significant release of protein and acid phosphatase from spermatozoa. Our results did not reveal any single characteristic of semen collected by catheter that could be used as a powerful predictor of cryopreservation success, presumably because all samples were of high quality.  相似文献   

7.
In this study, post‐mortem myofibril proteins change in myosin heavy chain (MHC) mass using sodium dodecyl sulphate‐polyacrylamide gel electrophoresis (SDS‐PAGE), texture properties such as hardness, stickiness, cohesiveness, elasticity, chewiness, gumminess and stress relaxation test parameters (Fmax, Fmin and Fmax‐Fmin) were determined and compared on the days 0, 3, 6 and 9 in three different trout species (Oncorhynchus mykiss, Salmo trutta fario and Salvelinus fontinalis). In terms of texture properties, post‐mortem hardness, cohesiveness, elasticity, chewiness and gumminess were decreased day by day, whereas stickiness, Fmax, Fmin and Fmax‐Fmin were significantly increased (p < .05). According to SDS‐PAGE results, a decreased value on MHC was determined (p < .05). Texture properties and SDS‐PAGE results were found different among groups (p < .05). Correlation between chewiness, gumminess, MHC and hardness; between chewiness and gumminess; between Fmin, Fmax‐Fmin and Fmax; between Fmin and Fmax‐Fmin were significant. As a result of the study, S. fontinalis exhibited least deterioration in comparison with other species and it was determined that SDS‐PAGE results and texture properties support each other.  相似文献   

8.
In rainbow trout, Oncorhynchus mykiss, parameters to determine semen fitness for cryopreservation and quality control of cryopreserved semen were investigated. The following parameters can be used to evaluate semen fitness for cryopreservation as they are statistically significant (P < 0.01) correlated to the post-thaw fertilization rate: motility rate of fresh semen (y = 4.996x - 0.0958x2 + 0.0006x3 - 5 1.7363); sperm velocity of fresh semen (y = 6.741x - 0.036x2 - 268.37); seminal plasma osmolality (y = 0.539x - 125.59); seminal plasma pH (y = -82.768x + 728.133); seminal plasma triglyceride levels (y = 0.069x + 29.863); seminal plasma ß-D-glucuronidase activity (y = -1.112x + 0.0058x2 + 82.229); seminal [lasma lactate dehydrogenase activity (y = -0.096x + 0.00006x2 + 583.80); spermatozoan acid phosphatase activity (y = -132.51x + 126.38x2 + 66.48); spermatozoan adenylate kinase activity (y = 3.474x + 4.925). Quality of deep-frozen semen can be evaluated by motility parameters (P < 0.01): frozen/thawed semen motility rate and post-thaw fertilization rate: y = 1.943x + 28.002; sperm velocity and post-thaw fertilization rate: y = 0.8812x - 0.0059x2 + 24.9686.  相似文献   

9.
Abstract – Otoliths preserve a continuous record of the life cycle from the natal through the adult stage. For that reason, the morphological and chemical characteristics of otoliths of two nonnative Salmonids, brown trout (Salmo trutta) and brook charr (Salvelinus fontinalis) from populations on the Kerguelen Islands were compared. Several approaches were used to study the relationships between otolith morphometry, crystal morph and chemical elemental composition. These salmonids sampled in Kerguelen are well differentiated in terms of species through their otolith shape. The results indicate that ecotypes and river populations can be reasonably well differentiated on the basis of otolith shape. The crystallisation study has revealed the presence of a particular form: the vaterite, present at a high rate: 45% of S. fontinalis and 18% from Salmo trutta fario. Moreover, vaterite and aragonite otoliths presented differences in chemical composition.  相似文献   

10.
Abstract The potential to discriminate between wild and hatchery trout [Salmo trutta f. fario L. and Oncorhynchus mykiss (Walbaum)] by chronological microchemical patterns of different otolith regions was tested. Central and rim regions of otoliths were analysed for life stage specific elemental patterns of strontium (Sr), sodium (Na) and calcium (Ca) as well as of 87Sr/86Sr ratios using laser ablation inductively coupled plasma mass spectrometry (LA‐ICP‐MS). Classification to life stage specific habitats was achieved with 100% accuracy by 88Sr/43Ca, 23Na/43Ca and 87Sr/86Sr ratios resulting in a discrimination of hatchery fish from wild fish. 87Sr/86Sr ratios and Sr concentration in otoliths were positively correlated with the ratio and amount in the water. Na content in otoliths did not directly reflect the concentration in the water, although the 23Na/43Ca ratio in otoliths contributed to the discrimination. Clear differences in otolith chemistry were found for fish experiencing different geological units or a groundwater‐fed environment in a hatchery. Natural chronological microchemical patterns in otoliths proved to be a valuable tool for discriminating wild and hatchery fish without the necessity to use marking methods. LA‐ICP‐MS is well suited for this application because of its limited need for sample preparation, its potentially high sample throughput and the possibility of simultaneous measurement of multiple elemental and isotopic fingerprints.  相似文献   

11.
Sperm cryopreservation is an essential tool for long‐term storage of genetic resources for aquaculture fishes. The goal of this study was to develop an efficient and streamlined protocol for high‐throughput processing for sperm cryopreservation in Atlantic salmon, Salmo salar. The objectives were to evaluate: (1) osmolality of blood serum for determining extender osmolality, (2) effects of extenders for fresh sperm dilution and refrigerated storage, (3) effects of methanol and dimethyl sulfoxide (DMSO) on fresh sperm motility, and (4) motility and fertility after thawing. In this study, sperm samples were collected at a hatchery site in Canada and shipped to a freezing site located 2200 miles (3550 km) away in the USA. Evaluation of three extenders indicated that Mounib solution was suitable for diluting dry sperm for sample processing. Ten percent of methanol or DMSO was less toxic to sperm cells than was 15% within 30 min. Further testing with methanol at 5, 10, and 15%, and sperm solution : extender dilutions (v:v) of 1:1, 1:3, and 1:19 (at concentrations of 5 × 107, 3 × 108, and 1 × 109 cells/mL) indicated that methanol at 5 and 10% showed less toxicity to fresh sperm within 1 h at sperm:extender dilutions of 1:1 and 1:3. Post‐thaw motility of sperm cryopreserved with 10% methanol was significantly higher than that with 10% DMSO, and fertility reflected those results (0–1% in DMSO vs. 38–55% in methanol). Further evaluation of sperm cryopreservation with 10 and 15% methanol at sperm dilution ratios of 1:1, 1:3, and 1:19 indicated that post‐thaw motility in 10% methanol was significantly higher than that in 15% methanol, and post‐thaw fertility in 10% methanol at 1:1 and 1:3 dilution ratios had fertilization rates similar to that of fresh sperm controls. Sperm samples from 12 males cryopreserved with 10% methanol showed male‐to‐male variation in post‐thaw motility (0–36%). Overall, a simplified standard protocol was established for cryopreservation of shipped sperm of Atlantic salmon using extender without egg yolk and yielded satisfactory post‐thaw motility and fertilization rates. This procedure can be readily adopted by aquaculture facilities to take advantage of high‐throughput cryopreservation capabilities at remote service centers. Most importantly, this approach lays the groundwork for an alternative commercial model for commercial‐scale production, quality control, and development of industrial standards. Control of male variability and sperm quality remain important considerations for future work.  相似文献   

12.
The effects of extender composition and freezing rate on cryopreservation efficiency of refrigerated spermatozoa of rainbow trout Oncorhynchus mykiss (Walbaum) were evaluated in order to test the suitability of a computer-controlled ultrafreezer to cryopreserve milt samples obtained in field conditions and stored for several hours. A very highly significant first-order interaction between freezing rate and the type of extender was found. Six of the eight experimental variants did not differ significantly, resulting, after fertilization of eggs with cryopreserved sperm, in a range of 62.3–74.8% of eyed embryos. This procedure was effective for samples stored at 1 °C for 2 days.  相似文献   

13.
Cryopreservation of Arctic charr Salvelinus alpinus (L.) semen was investigated using three diluents, three cryoprotectants [10% dimethyl sulphoxide (DMSO), 10% dimethylacetamide (DMA) or 20% glycerol] and three sizes of straw. The three diluents and three cryoprotectants were combined, resulting in nine extenders. One part semen was added to three parts extender, and motility was evaluated to assess the toxicity of six of the extenders. Semen in nine extenders was frozen in 0.5‐mL straws using liquid nitrogen vapour. Semen extended in 0.3 m glucose and each of the cryoprotectants was also frozen in 0.5‐mL, 1.7‐mL (flat) or 2.5‐mL straws. The freezing rate in each size of straw was measured. Fertility trials were conducted to determine the post‐thaw viability of the frozen semen. The motility of activated spermatozoa was higher in the DMA and DMSO extenders than in the glycerol extender. For the trial using 0.5‐mL straws, post‐thaw fertility results were higher for all extenders containing DMSO, or 0.3 m glucose and DMA, than for all other combinations of diluent and cryoprotectant. For the straw size comparison, the highest fertility was obtained for the 1.7‐mL straw using either DMSO or DMA and for the 2.5‐mL straw using DMSO. For all cryopreservation trials, fertility was low for extenders containing glycerol.  相似文献   

14.
The present study investigated the influence of ovarian fluid (OV) on the gamete physiology in the brown trout (Salmo trutta f. fario). In comparison to water OV prolongs the duration of sperm motility for >'5 min and the fertilizability of eggs for >'10 min independent from the quality of the egg batch from which the OV derived. These stabilizing effects are related to the inorganic composition of OV as similar results are obtained in buffered artificial saline solution (AFS). OV compensates also for sub-optimal environmental conditions as demonstrated in fertilization experiments with mixtures of OV and buffer solutions of different pH. At a dilution ratio of OV: water of 1:1 the stabilizing effect on gametes is significantly decreased and at a dilution ratio of 1:8 it is completely lost. Due to the described effects significantly higher fertilization rates are obtained in OV than in water. In OV the fertilization rates are also higher than in AFS but only at low sperm to egg ratios or when semen with low motility rates and swimming velocities is used. This is considered to be due to a semen dependent egg activation which is partly suppressed by a OV protein with a molecular weight between 20 and 62 kdaltons. The phenomen is discussed in detail.  相似文献   

15.
In two trials, Arctic char (Salvelinus alpinus) semen was frozen in 0.5 mL straws using extenders consisting of 0.3 M glucose and 10%, 12.5% or 15% methanol. Cryopreserved semen was thawed by immersing straws in 25 °C water for 17 s (11.6 °C s?1) or in 5 °C water for 60 s (3.3 °C s?1). The viability of the frozen–thawed semen was measured by determining post‐thaw motility and sperm membrane integrity. Two fertility trials were also conducted. There was no effect of trial or thaw rate on post‐thaw sperm viability or fertility. Use of 15% methanol in the extender resulted in the highest overall percentage of sperm motility and fertility. Use of 12.5% methanol as a cryoprotectant resulted in a higher per cent post‐thaw motility and a lower percentage of dead cells than did 10% methanol. Thus, levels of methanol higher than the commonly used 10% are beneficial for cryopreserving Arctic char sperm.  相似文献   

16.
The first evidence of proliferative kidney disease (PKD) in an Austrian river (the River Kamp) was documented in 2016, and no information on the PKD infection status of trout in other rivers was available. Since then, brown trout (Salmo trutta fario) and rainbow trout (Oncorhynchus mykiss) have been collected from rivers in Upper and Lower Austria for different diagnostic purposes. In this study, we summarize the recent findings of a first survey concerning the distribution of Tetracapsuloides bryosalmonae, the causative agent of proliferative kidney disease (PKD), from these samples. Between September 2015 and October 2017, a total of 280 brown trout and 39 rainbow trout were collected from 21 rivers in the provinces of Upper and Lower Austria. T. bryosalmonae was detected by PCR of kidney tissue in 17 of 21 sampled rivers and in 138 of 280 brown trout as well as in 11 of 39 rainbow trout. Pathological signs of PKD (e.g., hypertrophy of the kidney) were observed in 33 analysed brown trout and six rainbow trout samples. No correlations between fish infected by T. bryosalmonae and the parameters size and age class, condition factor, geological origin of the streams and distribution within the river course were found, while positively tested fish are significantly increased at sampling sites exceeding water temperatures of 15°C for median periods of 115 days. The prevalence within the affected streams or stream sections is highly variable, and in single rivers, infection rates of up to 90% are confirmed.  相似文献   

17.
Proliferative kidney disease (PKD) of salmonids caused by Tetracapsuloides bryosalmonae causes high mortalities of wild brown trout (Salmo trutta fario) and farmed rainbow trout (Oncorhynchus mykiss) at elevated water temperatures. Here the aim was to compare the temperature‐dependent modulation of T. bryosalmonae in the two salmonid host species, which display different temperature optima. We used a novel experimental set‐up in which we exposed brown trout and rainbow trout to an identical quantified low concentration of T. bryosalmonae for a short time period (1 hr). We followed the development of the parasite in the fish hosts for 70 days. PKD prevalence and parasite kinetics were assessed using qPCR. Exposures were performed at temperatures (12°C and 15°C) that reflect an environmental scenario that may occur in the natural habitat of salmonids. T. bryosalmonae infection was confirmed earliest in brown trout kept at 15°C (day 7 post‐exposure) while, in all other groups, T. bryosalmonae was not confirmed until day 15 post‐exposure. Moreover, significantly greater infection prevalence and a faster increase of parasite intensity were observed in brown trout kept at 15°C than in all other groups. These results indicate that PKD is differentially modulated by water temperature in related host species.  相似文献   

18.
The aim of this study was to optimize the cryopreservation protocols for the sperm of red seabream, Pagrus major. The 2‐mL cryovials and programmable freezer were employed for cryopreservation. Six extenders, six cryoprotectants in various concentrations ranging from 6 to 20% (v/v), four cooling rates, and three thawing temperatures were evaluated by postthaw sperm motility and fertility. The ratio of sperm to egg for postthaw sperm fertilization trials was experimentally standardized and was optimal at 500:1. The best motility of postthaw sperm (79.4 ± 4.7% to 88.6 ± 8.0%), fertilization rates (89.6 ± 2.9 to 95.6 ± 1.9%), and hatching rates (85.3 ± 5.1% to 91.4 ± 4.3%) were achieved when Cortland extender, dimethyl sulfoxide (15, 18, and 20%) or ethylene glycol (9, 12%) as cryoprotectants, 20 C/min as the cooling rate, and 40 C as the thawing temperature were employed. Moreover, the results on embryonic development were not significantly different between cryopreserved sperm and fresh sperm during incubation process. In conclusion, these methods of cryopreservation of red seabream sperm are suitable for routine aquaculture application and preservation of genetic resources.  相似文献   

19.
《水生生物资源》1998,11(6):387-394
A sperm cryopreservation protocol adapted from turbot, was tested on sea bass using either 250-μL straws or 1.5-mL cryovials. A dilution to 1/3 in Mounib s extender and a cooling rate of −65 °C·min−1 allowed frozen sperm to recover an initial motility similar to that of fresh sperm at thawing; however, significant differences in motility (P < 0.001, n = 10 fish semen) were observed at further post-activation times, the motility decrease being faster in thawed sperm. At the experimental scale, triplicate inseminations of 2-mL aliquots (approximately 2 000 eggs) showed a significant fertility decay of thawed sperm compared to that of fresh sperm (P < 0.01, n = 12 fish semen) when a discriminating 35·103 spermatozoa to egg ratio was applied. When 70·103 and 200·103 spermatozoa per egg were provided in the same experimental conditions, no significant difference appeared between the fertilisation rates of fresh and thawed sperm. In order to validate the procedure for production or cryobank purpose, a scaled-up protocol was established. Two and 50 mL batches of eggs (approximately 2·103 and 50·103 eggs, respectively) were inseminated in triplicate using either fresh or thawed individual sperms of 5 males with 200·103 spermatozoa per egg. The mean fertility decreased by 23.5 % due to cryopreservation. This decline was explained by the loss of fertility of only one sperm, and only in large-volume conditions, probably due to the delay of use after thawing.  相似文献   

20.
基于线粒体COI基因序列的亚东鲑DNA条形码研究   总被引：7，自引：0，他引：7  
以亚东鲑为研究对象,测定分析了线粒体COI基因638bp碱基序列,探讨了该基因作为DNA条形码在鲑科三种鱼类(亚东鲑、大西洋鲑和红点鲑)鉴定方面的可行性和有效性。亚东鲑33个个体共享1种单倍型。利用Kimura-2模型分析得到亚东鲑与大西洋鲑遗传距离最小,为0.079;大西洋鲑与红点鲑遗传距离最大,为0.141;亚东鲑与红点鲑遗传距离为0.120。基于COI基因片段序列构建的NJ显示,亚东鲑和大西洋鲑首先聚到一起,然后再与红点鲑相聚。结果表明COI基因片段作为三种鱼类DNA条形码进行分类鉴定具有一定可行性。  相似文献   

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