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1.
To develop a suitable cryopreservation diluent for spermatozoa of the endangered Sakhalin taimen Hucho perryi, all possible combinations of cryoprotectants (glycerol, dimethyl sulfoxide [DMSO], methanol) and extenders (fetal bovine serum [FBS], 300 mM glucose solution [GS], artificial seminal plasma for masu salmon) were examined by observing sperm motility 10 s after thawing. Spermatozoa cryopreserved with diluents such as mixtures of 10% glycerol plus 90% FBS, 10% DMSO plus 90% FBS, and 10% methanol plus 90% GS showed the highest motility. The maximal post-thaw motility was observed at 10% among all concentrations (0, 5, 10, 15 and 20%) of these three cryoprotectants. No significant difference among three diluents was observed in motility at 10 s. Mixtures of 10% glycerol plus 90% FBS, 10% DMSO plus 90% FBS, and 10% methanol plus 90% GS are suitable cryopreservation diluents for Sakhalin taimen spermatozoa.  相似文献   

2.
鲤、鲢、鳙精子低温短期保存研究   总被引：8，自引：0，他引：8  
本文对鲤(Cygrinus carpio)、鲢(Hypophthalmichthys molitrix)和鳙(Aristichthys nobilis)精液在2—4℃的短期保存进行了实验研究。筛选出了几种较为理想的稀释保护液配方;确定了精液与稀释液的适宜稀释比例为1:1;研究了抗冻剂二甲亚砜(DMSO)对低温保存条件下精于活力的影响,确定了DMSO的适宜添加浓度为6％左右。鲤精在2—4℃保存10天,活力仍高达70％,少数精子存活时间长达12天;鲢和鳙精子在2—4℃保存7天后活力仍高达60％,达到了生产应用的水平。为水产养殖和鱼类育种研究提供了一种简便易行的精液短期保存技术。  相似文献   

3.
The addition of cryoprotectants during the freezing of semen in liquid nitrogen protects spermatozoa from the negative influence of freezing. Every species needs an appropriate cryoprotectant that has to be experimentally selected. Semen obtained from five perches was diluted with the Kobayashi buffer solution at 1:9 ratio. To determine the influence of cryoprotectants on spermatozoa motility parameters, the same type of buffer solution was applied with the addition of methanol, dimethyl sulfoxide (DMSO) and dimethylacetamide (DMA) using the concentration of 10, 5, 2.5 %, respectively, glycerol (15; 7.5 %), sucrose and trehalose (0.45; 0.225; 0.113 M). After the preparation of such tests, parameters of spermatozoa motility were measured, using the CASA system (Image House CRISMAS Company Ltd.). Among used cryoprotectants, methanol did not cause any effect on the sperm motility parameters. The lowest percentage concentrations of DMA, DMSO, glycerol, sucrose and trehalose did not significantly influence the percentage of motile spermatozoa. Higher concentrations of these compounds considerably lowered all motility parameters. As for glycerol and saccharides, their addition resulted in the lowering of the spermatozoa motility possibly due to a higher viscosity of the solution. However, DMA and DMSO were most probably toxic to perch sperm cells. The obtained results indicate that the best cryoprotectant to be used with perch spermatozoa is methanol.  相似文献   

4.
With the aim of improving artificial fertilization (AF) in turbot, Scophthalmus maximus (L.), a series of fertilization experiments was carried out under dry conditions and different wet conditions (eggs/sea water: 2V/V and V/V). Another series of fertilization experiments was carried out with different quantities of sperm pool to determine the optimal ratio of spermatozoa to eggs for each AF method. Sperm pool from two males and eggs from spawns with a viability rate of > 70% were used. The sperm pool’s density (0.4–5.18 × 109 sperm mL–1) and motility (1–5) had been assessed previously. Significantly different fertilization rates were found when comparing 2V/V and V/V wet conditions. Significantly higher fertilization rates were found in dry fertilization when the sperm–egg ratio was > 9000 spermatozoa per egg and, under wet condition V/V, at 3000–4000 spermatozoa per egg.  相似文献   

5.
The commercial‐scale production of fish by use of artificial (induced) spawning would require reliable, large‐volume sources of sperm. Cryopreservation can be used to preserve and store sperm within commercial and research germplasm repositories, but is limited in its application to aquaculture. Straw volume and cooling chamber size restrict the quantity of sperm that can be frozen, and straws must be filled by hand. In contrast, the dairy industry has refined methods for freezing of bull sperm, including automation of straw filling and the use of large cooling chambers. These methods could be used for commercial‐scale cryopreservation of fish sperm, although application would require testing. To supply sperm in large volumes, bags originally developed for swine semen could be cooled using dairy protocols and used as a container for fish sperm. The current study documented the use of commercial‐scale dairy cryopreservation techniques for the production of hybrids of channel catfish Ictalurus punctatus (female) by blue catfish Ictalurus furcarus. Four cryoprotectants (methanol, dimethyl sulfoxide, dimethyl acetamide, and glycerol) were initially evaluated for use with blue catfish sperm. During May 2000 and March to April 2001, suspensions of blue catfish sperm were cryopreserved with 10% methanol in 0.5‐mL French straws and in commercial swine semen bags (Cochette* bags, IMV International. Minneapolis, Minnesota, USA). Cryopreservation took place at a dairy breeding cooperative, using technology employed for bull semen. Sperm motility before freezing was 26 ± 18% during Year 1 (2000) and 62 ± 30% during 2001. Sperm were thawed at 40 C and used to fertilize the eggs of channel catfish (yielding hybrids). Motility after thawing for sperm frozen in 0.5‐mL straws was 11 ± 10% during 2000 and 50 ± 24% during 2001. Motility after thawing was 41 ± 17% for sperm frozen in swine semen bags in 5‐mL aliquots and 43 ± 10% for sperm frozen in 10‐mL aliquots. Neurulation of eggs fertilized with thawed sperm from straws was 83 ± 13% during 2000 and 54 ± 27% during 2001. Neurulation was 57 ± 24% using sperm frozen in swine semen bags in 5‐mL aliquots and 55 ± 10% using sperm frozen in 10‐mL aliquots. There was no correlation between sperm motility before freezing (in 0.5‐mL straws) and after thawing during 2000 (r= 0.52) or during 2001 (r= 0.49). In addition, there was no correlation between initial motility and neurulation of channel catfish eggs fertilized using thawed sperm during 2000 (r= 0.14) or during 2001 (r= 0.29). Sperm of blue catfish can thus be cryopreserved at a commercial scale using dairy protocols and can be made available for the production of hybrid catfish when viable eggs are available.  相似文献   

6.
《水生生物资源》2003,16(5):457-460
Experiments were carried out to investigate the effect of five extenders (sucrose, glucose, fructose, KCl and a saline carp sperm extender) and two cryoprotectants (dimethyl-sulfoxide (DMSO) and methanol) on the cryopreservation of common carp sperm. Freezing of sperm using glucose extender and methanol as cryoprotectant resulted in the highest post-thaw motility, fertilization as well as hatching rates (63 ± 9%, 74 ± 15% and 67 ± 17% vs. 87 ± 5%, 84 ± 14% and 69 ± 14% using fresh sperm, respectively). In general, sugar-based extenders combined with methanol as cryoprotectant yielded higher motility, fertilization and hatching rates than ionic extenders in combination with DMSO. The jelly-like agglutination observed after thawing in samples frozen with sugar-based extenders did not reduce fertilization and hatching rates. Frozen–thawed sperm samples were able to successfully fertilize 10 g (8000) eggs.  相似文献   

7.
Sperm were collected in Florida from wild common snook, Centropomus undecimalis (Bloch), and were shipped to Louisiana State University for analysis and cryopreservation. Threshold activation of sperm (10% motility) occurred at 370 mOsmol kg?1, and complete activation occurred at 680 mOsmol kg?1. These values were significantly different. Sperm samples stored at 1°C in Hanks' balanced salt solution (HBSS) or in 0.6% NaCl solution at 200 mOsmol kg?1 retained motility for as long as 22 days. Mean motility remained above 50% for 9 days for sperm stored in HBSS and for 7 days for sperm stored in NaCl solution. Sperm exposed to 5% dimethyl acetamide (62±10%; mean±SD), 10% dimethyl sulphoxide (DMSO) (39±16%), 5% glycerol (26±5%) or 10% glycerol (6±2%) for 30 min had significantly lower motility than did unexposed sperm (89±9%). When used as a cryoprotectant, samples frozen with 5% or 10% DMSO or 5% methanol had significantly higher post‐thaw motility than did samples frozen with other cryoprotectants. Sperm cryopreserved with 10% DMSO (38±12%) had significantly higher post‐thaw motility than did sperm cryopreserved with 15% DMSO (19±10%) or 20% DMSO (4±4%). There were no significant differences in hatch rates of eggs fertilized with fresh sperm (54±29%) or cryopreserved sperm (41±35%). Survival to first feeding was not different between fish produced with fresh sperm (37±30%; range, 0–86%) or with thawed sperm (24±29%; 0–77%). Transport of sperm to a cryopreservation laboratory and back to a hatchery for thawing and use enabled collaboration between groups with specific expertise and provides a model for the application of cryopreservation by transport of fresh and frozen samples.  相似文献   

8.
The yamú Brycon siebenthalae is an endemic fish of the Orinoco river basin, but wild stocks are decreasing because of the disruption of their habitat. We evaluated a protocol for the cryopreservation of yamú sperm to contribute to the preservation of this endangered genetic resource. Milt was mixed with a cryoprotectant medium (5.5% glucose, 12% egg yolk, and 5%, 10%, or 15% dimethyl sulfoxide - DMSO) in a ratio 1:4 (milt:medium), stored in 0.5-mL French straws, frozen in nitrogen liquid vapor (-76 C), then immersed and stored in liquid nitrogen for 10 d or 12 mo. Motility of thawed spermatozoa was higher ( P < 0.001) in 10% DMSO medium than 5% DMSO or 15% DMSO mediums; but lower than the control ( P < 0.001). With sperm cryopreserved, the highest level of fertilization was achieved with 10% DMSO ( P < 0.001) after 10 d or 12 mo of cryopreservation. Fertilization of eggs inseminated with 6.4 × 109 spermatozoa per g of eggs was higher ( P <0.05) than with 1.6 × 109 spermatozoa per g of eggs. There was no difference (P > 0.05) in fertilization between insemination doses of 3.2 × 109 and 6.4 × 109 spermatozoa per g of eggs. Cryopreservation of yamu milt can be performed successfully with a simple medium combined with 10% of DMSO as cryoprotectant. The highest level of fertility was achieved using between 3 × 109 and 6 × 109 spermatozoa per g of fresh eggs.  相似文献   

9.
半滑舌鳎精子冷冻保存   总被引：4，自引：0，他引：4       下载免费PDF全文
半滑舌鳎精子冷冻保存对于人工繁殖育苗、杂交育种、雌核发育及其性别控制研究具有重要的意义,为此,本文对半滑舌鳎精子冷冻保存方法进行了研究。分别利用2．8mol／L的二甲基亚砜（DMSO）、甘油（Gly）和1,2-丙二醇（PG）冷冻保存该鱼精子。结果显示,DMSO冷冻保存精子的活力较高。利用MPRS＋2．8mol／L DMSO以1：0．5、1：1、1：1．5和1：2的比例稀释并冷冻精子,1：1比例在冻前能够抑制精子的运动,冻后活力可达82．50±3．54％,显著高于其他稀释比例（P〈0．05）。分别利用冷冻保存液A（MPRS＋2．8mol／L DMSO）和B（TS-2＋2．8mol／LDMSO）稀释平衡精子,精子在A中的冻前快速运动时间、寿命分别为37．75±6．45S和145．00±78．98S,与鲜精无显著差异（P〉0．05）。利用以上两种冷冻稀释液冷冻保存精子,精子在A液中的冻后活力和寿命分别可达53．50±6．69％和98．00±13．51s,冷冻效果优于B液（P〈0．05）。冷冻后精子的受精率和孵化率分别为55．00±5．00％和35．00±13．23％,受精率与鲜精无显著性差异（P〉0．05）,因此认为MPRS＋2．8mol／L DMSO可用于半滑舌鳎精子的冷冻保存。  相似文献   

10.
《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   

11.
Effectiveness and efficiency of frozen sperm on fertilization and hatching success of eggs from silver barb was examined in relation to cryoprotectants, freezing rate and storage period. Sperm was diluted in calcium‐free Hank's balanced salt solution, equilibrated with dimethylsulphoxide (DMSO), propylene glycol, sucrose or methanol at 5%, 10%, 15% or 20% final concentrations, and frozen in 250‐μL straws using a one‐step freezing procedure (1, 5 and 8°C min?1 from 25 to ?40°C). Highest post‐thaw sperm motility was found from a treatment using 10% DMSO and 5°C min?1 (82.2 ± 2.1%), similar to that of 10% DMSO and 8°C min?1 (87.8 ± 3.2%). Post‐thaw motility of sperm frozen at 5 or 8°C min?1 was significantly higher than 1°C min?1. Relative sperm motility declined significantly after 10 months of cryostorage while viability did not change during a 12‐month cryostorage. Average fertilization rates of sperm after 1 and 4 months of storage were 64.5 ± 4.6% and 61.3 ± 3.4%, respectively, similar to those of fresh sperm (69.6–72.3%). Hatching rates of cryopreserved sperm (45.4–51.2%) were similar to those of fresh sperm (51.8–57.8%). This study developed suitable methods for cryopreservation of silver barb sperm that can be used to facilitate hatchery operation.  相似文献   

12.
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   

13.
The primary objective was to compare thyroid hormones and levels of carp pituitary extract for the artificial production of female Ictalurus punctatus × male I. furcatus hybrid catfish. The effects of different carp pituitary extract dosage rates (5, 6, 9, and 10 mg/kg), carp pituitary extract (6 and 10 mg/kg) supplemented with the thyroid hormones thyroxine (T4) and triiodothyronine (T3), or pregnenolone (DHP) were determined for inducing ovulation of female channel catfish, fertilization of channel catfish eggs with male blue catfish sperm, and hatching rate of these embryos. Hormone treatments thyroxine and tri-iodothyronine with carp pituitary extract, carp pituitary extract alone, and pregnenolone with carp pituitary extract used to artificially produce hybrid catfish were not different in terms of ovulation rates, eggs/kg, fry/kg body weight of female channel catfish, fertilization rates, or hatching rates (P > 0.05). These findings suggest that lower amounts of carp pituitary extract may be used to induce spawn of female channel catfish for production of channel-blue catfish hybrids and the addition of thyroid and steroid hormones is ineffective at the rates used in this study.  相似文献   

14.
The effects of different concentrations of cryoprotectant (dimethyl sulfoxide; DMSO), cooling rate and straw size on the post-thaw motility of frozen sperm from spotted wolffish, Anarhichas minor, were studied. There was no significant difference in the post-thaw motility of sperm treated with three different concentrations of DMSO (10, 20 and 30%). Similarly, there was no significant difference in the post-thaw motility of spermatozoa when using different freezing rates (i.e. distance of straws from the surface of liquid N2, 4.7, 5.5 and 7.1°C min−1) and the straw size (0.5 and 1.0 ml) did not affect survival. The cryopreservation of sperm can be used to make up for the frequent lack of sperm and/or the unsynchronised timing of sperm production in spotted wolffish males and the ovulation time in females. The results show that sperm from spotted wolffish can be frozen to secure access to viable sperm, but further experiments are needed in order to reveal the effect of different parameters on the post-thawing mortality and define the optimum conditions for cryopreservation.  相似文献   

15.
5月中下旬繁殖季节,用方正银鲫（Carassius auratus gibelio）的卵子分别与麦穗鱼（Pseudorasbora parva）（方正银鲫♀×麦穗鱼♂,简称FM）、方正银鲫（方正银鲫♀×方正银鲫♂,FF）和荷包红鲤（Cyprinus carpio）（方正银鲫♀×荷包红鲤♂,FH）的精子进行人工授精,孵出的仔鱼在网箱中饲养,测定和统计各组子代的存活率、绝对增重率和性比等。结果表明：FF子代的成活率（91.15±1.78%）极显著地高于FM（87.50±2.13%）和FH（85.00±1.04%）（P〈0.01）,FM和FH之间差异不显著（P〉0.05）;FM（20.17±4.33 g）、FH（23.13±3.58 g）子代的出池体质量显著高于FF（18.90±3.82 g）（P〈0.05）,无论雌性还是雄性子代,组间差异均极显著（P〈0.01）,而组内、性别间生长差异不显著（P〉0.05）;FM（99%）、FF（73%）和FH（99%）子代的雌性百分比明显偏向雌性（P〈0.01）,且组间差异极显著,异源精子受精后子代中雌鱼比例明显增高（P〈0.01）。本研究结果证明：银鲫生长性状中存在＂异精效应＂,异源精子对子代的存活率和性别比例有显著影响,对生产中积极利用异源精子提供了数据支持。  相似文献   

16.
Concentration, ability to motility, motility during the second activation (reactivation), and endogenous respiration were studied in sperm from two experimental groups of carp males. Group 1 was maintained for 7 days at 15°C (cold water (CW) group), whereas the second group was subjected to a temperature of 20°C (warm water (WW) group) before sperm sampling. Reactivation were achieved after incubation of firstly activated sperm in media with osmotic pressure adjusted up to 300 mOsm*kg−1 by increasing K+ concentration. Statistically significant reduction of spermatozoa concentration in CW samples versus WW (from 46.0 ± 12.5 (15°C) to 59.3 ± 7 109 (20°C) spermatozoa /ml) have been observed. The sperm of the CW group required a significantly longer incubation time (37 min) under isotonic conditions to achieve a maximum percentage of potent motility at repeated activation than the WW group (23 min). After activation of sperm motility, an increase of respiration rate up to maximum level has been found, this level remained the same under condition of recovering the potential to repeated activation. During the sperm movement respiration rate, in CW group (6.1 nmolO2/min/109spermatozoa) and WW (3.9 nmolO2/min/109spermatozoa), was significant higher compared to nonactivated sperm (2.4 nmolO2/min/109spermatozoa for CW and 1.1 nmolO2 /min/109spermatozoa for WW). And keeping males for 7 days at 15°C increase the respiration rate of sperm.  相似文献   

17.
The effects of straws volume, cryoprotectants and thawing temperatures were evaluated on the sperm quality of cachama blanca Piaractus brachypomus (Cuvier), an important Colombian fish species. Sexually mature fish were induced to ovulation or spermiation with a carp pituitary extract. A pool of suitable sperm samples was diluted in glucose, egg yolk, dimethyl sulphoxide (DMSO‐10%), methanol (MET‐10%) or ethylene glycol (ETG‐5%) and packed in 0.5, 2.5 or 5.0 mL straws and frozen in nitrogen vapour. The thawing process was performed in a 35 or an 80 °C water bath. The fertility was evaluated after 6 h post fertilization. The highest motility percentage (33 ± 3%) was observed with sperm cryopreserved with DMSO, packed in 5 mL straws and thawed at 35 °C. The treatments with DMSO and MET packed in 0.5 and 5.0 mL straws and thawed at 35 °C showed the highest fertility (higher than 71%) and the lowest fertility was obtained with MET‐2.5 mL (9 ± 5%). In all the treatments, a significant decrease in the sperm quality was observed at 80 °C. Sperm cryopreserved with DMSO‐10% or MET‐10%, packed in 2.5 or 5.0 mL straws are suitable to achieve acceptable fertilization and to fertilize high amounts of eggs.  相似文献   

18.
In spite of the fact that egg yolk from different avian species has successfully been used as an additive for the cryopreservation of sperm in mammalian species, its efficacy for cryopreserving fish sperm has not previously been tested comparatively. Therefore, the present study was carried out to determine the effect of egg yolks from different avian species, namely domestic chicken (Gallus gallus domesticus), turkey (Meleagris gallopavo) and quail (Coturnix coturnix), on post-thaw motility and fertilization ability of cryopreserved common carp spermatozoa. Egg yolks from chicken, turkey and quail were analysed for moisture, total fat, protein, cholesterol and phospholipid profile. Total fat and cholesterol contents of the turkey egg yolk were higher than chicken and quail egg yolks (p < 0.05). Semen was frozen according to conventional slow freezing procedure. The extender contained 350 mM glucose, 30 mM Tris and 5 % glycerol supplemented with different ratios of avian egg yolk (10, 15 and 20 %). Semen was equilibrated at 4 °C for 15 min and placed into 0.25-ml straws and frozen in liquid nitrogen vapour (for 10 min at ?120 °C) and finally stored in liquid nitrogen (?196 °C) tank. The frozen spermatozoa were thawed in a water bath at 35 °C for 30 s. Fertilization was conducted using a ratio of 1 × 105 spermatozoa/egg. Cryopreservation experiments resulted in higher post-thaw motility and fertilization rates. Mean post-thaw motility of cryopreserved spermatozoa was between 45 and 80 %, and fertilization rates, expressed as the percentage of eyed embryos, ranged from 70 to 95 %. In conclusion, the present study showed that turkey and quail egg yolks are suitable alternatives to the chicken egg yolk for the cryopreservation of common carp spermatozoa.  相似文献   

19.
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   

20.
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   

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