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1.
Abstract. The effect of four environmental conditions was investigated upon sperm output in turbot, Scophthalmus maximus (L.), submitted to three different rhythms of stripping. Males kept under a natural light cycle and under a 6-month contracted light programme released a similar sperm output in terms of total volume of semen produced per fish during the experimental period (4·9 ± 0·9ml), mean sperm concentration (29·4 ± 2·8 × 109 spermatozoa/ml) and total sperm number (163·2 ± 40·5 × 109 spermatozoa). Attempts to stimulate spermiation for a second time just after the end of the natural reproduction period resulted in the release of low sperm output (total volume of semen: 1·6 ± 0·4 ml; mean sperm motility: 2 min 36s ± 0 min 47s; mean sperm concentration: 47·6 ± 10·2 × 109 spermatozoa/ml; total sperm number: 84·5 ± 25·3 × 109 spermatozoa). Stripping frequency had no effect on total volume of semen, mean sperm motility and total sperm number. Monthly collection did not modify sperm samples in relation to stripping rank. However, decreasing volume, motility and sperm concentration were observed when males were stripped fortnightly and weekly. During the natural spawning period, the presence of females in the tank enhanced mean sperm motility (from 3 min 27s + 0 min 52s to 6 min 38s ± 1 min 38s).  相似文献   

2.
The high sperm density, together with the short spermatozoa swimming time, makes European eel sperm manipulation and assessment for quality difficult. Two diluting media (K15 and K30) previously designed for Japanese eel sperm were tested. After 24 h, European eel sperm showed significant reduction in the percentage of motile spermatozoa after activation and different motility parameters (VAP, angular velocity; VCL, curvilinear velocity; VSL, straight line velocity; BCF, beating cross frequency), concluding that these media are not suitable to preserve the sperm of this species. After a hormonal treatment to induce spermiation, sperm volume, density and motility were recorded at weekly samplings. The variation of the osmolality (325–330 mOsm kg−1), pH (8.4–8.6) and the ionic composition (concentration of Na+, K+, Mg2+ and Ca2+) of the seminal plasma were registered. Physio-chemical results were related with sperm quality throughout the treatment, to determine which must be the suitable characteristics of one extender for the sperm of this species, and to find the best conditions to obtain suitable cryopreservation media for European eel sperm. K+ concentration increased, while Ca2+ and Mg2+ concentrations showed a progressive reduction in correlation with the sperm quality improvement. Na+ showed a decreasing, but not significant tendency. P1 and P2 freezing media were designed considering the physio-chemical parameters as well as the ionic composition shown by the best quality sperm samples, and then compared with the previously described solutions, TNK and K30. Sperm quality was determined, checking the percentage of motile spermatozoa and motility parameters using computer-assisted sperm analysis (CASA) software. Samples were frozen after dilution (1:5, 1:20, 1:100) in different freezing media supplemented with 10% dimethyl sulfoxide (DMSO). After thawing, samples frozen with low dilution ratio (1:5) in TNK and P1 media showed higher, although not significant, spermatozoa survival (35.5 ± 14.5 and 36.6 ± 6.7%). The addition of l-α-phosphatidylcholine to the media seems to have a positive effect, as reported in the Japanese eel.  相似文献   

3.
Different media and protocols were assayed for European eel sperm cryopreservation.  相似文献   

4.
The fertilization efficiency of cryopreserved sperm was compared with fresh sperm from striped catfish, Pangasius hypophthalmus . Of the two sets of experiments carried out, the first compared four sperm doses using fresh sperm and fresh eggs. The second experiment compared six concentrations of cryopreserved sperm ranging from 6.94 × 107 to 6.94 × 1010 to fertilize 100 eggs per batch. Fertilization, hatch and survival rates were compared between cryopreserved and fresh sperm. The highest fertilization rate (53.75±1.62%) was achieved with a sperm dose of 6.94 × 108. Increasing the sperm dose to 3.47 × 109 did not increase the fertilization rate, indicating that the optimum sperm:egg ratio lies between 6.94 × 106 and 3.47 × 107 sperm per egg. Both highest (6.94 × 1010) and the lowest (6.94 × 107) sperm doses resulted in lower fertilization rates (2.04% and 16.90% respectively). No significant differences were found among four fresh sperm doses compared. Mean hatch and survival rates resulting from fresh and cryopreserved sperm were similar. The experiment shows that while only 1.89 × 106 fresh spermatozoa was required to fertilize a fresh egg, 6.94 × 106 (or 3.67 times more) cryopreserved sperm was required to achieve the same level of fertilization. This provides important information for making decision to cryopreserve sperm for commercial and/or conservation purposes.  相似文献   

5.
Three groups of captive-reared striped bass Morone saxatilis ages 1, 3 and 12 yr, were examined for age-related changes of sperm characteristics including short-term storage. All groups had similar ranges of the following parameters (mean× SEM): expressible milt (5.6× 0.5 mI/kg body weight (BW) to 7.5× 2.1 mL/kg BW), percentage of motile sperm (55× 6% to 60× 2%), duration of sperm motility (69× 3 sec to 72× 5 sec) and percentage of viable sperm (91× 2% to 93× 2%). Compared to the 1 and 12-yr-old fish, the 3-yr-old fish produced the greatest number of spermatozoa (1,190× 370× 109 spermatozoa/kg), sperm concentration (120× 8 × 109 spermatozoa/mL) and spermatocrit (74× 4%). In addition, during short-term storage at 4 C, extender-preserved sperm samples of the 3-yr-old group showed a significantly higher ( P < 0.05) percentage of motile sperm and duration of sperm motility, compared to the other two groups. This suggests that short-term storage may be affected by the age of the male fish. Sperm longevity of the 3-yr-old group was successfully maintained for as long as 15 d, longer than that of the 1-yr-old group (9 d) and 12-yr-old group (7 d). Overall, the 3-yr-old fish appeared to have superior sperm quality than the 1 or 12-yr-old fish based on higher sperm production and increased sperm longevity.  相似文献   

6.
Four experiments were designed to evaluate the effects of osmolality, cryoprotectant, and equilibration time on striped bass sperm motility. In the first experiment, solutions of NaCI or KCI with osmolalities ranging from 0 to 700 mmol/kg were tested on sperm activation. Over 60% of the sperm were activated by isotonic NaCI and KCI solutions with a treatment osmolality of 350 mmol/kg. Sperm remained motile until osmolality increased to 600 mmol/ kg. In the second and third experiments, Extenders 1, 2 and 3 with osmolalities of 350, 500, and 600 mmol/kg, respectively, were tested. Sperm samples stored in Extender 2 showed significantly higher ( P 0.01) sperm motility after 10 min of exposure as well as greater ( P < 0.01) post-thaw motility when compared to samples stored in Extenders 1 and 3. In the fourth experiment, two trials were carried out to evaluate the effects of cryoprotectant and equilibration time. In the first trial, methanol with a concentration of 5% and 10% yielded the highest ( P < 0.05) sperm motility prior to freezing at all equilibration times examined. However, 5% DMSO yielded the highest ( P < 0.01) post-thaw motility (38 ± 3.6%). DMSO with concentrations of 10% and 15% resulted in 17 ± 2.3% and 6 ± 1.0% post-thaw motility, respectively. Both methanol and DMA, at all concentrations tested, resulted in less than 10% post-thaw motility. In the second trial, four DMSO concentrations with three different equilibration times were examined. We observed a significant ( P < 0.001) interaction effect between DMSO concentration and equilibration time. Post-thaw motility was significantly greater ( P < 0.01) with a concentration of 5% DMSO at all equilibration times examined, compared to 1.25, 2.5, and 10% DMSO. An average post-thaw motility of 40 ± 2.9% was achieved after 10 min equilibration using 5% DMSO.  相似文献   

7.
The loss of sperm quality in sterlet (Acipenser ruthenus) due to freeze-thaw process in cryopreservation was investigated in the present study. Two antifreeze proteins (AFPI or AFPIII) were used at different concentrations of 0.1, 1, 10, and 100 μg/mL. We compared motility, curvilinear velocity, and plasma membrane integrity of fresh, cryopreserved sperm, and sperm cryopreserved in the presence of antifreeze proteins. Fresh sperm (control) had 85?±?4% motility and 160?±?2 μm/s curvilinear velocity, respectively. After cryopreservation, the motility of frozen-thawed sperm without addition of antifreeze proteins significantly decreased (44?±?9%), compared to the control. The highest motility of frozen-thawed sperm was obtained in cryopreserved sperm with addition of 1 μg/mL of AFPIII (58?±?14%). No significant differences were observed in curvilinear velocity between fresh sperm and cryopreserved sperm with/without addition of AFPI or AFPIII. The flow cytometry analysis revealed that fresh sperm contained 94.5?±?6% live cells, while the cryopreserved sperm only contained 26.6?±?14% live cells. Supplementation of antifreeze proteins has significantly improved the percentage of live cells in frozen-thawed sperm, except 0.1 μg/ml of AFPI group. No significant difference in percentage of live cells was detected in the sperm cryopreserved with 10 μg/mL of AFPI or AFPIII, compared to fresh sperm. Thus, addition of antifreeze proteins to cryopreservation medium could be considered to improve the post-thawed sperm quality of sterlet.  相似文献   

8.
为研究适用于乌克兰鳞鲤精子的超低温冷冻保存方法,分析比较3种稀释液[Hank′s、Cortland、Freezefish冻精稀释液,精子与每种稀释液均设置3种比例(1∶1、1∶3、1∶5)]及3种体积分数为10%的抗冻剂(二甲基亚砜、1,2-丙二醇和丙三醇)对乌克兰鳞鲤精子低温(4 ℃)保存活力的影响;运用筛选出的冷冻保护液及稀释比例,分析比较3种“3步冷冻法”以及3种解冻温度(20、30、40 ℃)对乌克兰鳞鲤精子活力的影响。试验结果表明,采用Hank′s作为稀释液,10%二甲基亚砜为抗冻剂,精子与稀释液比例为1∶3,平均降温速率为12 ℃/min,解冻温度为30 ℃时,精子活力最高(>68%)。通过对稀释液、抗冻剂、稀释比例、降温速率和解冻温度的层层筛选,建立了适宜乌克兰鳞鲤精子超低温冷冻保存的方法,在其种质保护方面具有重要意义,为开展其他鱼类精子超低温冷冻保存提供参考。  相似文献   

9.
For the evaluation of hormonal control of spermiation in fish, a method to quanify the spermiation response of mature Rhynchocypris oxycephalus (Sauvage and Dabry) to hormonal therapy is described. Spermatocrit was determined after 7 min centrifugation at 18,000 ± g and sperm density was estimated by a standard hemocytomer method. Sperm density can be predicted from spermatocrit since their relationship is linear as described by the regression equation, Y = 3.68X - 27.18 ( R 2 = 0.82, N = 50). where Y is spermatocrit and × is sperm density. Milt production by mature R. oxycephalus was highest at 24 h after injection of 1,000 IU human chorionic gonadotropin (HCG) and 50 μg luteinizing hormone-releasing hormone analogue (LHRHa) per kg body weight. Increased milt production coincided with low spermatocrit and sperm density levels. These results demonstrate that spermiation in mature R. oxycephalus can be reliably evaluated by a spermatocrit method and that HCG and LHRHa are effective in stimulating of spermiation in this species.  相似文献   

10.
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   

11.
Crappie, Pomoxis spp., are popular game fish throughout North America and are produced by public and private hatcheries. However, production is limited by a lack of information on tank culture and induced spawning methods. Development of techniques for storage of sperm and in vitro fertilization would increase flexibility in spawning. Therefore, techniques for sperm cryopreservation were examined in white crappie, Pomoxis annularis. Sperm from adult wild white crappie were used to evaluate sperm extender, cryoprotectant agent and concentration, and cooling technique based on post‐thaw sperm motility. Percent egg fertilization was also compared between sperm stored in the two best cryopreservation protocols and two different osmotic activator solutions. Sperm were cryopreserved using treatment combinations of two extenders (350 mOsmol/kg Hanks' balanced salt solution [HBSS] and 350 mOsmol/kg Ca2+free HBSS) and two cryoprotectants (dimethyl sulfoxide [DMSO] and methanol) at concentrations of 5, 10, and 15% that were cooled at four different rates: 5, 10, 20, and 40 C/min. Post‐thaw sperm motility and fertilization rates indicated white crappie sperm can be cryopreserved using either extender, cryoprotectants of either 5% DMSO or 10% methanol, and cooling at 40 C/min. A follow‐up experiment demonstrated sperm in suspensions on ice retained viability after overnight transport.  相似文献   

12.
Artificially induced maturation of 10 male eels Anguilla anguilla L. weighing 170 ± 30-3 g was followed for 6 wk with in vivo CT scanning in a repeated manner. Since testis tissue has a well defined X-ray density value, the volumetric development could be described. Parallel with this process, a strong decrease of the abdominal fat volume was measured and depicted with the applied surface smoothing method. As an affect of the hCG treatment (250 IU/week per eel) at the third wk, the testis could clearly be recognized on the images. From that time the average testis volume values of the 10 eels were 4,975, 6345, 11,583 and 14,553 mmJ respectively. At the sixth wk, the relative testis volume in the abdomen was 25%. In the fillet a characteristic shift (from -10 to -20 Hounsfield units) of the density histogram towards the fat interval was detected. The average density value of the fillet was found to be zero; this was supported furthermore by the histological analysis of the fillet (fat to muscle rate 0.35-0.40). On the basis of the above findings, the applied CT scanning method was proven to be suitable to follow in vivo the maturation process of male eels.  相似文献   

13.
ABSTRACT:   In order to find out the role of sodium bicarbonate (NaHCO3) on the initiation of sperm motility in the Japanese eel Anguilla japonica , interactions were investigated between NaHCO3 and various reagents (K+ channel blocker 4-aminopyridine [4-AP], ammonium chloride [NH4Cl], sodium acetate and calcium chloride [CaCl2]) that could regulate internal factors (intracellular K+, intracellular pH [[pH]i] and intracellular Ca2+) in sperm motility. Contradictory effects of NaHCO3 were observed (i.e. an inhibitory effect when 4-AP was absent and a promoting effect when 4-AP was present). Sodium bicarbonate inhibited the initiation of sperm motility in the Japanese eel. However, NaHCO3 restored the motility of immotile sperm that 4-AP inhibited. The inhibitory effect of NaHCO3 disappeared with the addition of NH4Cl, which raised [pH]i, but the promoting effect was not affected by [pH]i. Although NaHCO3 recovered motility in the presence of 4-AP, this recovery was also observed with the addition of CaCl2 instead of NaHCO3. In the initiation of sperm motility in the Japanese eel, two roles for NaHCO3 are suggested: an inhibitory role relating to the regulation of [pH]i and a promoting role relating to the uptake of another initiation factor, which could be Ca2+.  相似文献   

14.
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   

15.
The Southern flounder, Paralichthys lethostigma, is a valuable aquaculture fish with established markets in the USA. All‐female production in this species is an important technology for aquaculture because the females usually have body sizes twice those of males at the same age, and sex‐reversed males (genotypic XX neomales) are used for all‐female production by crossing with genetically normal females. However, sperm volume from the neomales is usually small (<0.5 mL) and limits their application for all‐female fish production. Cryopreservation of sperm from these sex‐reversed neomales will provide access on demand with increased efficiency to extend the application of neomales. The goal of this study was to develop a protocol for cryopreservation of sperm from the Southern flounder by using an automated high‐throughput processing system. The objectives were to: (1) determine the effect of osmolality on activation of sperm motility; (2) evaluate the effect of extender solutions on sperm motility capacity; (3) evaluate the acute toxicity of cryoprotectants (dimethyl sulfoxide [DMSO], propylene glycol, and polyethylene glycol) on sperm motility, and (4) estimate the effect of cooling rate on sperm cryopreservation and post‐thaw fertilization. Sperm motility was activated when osmolality was 400 mOsmol/kg or higher. Of the three extender buffers tested, HEPES4‐(2‐hydroxyethyl)‐1‐piperazineethanesulfonic acid (HEPES) at 300 mOsmol/kg resulted in better protection for sperm motility than did Hanks' balanced salt solution and Mounib solution at 300 mOsmol/kg during 7 d of refrigerated storage. After 30 min equilibration with the cryoprotectant of 15% DMSO, sperm motility was 24 ± 21% (fresh sperm motility without any cryoprotectants was 42%). After cooling at a rate of 20 C/min, post‐thaw sperm motility was 8 ± 5% and fertilization was 63 ± 40% evaluated at the 32–64 cell stage (5 × 105 sperm per egg). Overall, a protocol was developed for sperm cryopreservation in the Southern flounder with high‐throughput processing, which provides a tool to preserve the valuable genetic resources from neomale flounders, and enables germplasm repository development for the Southern flounder.  相似文献   

16.
日本黄姑鱼精子生理特性及超低温冷冻保存研究   总被引：5，自引：1，他引：4  
对日本黄姑鱼精子部分生理特性及其超低温冷冻保存技术进行了研究。结果表明,日本黄姑鱼精液pH值为7.0~7.5,精子密度为(11.23±2.78)×109/m l,精子寿命(229.33±17.16)s。在盐度为30~35,pH为7.5~8.5时,精子的活力最高,分别达到(88.33±2.89)%和(88±4.33)%。精子在室温(25℃)条件下,可存活24 h;在低温(4℃)条件下可以存活36 h。以D液作为稀释液,20%的乙二醇作为抗冻剂,利用快速降温法对精子进行超低温冷冻保存,38℃水浴快速解冻,解冻后的精子获得最高的活力为(47±5.78)%。  相似文献   

17.
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   

18.
This study developed a technique of sperm cryopreservation using liquid nitrogen (LN) vapour in farmed blacklip abalone Haliotis rubra through evaluating the following five key factors: (1) cryoprotectant agent (CPA) toxicity; (2) cooling temperature; (3) thawing temperature; (4) sperm to egg ratio and (5) sugar addition, using sperm motility or fertilization rate as quality assessment indicators. The results demonstrated that 6% dimethyl sulfoxide (DMSO) was the best single CPA for sperm cryopreservation in this species. The highest post‐thaw sperm motility was achieved when sperm were exposed to LN vapour for 10 min at 5.2 cm above the LN surface and thawed at 60°C and recovered at 16°C in seawater baths. Post‐thaw sperm motility was found to be significantly higher when 6% DMSO was used in combination with 1% or 2% glucose than 6% DMSO alone. Further evaluation of fertilization rate between these CPAs showed that 6% DMSO+2% glucose achieved the highest fertilization rate of 70% at a sperm to egg ratio of 10 000:1.  相似文献   

19.
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   

20.
This study investigated factors key to the development of sperm cryopreservation in the greenlip abalone Haliotis laevigata using a programmable freezing technique, including (1) permeable cryoprotectant agent (CPA) selection; (2) cooling rate; (3) endpoint temperature; (4) thawing temperature; (5) sperm to egg ratio and (6) sugar, vitamin and amino acid supplementation, using sperm motility, fertilization rate, plasma membrane integrity, mitochondrial membrane potential or acrosome integrity as quality assessment indicators. Results showed that among the permeable CPAs evaluated, 10% dimethyl sulfoxide was the most suitable for greenlip abalone sperm cryopreservation. The highest post‐thaw sperm motility was achieved with the sperm being frozen at a cooling rate of ?5°C min?1 to ?30°C from 0°C and thawed and recovered in 40°C and 18°C seawater baths respectively. The addition of sugars in 10% dimethyl sulfoxide did not significantly improve the post‐thaw sperm motility and fertilization rate. The addition of 0.6% glycine, 0.2% taurine or 0.02% L‐ascorbic acid, on the other hand, significantly improved the post‐thaw sperm motility. However, only the addition of 0.6% glycine improved the post‐thaw sperm fertilization rate, which was further confirmed by the improvement of the post‐thaw sperm mitochondrial membrane potential and acrosome integrity through flow cytometry analysis.  相似文献   

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