Some properties of bream Abramis brama L. sperm and its cryopreservation

Concentration and motility of spermatozoa, total protein content and its electrophoretic profile, glucose content, activity of aspartate aminotransferase (AspAT) and acid phosphatase (AcP) were assessed in 18 samples of semen from common bream Abramis brama L. males, which were hormonally stimulated to spermiation. Also, milt pooled from four donors was cryopreserved as pellets in vapours of liquid nitrogen (?80 °C) using four extenders (each with or without the addition of hen egg yolk). Mean spermatozoa concentration was 11.68 × 10⁹ mL^?1, and mean spermatozoa motility was about 60%. Protein content in seminal plasma was 2.08 mg mL^?1; both PAGE and SDS–PAGE showed considerable heterogeneity of protein fractions. Mean glucose content was over 11 mg%. AspAT and AcP activities were detected in both seminal plasma and spermatozoa extracts. As calculated to 1 × 10⁹ spermatozoa, AcP and AspAT activities were almost sixfold and 46-fold higher in spermatozoa than in seminal plasma respectively. In the best variant, cryopreservation attempts resulted in 66.6% of eyed embryos (compared with control fertilization) obtained after fertilization of eggs with cryopreserved semen.     

Effect of individual male variability on cryopreservation of northern pike, Esox lucius L., sperm

Milt from individual males of northern pike, Esox lucius L., was separately cryopreserved. Concentration of spermatozoa in fresh milt and spermatozoa motility before freezing and after thawing was evaluated. Activity of aspartate aminotransferase (AspAT, E.C. 2.6.1.1.) and acid phosphatase (AcP, E.C. 3.1.2.2.) in fresh and thawed sperm were determined. In comparison with the control group, egg fertilization with cryopreserved milt varied from 6.6% to 96.0%, depending on the donor male. Fertilization success with cryopreserved pooled milt was 71.8%. Freezing and thawing procedure caused loss of proteins from injured spermatozoa, resulting in significantly lower enzymatic activity in spermatozoa. Intensity of enzyme leakage in thawed milt correlated negatively with fertilization success. Concentration of spermatozoa could be a possible accessory quality indication, useful when selecting sperm samples appropriate for cryopreservation.     

Motility Parameters of Rainbow Trout (<Emphasis Type="Italic">Oncorhynchus mykiss</Emphasis>) Spermatozoa in Relation to Sequential Collection of Milt,Time of <Emphasis Type="Italic">Post-mortem</Emphasis> Storage and Anesthesia

The present study investigated the effects of sequential collection of milt, time of post-mortem storage and anesthesia on rainbow trout (Oncorhynchus mykiss) sperm motility parameters (using computer-assisted sperm analysis – CASA) as well as seminal plasma osmolality and sperm concentration. The post-mortem storage and time of anesthesia altered motility characteristics of rainbow trout sperm to different extents. The moderate impact of time of anesthesia was manifested in a shortened duration of sperm motility after 10 min exposure of fish to anesthetic. The prolonged post-mortem storage (≥40–60 min), in addition to lowering sperm motility duration, also significantly influenced sperm motility parameters, such as sperm velocities, percentage of motile sperm and sperm trajectory parameters. These results clearly demonstrate that when milt from sacrificed fish is used for sperm motility studies, the time of post-mortem storage significantly alters sperm motility characteristics. Since sperm motility rate and swimming velocity could predict fertilizing ability, detrimental effects of prolonged post-mortem storage may lead to reduced fertilization success. Sperm concentration and seminal plasma osmolality were lower in the first fractions and increased with successive collections of milt. It suggests the presence of urine contamination of the first milt fractions which were collected by stripping. Therefore, testing of sperm concentration and/or seminal plasma osmolality should be mandatory while handling stored milt.     

Physiological and Biochemical Determination of Rainbow Trout,Oncorhynchus mykiss,Semen Quality for Cryopreservation

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.     

Relationship between biochemical constituents of fish semen and fertility: the effect of short-term storage

Spermatozoa and seminal plasma obtained from rainbow trout and whitefish were analyzed in respect to their aspartate aminotransferase (AspAT) and alkaline phosphatase activities. In particular, the experiments characterized AspAT optimum pH, optimization of assay conditions and action of coenzyme, pyridoxal 5-phosphate (vitamin B₆). The effect of short-term semen storage at 0°C on biochemical indicators and fertilization rate was examined in both species. The concentrations of reduced and oxidized ascorbic acid in seminal plasma of both species were several folds higher than in spermatozoa and blood plasma of fish. Highly significant correlations were found for both species between AspAT activity (sperm or seminal plasma) and fertilization rate (% of eyed-stage or hatched embryos). For rainbow trout, highly significant correlations were found between sperm concentration, motility and fertilization rate. These results suggest that several biochemical indicators of seminal plasma can be used as measures of sperm quality of fish. Some common biochemical parameters for fish and mammal's semen provide evidence for using fish sperm as a model in biomedical research.     

Spawning of ocean pout (Macrozoarces americanus L.): evidence in favour of internal fertilization of eggs

Sperm physiology, in vivo artificial insemination and spawning of the ocean pout (Macrozoarces americanus L.), a marine bottom fish, were studied. Milt was collected from the reproductive tract of mature males by suction using a catheter. The uncontaminated milt, having a very low sperm concentration, contains highly motile spermatozoa and sperm motility was retained in vitro at 4 °C for at least 24 h in both seminal plasma and ovarian slime collected from the oviduct of pre-spawning females. Instead of activating sperm, dilution in sea water instantly immobilized the spermatozoa of ocean pout. Osmolarity and pH of ocean pout seminal plasma were in the ranges 365–406 mOsM and 7.2–7.5, respectively. A study of the ionic composition of ocean pout seminal plasma demonstrated the presence of various ions including Na⁺, K⁺, Ca²⁺, Mg²⁺, and Cl⁻, with a remarkably lower K⁺ concentration compared to that from other fish species. Since injections of milt containing motile sperm into the ovaries of pre-spawning females, which spawned in the absence of males, yielded fertilized ocean pout eggs, it is concluded that the ocean pout exhibits internal fertilization. The larvae hatched after 3 months of egg incubation in ambient sea water (9–10 °C). With proper timing of in vivo artificial insemination of mature females, fertilized ocean pout eggs can be obtained from fish reared in captivity.     

Dry shipper cryopreservation of seven‐band grouper (Epinephelus septemfasciatus Thunberg) spermatozoa

This study examined the usage of a dry shipper for cryopreservation of Epinephelus septemfasciatus (Thunberg) spermatozoa. Milt was diluted 1:49 with 5% dimethyl sulfoxide plus 95% foetal bovine serum for cryopreservation. Computer‐assisted sperm analysis was used to analyse sperm motility, while fertilization and hatching trials were conducted to gauge the applicability of the cryopreservation method for aquaculture. We showed that cooling rates of the dry shipper were stable for 14 days and could be manipulated by the use of different sized freezing straws and use of a simple polystyrene foam container (5 × 5 × 12 cm and 1 cm thickness on all sides with the upper layer exposed). Dry shipper cryopreserved spermatozoa had significantly lower post‐thaw per cent motility and velocity than fresh sperm, but linearity of movement was unchanged. Fertilization and hatching rates were not significantly different at all tested sperm to egg ratios (3000:1–243000:1). The results indicated that 0.33 mL of milt when cryopreserved was sufficient to fertilize up to 450 g of oocytes. Application of this technology will help improve seed production in aquaculture and further develop breeding and genetics studies.     

Cryopreservation of silver barb Puntius gonionotus (Bleeker) spermatozoa: effect of extender composition,cryoprotective agents and freezing rate on their postthawing fertilization ability

The effects of extender composition, cryoprotectant concentration and freezing and thawing on the fertilization efficiency of cryopreserved spermatozoa of Puntius gonionotus were evaluated. Computer‐aided motility analysis of semen was conducted to check the suitability of spermatozoa for cryopreservation after mixing with different extenders and cryoprotective agents (CPAs). Extender‐4 with an osmolality 260 mOsmol kg⁻¹and pH 7.6 was used for the cryopreservation study. Among the CPAs, dimethyl sulphoxide (DMSO) was least toxic and more than 60% fertilization was achieved when used at 1.4 M at 0 °C for 10 and 30 min, whereas the toxicity of all CPAs to spermatozoa was evident when tested at 30 °C. Semen frozen at −16 °C min⁻¹ with 1.4 M DMSO showed 70% fertilization, which was significantly higher (P<0.05) than other freezing rates. Samples thawed at 35 °C water showed a fertilization rate comparable with that of fresh semen. Computer‐assisted semen analysis of fresh and frozen semen after thawing showed variations in different types of motility in spermatozoa and in their class. There was no significant difference in motility before or after cryopreservation; however, significant differences could be observed in the average path velocity (VAP), straight line velocity (VSL) and curve linear velocity (VCL). Semen of silver barb could be cryopreserved with extender‐4 by addition of 1.4 M DMSO to a final cryopreservation medium (MED 2) cooled at a rate of −16 °C min⁻¹, stored in liquid nitrogen (−196 °C) and utilized after thawing at 35±2 °C.     

Computer-controlled freezing of rainbow trout Oncorhynchus mykiss (Walbaum) spermatozoa for routine programmes

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.     

Cryopreservation of sperm in marine fish

Since the first work of Blaxter in 1953, fish sperm cryopreservation has been attempted on about 30 marine species. The present paper reviews the techniques used and the results published in these species. Particular attention is paid to the handling procedure of sperm before freezing, the problems of semen ageing and semen contamination with urine. The quality of frozen–thawed semen was evaluated using previously standardized biotests, such as a two‐step motility activation technique adapted for the different species and fertilization assays using a discriminating insemination technique. Most extenders used in marine fish are saline or sugar solutions. From the investigated cryoprotectants, dimethyl sulphoxide (DMSO) generally leads to the best results. Cooling rates range from 8 °C to 99 °C min^?1; the thawing rate is generally high. Compared with freshwater species, a high percentage of spermatozoa survives cryopreservation. Therefore, and because of the simplicity of the techniques, the cryopreservation of marine fish sperm is suited for application in aquaculture.     

An efficient method for cryopreservation of testicular sperm from the Northern pike, Esox lucius L.

The cryopreservation of semen from the Northern pike, Esox lucius L., was investigated with a method that was originally developed for the Salmonidae. Because the amounts of semen obtained by stripping were insufficient, the suitability of testicular sperm was tested for cryopreservation. Frozen-thawed testicular sperm had fertilization rates similar to frozen-thawed semen obtained by stripping (74.2-84.7%), and at sperm to egg ratios of S= 4.5 × 10⁵ spermatozoa per egg, the post-thaw fertilization rates were also similar to fresh, untreated semen controls. Out of all the fertilization solutions investigated, a 100-mm NaCl, 10-mm Tris (pH 9) solution resulted in the highest post-thaw fertilization rates. To facilitate the fertilization of large egg batches, 1.2-mL straws were used for cryopreservation with a similar efficiency to 0.5-mL straws.     

Biochemical Composition of Seminal Plasma and Annual Variations in Semen Characteristics of Jundiá <Emphasis Type="Italic">Rhamdia quelen</Emphasis> (Quoy and Gaimard,Pimelodidae)

This study investigated the composition of milt of the South American silver catfish (Rhamdia quelen) or jundiá. The semen was taken from jundiá in different periods during the four seasons. The biochemical composition of seminal fluid and the characteristics of sperm were analyzed. The semen quantity which can be extracted per fish in one day was 0.95 ± 0.08 ml during spring (maximum) and 0.24 ± 0.03 ml during winter (minimum). Sperm density (spermatocrit) showed higher values in the spring (75.1 ± 1.3%) decreasing slightly afterwards and reaching 63.0 ± 2.4% to 65.0 ± 2.2% in the fall and winter. Immediately after water dilution, 90–100% of the spermatozoa presented vigorous straightforward motility that remained for at least 20 s. The total duration of the motility was 47.9 ± 1.3 s in the spring and 38.6 ± 0.6 s in the other seasons (P < 0.05). This pattern of motility is maintained for more than 2 h after storage of the milt at room temperature. The pH from 5 to 10 of the water dilution does not influence the sperm motility. The mean seminal pH and osmolality values were 8.7 ± 0.07 and 274.8 ± 11.2 (mOsm/kg), respectively. The ion concentration was: Na 153.7 ± 2.4, K 10.7 ± 2.4, Cl 139.4 ± 2.1, Ca 4.2 ± 0.2, Mg 0.9 ± 0.05, P 0.9 ± 0.08 (mEq/l). The total protein was 0.6 ± 0.05 mg/dl and cholesterol concentration was 13.9 ± 0.9 mg/dl.     

Influence of Egg Yolk on the Quality of Cryopreserved Spermatozoa of Common Carp,Cyprinus carpio

Cryopreservation of fish gametes can help in producing quality fish seeds. Success of cryopreservation is evaluated by the post-thaw motility of the spermatozoa. The changes in the seminal plasma during cryopreservation would alter the energy supply for the motility of the spermatozoa, and thus energy supplementation is found to be useful during cryopreservation. Cyprinus carpio spermatozoa were cryopreserved along with egg yolk as a co-cryoprotectant after 1:100 dilution with 0.85% physiological saline as extender and DMSO as cryoprotectant (85:15). The diluents contained egg yolk at three different concentrations, viz., T₁ (5%), T₂ (10%), and T₃ (15%). The diluted milt was equilibrated for 10 min at 5°C and loaded into 0.25 ml straws. The loaded straws were then frozen with LN₂ vapor for 5 min and immersed in liquid nitrogen. Observations were made once in 7 days for 42 days on motility parameters based on which the duration, score, pattern, and percentage were determined. There were significant differences in the motility duration between treatments, and egg yolk at 5% (T₁) concentration was found to support the cryopreserved spermatozoa better than the other concentrations; the difference in motility duration was statistically significant (P > 0.005).     

Cryopreservation of summer flounder,Paralichthys dentatus L., sperm

The summer flounder, Paralichthys dentatus L., is a high‐value species and considerable research has been conducted to determine practices conducive for its culture. As milt can be limited in this species, experiments were conducted to develop a practical sperm cryopreservation protocol for hatchery use. Two dilution ratios (1:2 and 1:4; sperm:extender), 2 diluents (saline and sucrose‐based), 2 cryoprotectants (10% DMSO and 12% glycerol) and 3 freezing rates (?5, ?10 and ?15°C min^?1) were evaluated using differential staining to assess post‐thaw sperm survival. Seven combinations of the factors examined reduced post‐thaw viability by less than 30%. The average viability of sperm from fresh, pooled flounder milt (67.2 ± 2.9%) was not different from that of thawed milt diluted 1:4 with sucrose diluent (10% DMSO) frozen at ?5°C min^?1 (38.4 ± 7.7%) and fertilization and hatch success were not different in trials using fresh or thawed, cryopreserved sperm. From these experiments a practical sperm cryopreservation method was developed, but further refinement of the freezing protocol is necessary to optimize results.     

Quality of testicular semen of the African catfish Clarias gariepinus (Burchell, 1822) and its relationship with fertilization and hatching success

Quality differences of testicular semen of the African catfish, Clarias gariepinus, and their influence on fertilization and hatching success were investigated. In accordance with an earlier study, two semen types of the African catfish were distinguished according to testicular maturity stage. Semen type I derived from males with white mature testes whereas type II semen derived from males with grey, partly mature testes. Semen volume, sperm cell concentration and seminal plasma pH was significantly higher in type I semen than in type II semen, while sperm motility was similar. Similar fertilization percentages were obtained with semen type I and semen type II. However, the hatching percentage was higher and the percentage of deformed hatched larvae was lower for type I semen. There were significant (P<0.01) positive correlations between sperm motility and fertilization percentage, seminal plasma pH and hatching percentage and a negative correlation between seminal plasma pH and percentage of deformed larvae. Therefore seminal plasma pH and sperm motility are useful to predict semen quality of the African catfish.     

羊驼人工采精及授精技术的研究和应用

羊驼常用的人工采精方法主要是假阴道法采精和电刺激法采精。其精液特性不同于其他家畜及野生动物,主要表现为精液量少、精子密度低及精液呈高粘性,这些因素阻碍了羊驼人工采精及授精技术的发展。采精的精液用于授精时要考虑诱导排卵的方法和时间、精液质量及输精时间等因素。重点讨论了羊驼人工采精和授精技术的研究进展及其应用。     

Seasonal Changes in the Milt Quality of Waigieu Seaperch,Psammoperca waigiensis: Implications for Artificial Propagation

Milt quality of Waigieu seaperch was sampled at the beginning, the middle, and the end of the spawning season to assess if the sampling season has an effect on milt quality parameters. The milt volume and total sperm production were higher at the middle of the spawning season while the sperm concentration in milt was significantly higher at the beginning and the end of the spawning season. Sperm morphology and the major parameters of seminal plasma (pH, total protein, Mg²⁺, or Ca²⁺ concentrations) did not differ throughout the spawning season. The concentrations of Na⁺ and the osmolality increased throughout the spawning season. The percentage of motile cells did not differ during the spawning season while the duration of sperm motility (swimming duration) and velocity (swimming speed) were significantly different during the spawning season. The fertilization rate (75.7 ± 6.8%) and hatching rate (56.5 ± 3.1%) at the middle of the spawning season were higher than at the beginning and end periods. Results indicated that milt quality parameters and fertility success in the middle of the spawning season were higher compared to earlier and later sampling dates. Thus, we recommend collecting milt during this time for maximal fertilization and hatching success.     

Changes in Sperm Production,Sperm Motility,and Composition of Seminal Fluid in Caspian Brown Trout,Salmo trutta caspius,Over the Course of a Spawning Season

This study was carried out to evaluate milt quality in male Caspian brown trout (Salmo trutta caspius) over the course of the winter spawning season. Milt samples were collected biweekly during December and January. Chemical composition of seminal fluid, sperm production (milt volume, sperm density, spermatocrit,) and sperm motility characteristics (percentage and duration of motility) were measured. Milt volume, sperm density, osmolality, seminal minerals (Ca²⁺, Mg²⁺, K⁺, Na⁺, Cl^?), and total protein gradually decreased over the spawning season. Glucose and triglyceride content of milt did not show significant changes over the spawning season. Milt pH and the percentage and duration of motility were comparatively stable, declining only at the end of the season. Significant positive correlations were found between sperm density and seminal minerals, total protein and spermatocrit; percentage of motile spermatozoa and seminal minerals, total protein; and duration of motility and K⁺, Cl^?, total protein, and pH. Results show that season has a significant influence on milt quality in male Caspian brown trout, with the best milt being available at the beginning of spawning season.     

Cryopreservation of YamúBrycon siebenthalae Milt

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.     

Testicular structure, spermatogenesis and sperm cryopreservation in the African clariid catfish Heterobranchus longifilis (Valenciennes, 1840)

The morphological and physiological characteristics of the testes and the sperm of the catfish Heterobranchus longifilis (Val.) are presented. The effect of cryopreservation on the fertilizing capacity of the sperm was also evaluated. Testicular structure and spermatogenesis are described using histological techniques. The coexistence in the lobules of spermatozoa and all the spermatogenic stages indicates that this species is able to perform continuous reproduction. No seasonal trend was noticed in the evolution of the gonadosomatic index and in the quantity of the sperm produced over a year’period. However, maximum sperm production was observed in April and September. Different cryopreservation trials were conducted using a cryoprotective solution composed of 5% dimethyl sulphoxide (DMSO), 5% glycerol, 10% hen's eggyolk and 80% Mounib's solution. Fresh and cryopreserved semen gave equivalent hatching rates (81.1%, 83.4% and 78.9% respectively for the fresh, the 1-hour cryopreserved and the 8-month cryopreserved sperm). Percentages of normal and deformed larvae were not affected by sperm cryopreservation.