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1.
The aims of this study were to describe the fresh spermatozoon ultrastructure using scanning and transmission electron microscopy and to improve the sperm cryopreservation methodology for the freshwater fish pirapitinga Brycon nattereri. Extenders (BTS? and NaCl), straw volumes (0.5 and 4.0 mL), thawing temperatures (30 and 60 °C) and activating agents (0.29% NaCl and 1% NaHCO3) were tested. Methylglycol was used as a cryoprotectant agent and sperm was frozen in nitrogen vapour (dry‐shipper). Post‐thawed sperm motility rate, motility quality (score 0=no movement; 5=rapidly swimming spermatozoa), duration of motility and spermatozoon morphology were evaluated. Fresh spermatozoon was 35.06 μm long, the head was ovoid (2.00 × 1.22 μm) with no acrosome, the midpiece was 2.15 μm long and the flagellum was 30.90 μm long with the typical 9+2 axoneme arrangement. Post‐thawed sperm motility rate (70–79% motile sperm), motility quality (score 3.1–3.7) and morphology (9.3–11.6% abnormal spermatozoa) were not affected by any of the parameters tested. The duration of sperm motility was longer when triggered in 1% NaHCO3 (392–1031 s) compared with 0.29% NaCl (144–338 s). Brycon nattereri sperm cryopreserved under the conditions described above yields over 70% motility and should last long enough to fertilize oocytes, even after 2 years of freezing.  相似文献   

2.
Cryopreservation of sea cucumber Apostichopus japonicus (Selenka) sperm   总被引：1，自引：0，他引：1  
A simple and convenient method for the cryopreservation of sea cucumber Apostichopus japonicus (Selenka) sperm was tested in the present study. The highest motility (76.7±2.9%) of post‐thawing sperm was obtained in 15% dimethyl sulphoxide (DMSO) with a 1:9 dilution (semen volume to DMSO volume) when 0.5 mL semen–DMSO mixture was frozen at 6 cm above liquid N2 in a closed styrofoam box. After thawing, sperm cryopreserved in glycerol almost lost motility entirely. Although there was no significant difference in percentage of motile sperm between 15% and 20% DMSO, the duration of sperm motility of 15% DMSO group was longer than that of 20% DMSO group. The motility of post‐thawing sperm enhanced when the dilution ratio of semen increased from 1:1 to 1:9. Morphological changes such as the loss of mitochondria, swollen plasma membrane and broken or rolled‐up tails were observed in post‐thawing sperm using an eosin–nigrosin staining. The fertility of cryopreserved sperm was significantly lower than that of unfrozen sperm. The 10‐fold increase in sperm to egg ratio resulted in double fertility for cryopreserved sperm, and about 70% fertility relative to the control.  相似文献   

3.
The aim of this study was to test the effects of cryoprotectants [dimethyl sulphoxide (DMSO) and methylglycol], extenders (0.9% NaCl, 5% glucose, Beltsville Thawing Solution? and Merck III?), thawing temperatures (30 and 60 °C) and activating agents (0.29% NaCl and 1% NaHCO3) on the cryopreservation process of tiete tetra Brycon insignis sperm. Sperm was loaded in 0.5 mL straws, frozen in nitrogen vapour at ?170 °C and stored in liquid nitrogen. Post‐thaw sperm quality was evaluated in terms of subjective motility rate, quality motility score (0=no movement; 5=rapidly swimming spermatozoa), duration of motility and vitality (eosin–nigrosin staining). Post‐thaw sperm motility rate was greater in methylglycol (76–88%), compared with DMSO (23–59%). In general, the highest quality motility scores were observed when sperm was thawed at 30 °C and triggered in 1% NaHCO3 (3.5–4.3). Duration of motility was longer when triggered in 1% NaHCO3 (95–120 s) compared with 0.29% NaCl (69–107 s). Sperm vitality was not affected by any of the parameters tested and varied from 51% to 69% intact sperm. Brycon insignis sperm frozen in methylglycol combined with any of the extenders tested and using the methods described above yields motility above 57% and that should last long enough to fertilize oocytes.  相似文献   

4.
Abstract Four parameters were examined in order to define sperm quality in turbot Scophthalmus maximus L., sperm: (1) sperm motility, measured by direct counts of the number of active spermatozoa, expressed as % of total spermatozoa; (2) retention of motility after activation, measured by direct counts, 0–60min after activation, expressed as a % of the initial level of activity; (3) resistance to thermal stress, measured as change in retention of motility, and (4) adenosine phosphate (ATP) concentration, determined for samples of non-activated sperm. The proportion of motile spermatozoa at activation ranged from 34·8% to 97·6% (mean 76·3%) for the individual males tested. Turbot sperm retained on average 52% (range 27–90%) of its initial activity one hour after activation. Sperm samples which were stressed by cooling to –27°C retained only 8·6% (range 0–25%) of initial activity, compared to control samples which retained 49% (range 38–63%) of initial activity. The retention of motility after activation was not significantly related to the initial motility or the levels of ATP. Concentrations of ATP in turbot sperm (mean 0·46mg ATP/106 spermatozoa, equivalent to 9·2nmol ATP/108 spermatozoa) were comparable to those measured in mammals.  相似文献   

5.
To clarify factors reducing the motility and fertility of cryopreserved spermatozoa of the Japanese pearl oyster Pinctada fucata martensii, the structure of spermatozoa before and after cryopreservation was observed by scanning electron microscopy. Testicular spermatozoa were diluted with cryopreservation diluent (10% methanol+18% fetal bovine serum+72% sea water), and dispensed into 0.25-mL straws. The straws were cooled at a rate of approximately −20 °C/min to −50°C, and subsequently immersed in liquid nitrogen. Percentage motility of spermatozoa before cryopreservation was 69.9±4.2%, and that of cryopreserved spermatozoa was 24.0±1.8%, respectively. In cryopreserved spermatozoa, the percentage that lacked or had a deformed flagellum was 56.6±3.9%, while in fresh spermatozoa this was 8.7±2.0%. In cryopreserved spermatozoa, the percentage of deformed acrosomes was 76.6±5.2%, while in fresh spermatozoa this was only 0.9±0.3%. Cryopreserved spermatozoa with a normal acrosome and flagellum were only 15.4±3.5% of those in fresh spermatozoa. These results indicate that lesion of the flagellum and deformation of the acrosome occurred through the cryopreservation procedure, and both types of damage lead to loss of the motility and fertility in thawed spermatozoa.  相似文献   

6.
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   

7.
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.  相似文献   

8.
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.  相似文献   

9.
Three cryoprotectants (dimethyl sulphoxide, propylene glycol and glycerol) and two diluents (sucrose based and saline based) were mixed (9 parts diluent–1 part cryoprotectant) factorially to produce six extenders that were tested to develop an effective sperm cryopreservation protocol for winter flounder Pseudopleuronectes americanus (Walbaum). Sperm were diluted 1:3 with each extender and frozen by flotation on liquid nitrogen before being submerged and stored for 30 days. Sperm left unfrozen in each extender for 20 min showed no toxic effects on motility. Extenders containing propylene glycol (PG) as cryoprotectant yielded higher post‐thaw sperm motilities than those containing dimethyl sulphoxide (DMSO) or glycerol. The sucrose‐based diluent performed better than the saline‐based diluent when DMSO was used as cryoprotectant, but there were no differences in post‐thaw motility between diluents for the other cryoprotectants. Activating sperm with ovarian fluid and sea water instead of sea water alone had no effect on post‐thaw motility. In fertilization trials, no differences were observed between any of the extenders and fresh milt when milt, eggs and sea water were left in contact for 1 h. When sperm were forced to compete for eggs by reducing contact time to 20 s, fertilization results followed those of sperm motility rates. Percentage hatch and morphology of larvae at hatching did not differ for eggs fertilized by cryopreserved and fresh sperm. This study represents the first reported successful attempt at cryopreserving winter flounder sperm and should improve gamete and broodstock management protocols for this species.  相似文献   

10.
Sperm cryopreservation has led to transcendental changes in the reproductive biotechnology of both mammals and fish, and is a basic tool for animal improvement. However, these protocols generate damage to cell structure and physiology, altering sperm function as a result of cryoinjuries during freezing and thawing. This review is a compilation of the techniques developed and standardised for assessing sperm function in cryopreserved fish semen. Recent studies have analysed sperm function objectively, applying cellular and molecular techniques to characterise cryodamage. The Computer Assisted Sperm Analysis system has facilitated the assessment of motility, while electron microscopy (SEM and TEM) and cryo‐microscopy have made it possible to study sperm morphology and ultra‐structure. The effects of cryodamage on nuclear DNA have also been analysed using various methods, including the comet Fluorescence in situ Hybridization test, TUNEL, Sperm Chromatin Structure Assay, specific DNA sequences using RT‐PCR and specific genes by qPCR. The latter technique is used to study the mitochondrial genome (mtDNA), together with some candidate genes which are associated with bioenergy activity and sperm motility. Other parameters assessed are mitochondrial membrane potential and ATP content using high performance liquid chromatography, nuclear magnetic resonance spectrometry and cell respiration. All this information makes it possible to establish study and assessment criteria for cryopreserved fish spermatozoa. This work focuses on the use of technologies to study of quality of fish spermatozoa during cryopreservation.  相似文献   

11.
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.  相似文献   

12.
Abstract.— Two experiments were designed to improve upon existing methods for cryopreserving striped bass Morone saxatilis , semen. In the first experiment, two extenders, two cryoprotectant concentrations, and two freezing rates were evaluated on the basis of post-thaw semen motility after 1, 7, and 30 d of storage at −196 C. Semen samples cryopreserved at a freezing rate of −40 C/m resulted in a significantly higher percentage of motile sperm ( P < 0.001) and longer duration of spermatozoa motility ( P < 0.001) than samples cryopreserved at a freezing rate of -30 Chin. Also, the cryoprotectant dimethyl-sulfoxide yielded a significantly higher percentage of motile sperm ( P < 0.001) and longer duration of spermatozoa motility ( P < 0.001) when a 5% concentration was used instead of 7.5%. In the second experiment, the two extenders from Experiment I were re-evaluated and a new extender, which was a modified version of Extender 1, was tested. The samples were cryopreserved at -40 C/min with 5% DMSO and thawed in a 25 C water bath. Spermatozoa motility and fertilization ability were evaluated, and semen cryopreserved in Extender 2 yielded the longest duration of spermatozoa motility ( P < 0.001). the highest percentage of motile sperm ( P < 0.001). and the highest percentage of fertilized eggs ( P < 0.002) in comparison to Extenders I and 3.  相似文献   

13.
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   

14.
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   

15.
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).  相似文献   

16.
ABSTRACT

In this study, the cryopreserved spermatozoa of Epinephelus lanceolatus were transported using a novel method involving dry ice as the medium of preservation and a Styrofoam box. Five conditions were investigated for the cryopreserved sperm under different dry ice exposure times of (24, 48, and 72) h corresponding to treatment 1 (T1), 3 (T3), and 5 (T5), respectively. Meanwhile, the remaining treatments (T2 and T4) involved the same exposure to dry ice for (24 and 48) h followed by re-immersion into liquid nitrogen (LN). The performance of the cryopreserved spermatozoa of the hybrid grouper (E. fuscoguttatus ♀ × E. lanceolatus ♂) was evaluated through fertilization and hatching trials. The results showed no significant difference in fertilization for all five treatments. However, significantly poorer hatching rates than the fresh sperm were observed for spermatozoa exposed to dry ice after 48 h. This study recommends the use of the proposed method to successfully transport E. lanceolatus spermatozoa for the production of hybrid groupers via artificial insemination.  相似文献   

17.
Fertilizability of cryopreserved and cadaveric fish spermatozoa was attempted in the freshwater catfish Pangasius sutchi. Cryopreservation of spermatozoa was done with three cryoprotectants for short time storage (30 days). Whereas the spermatozoa obtained from the cadaveric fish were stored at ?20°C (30 days) without any cryoprotectants. Cryoprotectant toxicity assay showed maximum motility of 88.53 ± 2.01% and viability of spermatozoa (96.19 ± 4.92%) with 15% of Dimethyl acetamide (DMA) at 15 min equilibration time. Whereas Dimethyl sulfoxide (DMSO) (15%) registered moderate level of motility and viability 79.23 ± 2.02% and 80.89 ± 2.1%, respectively. However, the methanol (MeOH) (20%) resulted in low percentage of motility (58.6 ± 0.9%) and viability (68.6 ± 0.9%). Scanning electron micrographs further showed no significant deformity on the surface topography of spermatozoa of cadaveric fish as well as cryopreserved with DMA (15%). The results indicated that 15% of DMA with hanks balanced salt solution (HBSS) extender at a dilution ratio of 1:10 at ?80°C proved to be suitable for cryopreservation of spermatozoa in P. sutchi. This may be due to the osmolality of HBSS similar to seminal plasma of P. sutchi. Further studies on motility, viability and fertility potential of spermatozoa revealed 73.62 ± 1.61%, 88.34 ± 1.05% and 54 ± 2.2%, respectively, with DMA (15%). On the other hand, cadaveric fish sperm registered 57.12 ± 2.32%, 63.45 ± 0.94% and 25.33 ± 1.53% of motility, viability and fertilizability respectively. Thus, this study augments the feasibility of using cryopreserved as well as cadaveric fish spermatozoa for the seedling production in the fresh water catfish P. sutchi.  相似文献   

18.
The post-thaw motility and fertility of Japanese pearl oyster sperm show large variances, even among sperm samples obtained from the same individuals. This study aimed to clarify the factors that cause such differences. Spermatozoa were diluted 50 times with diluent comprising 10 % methanol, 18 % fetal bovine serum, and 72 % seawater, and dispensed into 0.25 ml straws. A total of 59 straws were cooled, one by one, at 11 different heights from the surface of liquid nitrogen (LN) to −50 °C, and then immediately immersed in LN. After thawing the straws, the relationships between the cooling rate and the post-thaw motility and post-thaw fertility of the spermatozoa were examined. Both the post-thaw motility and the post-thaw fertility showed a sharp peak when the straws were cooled at around −20 °C/min. There was a strong correlation between post-thaw motility and fertility (P < 0.001). There was a large difference in the cooling rates and the post-thaw motilities and fertilities of the spermatozoa, even between straws cooled at the same height. These results indicate that the optimum range for the cooling rate of oyster spermatozoa is quite narrow, and the method of cooling straws at a fixed distance from the LN surface is unsuitable for the cryopreservation of Japanese pearl oyster spermatozoa.  相似文献   

19.
The objective of this study was to describe the morphometry and motility parameters of vendace (Coregonus albula) spermatozoa. Morphometric parameters of vendace sperm head and tail were of values similar to rainbow trout. The effects of pH, sodium, potassium and calcium ion concentrations on computer-assisted sperm analysis (CASA) sperm motility characteristics were tested. Vendace sperm was motile in a wide pH range of 6.0–10.5 with the optimum pH established at 9.0. Increases in potassium and calcium ions caused decreases in the percentage of motile sperm. The CASA parameters and erratic sperm movement pattern of vendace spermatozoa were similar to whitefish (C. lavaretus) sperm motility, suggesting that there is a coregonid-specific sperm motility pattern.  相似文献   

20.
The aim of this study was to develop a protocol for semen storage of piracanjuba (Brycon orbignyanus) by both cool storage at 4 °C and cryopreservation at − 196 °C. Semen was diluted in some fish semen extenders (Exp. 1) or in extenders combined with the antibiotic gentamycin sulfate (Exp. 2) and stored at 4 °C. Sperm motility was estimated every 24 h. Then, the effects of egg yolk (0 and 5%), cryoprotectants (dimethyl sulphoxide — DMSO, methanol, and methylglycol) and extenders (NaCl 154 mM, BTS™ Minitub and M III™ Minitub) on semen cryopreservation were evaluated (Exp. 3). Semen was added to each of eighteen cryosolutions (2 yolk concentrations × 3 cryoprotectants × 3 extenders), aspirated into 0.5-mL straws, frozen in nitrogen vapor (Taylor-Wharton, CP 300, “dry shipper”) and stored at − 196 °C. Sperm motility was evaluated after thawing at 60 °C-water bath for 8 s. The three cryosolutions that produced the highest post-thaw sperm motility were used again to freeze semen. Post-thaw semen quality was then evaluated under three tests: sperm motility, the percentage of live spermatozoa and hatching rate (Exp. 4). Piracanjuba semen diluted (1:10 total volume) in NaCl 200 mM or in Saad solution (NaCl 200 mM, Tris 30 mM) maintained motility above 35% for as long as 7 days, at 4 °C. Motility of only 7% was observed on undiluted semen after 3 days at 4 °C. There was neither beneficial nor detrimental effect of gentamycin on sperm motility at 250 μg/mL. Egg yolk addition to the cryosolution was beneficial in samples cryopreserved in NaCl 154 mM and in M III™, but detrimental for samples cryopreserved in BTS™. Methylglycol was the most effective cryoprotector compared to DMSO and methanol. Motility and percentage of live spermatozoa were similar among semen cryopreserved in NaCl–yolk, M III™–yolk and BTS™, all containing 10% methylglycol, but lower than fresh control. Hatching rates of eggs fertilized with sperm cryopreserved in NaCl–yolk or BTS™ were higher than for eggs fertilized with sperm cryopreserved in M III™–yolk, but lower than control fertilizations. The semen cryopreservation protocols developed here will be used to set up a gene bank for endangered piracanjuba populations.  相似文献   

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