Sperm Survival Rate Research Articles

Cryopreservation of filefish (Thamnaconus septentrionalis Gunther, 1877) sperm

The present study examined the possibility of long-term storage, by cryopreservation in liquid nitrogen, of the sperm of filefish (Thamnaconus septentrionalis). Changes in motility, survival rate, ultrastructure and fertilization rate of the sperm after freezing and thawing were tested. For selection of the immobilizing solution, artificial seawater (ASW) of 250, 350 and 450 mOsmol kg−1 were tested. Sperm motility was significantly inhibited in 350 mOsmol kg−1 ASW, and restored entirely after 100% ASW (1200 mOsmol kg−1) was added. Two cryoprotectants, dimethyl sulphoxide and glycerol, were employed. The sperm was diluted at the ratio of 1:6 with the extenders, and frozen at a freezing rate of −40°C min−1 to −100°C after equilibration for 10 min at room temperature, followed by plunging into liquid nitrogen. The highest post-thawed sperm motility and survival rate were obtained with 5% glycerol. Afterwards, the effect of different freezing rates was examined using 5% glycerol as a cryoprotectant, and the rate of −30°C min−1 to −100°C showed the best result.

Cryo-banking of great ape spermatozoa using human protocols and variation among males

To develop a resource of great ape sperm cells and test the hypothesis that great ape males, like human males, differ in production of sperm cells and in rates of sperm cell survival after exposure to similar processing for cryopreservation. Prospective trial in a cohort of normal male chimpanzees. Seven male chimpanzees ranging in age from 9.9 to 24.2 years were trained to provide semen samples using an artificial vagina. Samples were removed from the plastic sleeve of the device and placed into conical centrifuge tubes. The liquid fraction of semen was removed within 30 minutes and mixed with an equal volume of human commercial transport medium containing egg yolk and buffer (Refrigeration Medium, Irvine Scientific). Samples were transported to the andrology lab for semen analysis and cryopreservation. Briefly, the diluted semen was assayed, then mixed with an equal volume of glycerol containing cryopreservation medium (Freezing Medium, Irvine Scientific) and loaded into half cc straws with labeled handles. Straws were placed into a controlled-rate, nitrogen vapor freezing machine for slow cooling to -7 degrees C, seeding, cooling to -80 degrees C at -10 degrees/min and plunging into liquid nitrogen. A straw from each sample was subsequently thawed, diluted with culture medium, concentrated by centrifugation, and diluted to the original volume of the straw for assay of motility via CASA system. During a 20-month interval 93 semen samples from seven males were obtained for analysis and potential cryopreservation. Individual males provided between 5 and 29 samples. Males differed significantly (pyr<0.000005) in the number of sperm cells provided in each sample from averages of 79, 88, 454, 1858, 1138, 678, and 1640 million and in the percentage of motile cells available for cryopreservation from averages of 48, 4, 32, 27, 13, 18, and 47 percent. Between 5 and 26 samples were cryopreserved for each male. The ratio of progressive motility after thaw and washing compared to pre-freeze levels differed (p=0.03) between males (0.43, 0.16, 0.33, 0.23, 0.41, 0.30, and 0.17) and was not related to number of sperm in the ejaculate or the initial motility. Currently, 531 straws with estimated yields after thaw of 3,686 million motile cells have been banked for research uses and seven males are trained to provide samples on demand. These procedures have been extended to another great ape species, the bonobo. Male chimpanzees of varying ages can be trained to provide semen samples for analysis, research, and cryopreservation. Once trained, animals can be induced with rewards to provide samples on a regular basis. A cryo-bank has been developed with sperm cells from seven males. Even with delays of 3 to 6 hours between collection and initiation of processing for cryopreservation, progressively motile sperm cells survive standardized cryopreservation and thawing protocols. However, sperm survival rates vary significantly among males.

Long-term cryopreservation of spermatophore of the giant freshwater prawn, Macrobrachium rosenbergii (de Man)

Long-term cryopreservation of the giant freshwater prawn, Macrobrachium rosenbergii, spermatophores using glycerol (Gly) and ethylene glycol (EG) as cryoprotective agents (CPAs) was studied. The tolerance of sperm to cryopreservation was evaluated on the basis of sperm survival and fertilizing ability. The survival of the sperm was determined by trypan blue staining, while the fertilizing ability was assessed from artificial insemination of the cryopreserved spermatophores. The rates of embryo survival on day 5 after spawning and of spermatophores capable of producing embryos survived to hatching were determined. Storage of spermatophores at −20°C without CPA for a short period of up of 1–5 days decreased the sperm survival significantly and did not preserve fertilizing ability. Preservation at −20°C in the presence of 10% or 20% Gly or of 10% or 20% EG offered a simple and efficient short-term storage up to 10 days. For a long-term storage, cryopreservation in the presence of 20% EG at −196°C was more efficient than at −20°C. High sperm survival rates and high fertilizing ability were recorded from those cryopreserved at −196°C for up to 150 days. High sperm survival rates with moderate levels of fertilizing ability were obtained from those cryopreserved at −20°C for not more than 30 days. The results indicate that preservation at −196°C with 20% EG is a suitable procedure for long-term storage of the giant freshwater prawn spermatophores.

Short-term storage and cryopreservation of Urechis unicinctus (Echiura: Urechidae) sperm

In the present study, attempts were made to preserve Urechis unicinctus sperm at 4°C. Cryopreservation procedures were optimized for various cryoprotectants and freezing rates, equilibration times and dilution ratios. During short-term storage, the motility of undiluted sperm was extended for 6 days of cold storage,and in 70% and 100% artificial seawater only persisted for 2 and 4 days respectively. The survival rate of undiluted sperm was maintained at a high level accordingly. After cryopreservation, the highest motility and survival rate (41.5±2.2%) were obtained in 15% dimethyl sulphoxide (Me2SO) using a freezing rate of 30°C min−1. After thawing the sperm cryopreserved in glycerol lost almost all motility. The motility and survival rate of post-thawing sperm did not show significant differences after 8 and 15 min equilibration using 15% Me2SO as cryoprotectant; the values were significantly higher than those of 2 min equilibration. Comparisons of motility and survival rate between treatments pooled by dilution ratio showed that the effect of 1:1 ratio (sperm volume to cryoprotectant volume) was best. There was no difference between 1:3 and 1:5, and other ratioswere significantly worse.

Sperm competition

Sperm competition

Survival rate and antioxidant enzyme activity of ram spermatozoa after dilution with different extenders or selection by a dextran swim-up procedure

The aim of this study was to evaluate the effect of four extenders (Sucrose (S), Galactose (G), milk–yolk (MY), and Fiser (F)) on the motility, membrane integrity, and functional integrity of ram spermatozoa during liquid storage at 15 °C. The use of either S or MY for the selection of high quality spermatozoa by a swim-up procedure was comparatively analyzed. Additionally, the activity of three antioxidant enzymes, superoxide dismutase (SOD), glutathione reductase (GR), and glutathione peroxidase (GPx) was evaluated in both swim-up selected samples maintained at 15 °C for 6 h. Sperm motility was better preserved in MY and was significantly higher after 6 h of incubation than in either S or F ( P<0.0001) and G ( P<0.0005). Likewise, the incidence of spermatozoa with integral and functional membranes was higher in samples diluted in MY, with no significant decrease after 6 h of incubation. The comparative analysis of the swim-up procedure performed with either MY or S revealed that not only was total sperm recovery significantly ( P<0.001) higher (67.3%±3.21 versus 47.6%±3.78), but also that the best survival rate of spermatozoa was found in the MY stored sample. Sperm motility, viability and response to a hypoosmotic swelling (HOS) test were also significantly higher in the MY extended sample, maintaining still significantly higher values after 6 h of incubation. In addition, this sample showed higher activity values for the antioxidant defense enzyme system.

Effects of exposure to dibutyltin dichloride on sperm density, viability and morphology in male mice

AbstractExperiments on the effects of 0.025–0.40 µg of dibutyltin dichloride (DBTCl) per kilogram body weight (kg bw), on sperm density, viability and morphology in mature mice were conducted by daily intraperitoneal injection for 7 days at 22 ± 2 °C and 12 h light–dark cycle conditions. The results demonstrated that DBTCl exhibited strong toxicity on sperm quality. Dosed with ≥ 0.05 µg DBTCl/kg bw groups, the testes weight, sperm density and the rate of survival of sperm decreased, whereas the rate of sperm abnormalities increased significantly. In addition, treatment with 0.05 µg DBTCl/kg bw resulted in increasing rate of sperm head abnormalities, whereas administration at 0.20 and 0.40 µg DBTCl/kg bw significantly increased the rate of sperm tail abnormalities. In the group treated at ≥ 0.10 µg DBTCl/kg bw, the mice body weights decreased. It appeared there was a noticeable dose–response relationship between DBTCl and the parameters studied. ED50 values (7 days) of DBTCl for survival rate of sperm and density were 0.17 µg/kg bw and 0.19 µg/kg bw respectively. The present study provides a possibility for early diagnostic indicators and methods for sperm quality induced by organotin compounds (DBTCl) in mammals. Copyright © 2001 John Wiley &amp; Sons, Ltd.

A Trial of Oocyte Maturation and In Vitro Fertilization by Frozen-Thawed Spermatozoa in the Red-Bellied Tamarin (Saguinus labiatus).

Freezing of epididymal spermatozoa from red-bellied tamarins (Saguinus labiatus) was achieved by using a semen-diluting medium called TTE (Tes, Tris, egg-yolk base) containing 5% glycerol. When the frozen spermatozoa were suspended and incubated in m-TYH medium (TYH medium supplemented with 1mM caffeine and 1mM dibutyryl cyclic AMP), the frozen-thawed spermatozoa showed a good sperm survival rate and hyperactive movement after 30 min of incubation. A total of 59 germinal vesicle (GV) stage oocytes were collected from 4 ovaries of 4 female red-bellied tamarins regardless of their menstrual cycles. At 24, 48, and 72 h after incubation in TCM-199 medium containing 10% fetal calf serum (FCS), 30%, 53%, and 71% oocytes, respectively, either showed germinal vesicle breakdown (GVBD) or had extruded the first polar body. Although the oocytes matured in vitro were inseminated by frozen-thawed spermatozoa, no eggs became fertilized.

Frozen storage of ram semen I. Processing, freezing, thawing and fertility after cervical insemination

Abstract Research up to 1993 is reviewed. Various methods for processing, freezing and thawing of semen have been elaborated and their effect on survival and fertility of spermatozoa have been examined in vitro or in fertility tests. Diluents initially used for freezing bull semen had their limitations and were modified or replaced by new media. The freezing diluents investigated included citrate-sugar-, milk-, lactose-, saccharose-, raffinose-, trisbased and other media containing protective agents (mainly glycerol and egg yolk), and other substances including antioxidants. Satisfactory sperm survival rates were obtained with a number of diluents and different combinations of the factors studied. Lambing results after cervical insemination varied depending on the parameters examined in the freezing technology or at insemination, but were low in comparison with those obtainable with fresh diluted semen. As a consequence, cervical insemination with frozen-thawed semen had a relatively limited application in sheep.

Cryogenic damage to boar spermatozoa frozen in pellets and in tubes

Abstract We studied the effect of the newly synthesized cryoprotectant EPG-20 (polyethylene glycol ester with polyunsaturated fatty acids, mol.mass 20 kD) on the freezability of boar spermatozoa. Semen samples were frozen by the pelleting method of Nagase-Niwa, as well as in 20-ml aluminum tubes. The addition of 0.5 % EPG-20 to the Serdica cryoprotective medium preserved the semen quality to a greater extent than the control medium containing no EPG-20. It was found that freezing in aluminum tubes resulted in higher sperm motility, survival rates and percentage of intact spermatozoa than when cells were frozen in the form of pellets, the differences being statistically significant (p

Cryopreservation of spermatozoa from cynomolgus monkeys (Macaca fascicularis)

Fertilization of eggs from cynomolgus monkeys (Macaca fascicularis) was achieved in vitro using frozen-thawed spermatozoa. The best sperm survival rate was obtained after freezing spermatozoa in a semen-diluting medium (TTE: Tes, Tris, egg yolk base) containing 5% glycerol with 30 min equilibrium time. Although frozen-thawed spermatozoa could fertilize eggs in vitro and some of the fertilized eggs developed into blastocysts or hatched blastocysts, a sperm fertility assay using salt-stored eggs indicated that frozen-thawed spermatozoa had a shorter lifespan in capacitation medium than did fresh, unfrozen spermatozoa. This may be attributable to damage to their plasma membranes.

Efficacy of the sperm survival test for the prediction of oocyte fertilization in culture

The present study was carried out to investigate the predictive value of the sperm survival test (SST) with respect to the fertilization of oocytes in culture. In general, our laboratory uses a total of 50,000-150,000 motile spermatozoa to inseminate each oocyte. The remaining material is evaluated for motility before and after 24 h of incubation at 37 degrees C in a 5% CO2 atmosphere. A total of 250 oocytes from 50 cases (mean +/- SD, 5.0 +/- 2.4 oocytes per retrieval) were inseminated and the final rate of cleaved embryos obtained was 52.5%. The SST (%) was considered normal when the ratio (final density of progressing spermatozoa after 24 h x 100/initial density of progressing spermatozoa) was 50% or more. Any other result was considered abnormal. Cases presenting one or more cleaved embryos (n = 40) were separated from those in which no embryo formation occurred (n = 10) and the results were compared in terms of the respective sperm survival rates over a period of 24 h: normal SST (one or more cleaved embryos, 37; none, five), abnormal SST (one or more cleaved embryos, three; none, five). The specificity of the SST was 0.92 and sensitivity 0.50, the predictive value of the abnormal test was 0.62 and the predictive value of the normal test 0.88. The efficacy of the test was estimated at 0.71, which was better than the conventional parameters of sperm analysis. A receiver-operating characteristics curve for SST confirmed that the test can be useful for the prediction of fertilizability of oocytes in the laboratory.

Survival of Bull Spermatozoa Seeded and Frozen at Different Rates in Egg Yolk-Tris and Whole Milk Extenders

Six factorially arranged experiments were designed to study effects of seeding, freezing, and thawing rates in whole milk and egg yolk-Tris extenders commonly used for commercial cryopreservation of bull sperm. In these extenders, semen normally is supercooled to –13 or –14°C unless the sperm are seeded. When sperm were supercooled or seeded, either mechanically or with immobilized silver iodide, and frozen to –196°C, the postthaw percentages of motile sperm were 59, 57, and 64%, respectively. Freezing rates of –15, –25, and –35°C/min gave similar sperm survival rates and were superior to –5°C/min. For milk, the critical freezing temperature extended to –75°C before transfer to liquid nitrogen gave good results. For egg yolk-Tris extender, transfer to liquid nitrogen was less critical once –50°C had been attained. Thawing of sperm in water baths at 25 and 45°C gave similar results, and both temperatures were superior to 5°C. The postthaw percentage of motile sperm in egg yolk-Tris was equal or superior to that of sperm frozen in milk. A freezing rate of –15°C/min to –100°C and thawing at 25°C consistently gave good results.

The hypoosmotic swelling test and cryosurvival of human spermatozoa

The hypoosmotic swelling test is a simple laboratory test to evaluate the functional integrity of the membrane of human spermatozoa. This test was performed on 83 samples of human semen before cryopreservation to determine whether it has any predictive value for the cryosurvival of human spermatozoa. Stepwise regression analysis demonstrated that conventional sperm characteristics, including the concentration, motility, normal morphology and viability, of pre-freeze semen samples were of limited value in predicting the cryosurvival of human spermatozoa. Further, the hypoosmotic swelling test results from pre-freeze semen samples did not correlate with the post-thaw motility or the survival rate of spermatozoa after cryopreservation.

Hamster egg penetration: comparison of preincubation periods

Seven-hour and 20-hour incubation periods were tested with donor specimens to be used in the hamster egg sperm penetration assay (SPA). Aliquots of the washed sperm were incubated in air and in 5% CO2 and 95% air at 37 degrees C for comparison of sperm survival rates. The mean penetration for the donors ranged from 0% to 64%, with group means of 21% and 36% for the 7- and 20-hour incubations, respectively. All donors had penetration rates in the fertile range after 20 hours of incubation, but 33% (3 of 9) had penetration rates less than 10% after 7 hours of incubation. Incubation of sperm in air reduced the motility by 9% after 20 hours. The results suggest that the 20-hour incubation in 5% CO2 is better because it reduces the possibility of false-negative results in the SPA. We conclude that penetration in the questionable range of 10% may require further SPA testing with different incubation periods and technique modifications, such as a sperm rise step to increase sperm motility and reduce the number of leukocytes.

The effect of controlling the interval from mating to ovulation on pregnancy rate in ewes treated with progesterone.

Oestrus and ovulation can be controlled in ewes during the breeding season by administering progestagens (Lamond, 1964). However, fertility at the controlled oestrus (40 to 60%) is generally low, regardless of the particular progestagen or the mode of administration (Robinson, 1968). The physiological basis for this subfertility at the controlled oestrus is not clear at present. Fertilized eggs transferred to ewes previously treated with progesterone developed normally (Schmidt, 1961; Shelton & Moore, 1966), indicating that the capability of such uteri to sustain normal growth and development of the embryo was not adversely affected. Quinlivan & Robinson (1969) found that the pattern of distribution and survival rate of spermatozoa became progressively altered at the first oestrus following progestagen withdrawal when compared with the pattern in non-treated control ewes. A significant decline in the cervical population of spermatozoa occurred