Recovery Of Motile Sperm Research Articles

Upstream or swim up processing technique: which one is more effective to select human sperm with high chromatin integrity

Sperm processing methods separate motile sperms with good morphology from dead and abnormal forms of sperms, immature germ cells, and non-sperm cells. The propose of this study was to compare the efficacy of upstream and swim-up processing techniques to separate sperms with the high quality especially in relation to sperm chromatin integrity. This experimental study used semen samples from 60 normozoospermic men. Specimens were divided into equal aliquots for processing by swim up (group A), and upstream (group B) methods and compare with control by raw semen (group C). Sperm concentration, morphology, motility, DNA fragmentation and chromatin maturation were measured in these three groups. The results revealed that sperm concentration in the swim up samples was significantly greater than upstream samples (p≤0.04). as addition, motile sperm recovery including the percentage of progressive motility and a total number of motile sperm was better in the swim-up compared to an upstream method and raw semen (p≤0.001). The cell debris and seminal fluid were equally removed by both methods and the percentage of normal forms was also similar in both procedures (p≥0.4). In addition, sperm DNA fragmentation and chromatin maturation were not significantly different between the three groups (p≥0.1). According to results, apparently the upstream method had no significant efficiency to separate good quality sperms compare to swim up. Therefore, swim up seems to be a simple, inexpensive, reliable and widely available method with an efficient yield to separate motile sperm with good morphology and better chromatin integrity for insemination in the infertility clinics.

Using two different thawing temperatures and their effect on the motility recovery of human cryopreserved sperms in cancer patients

Abstract Human sperm cryopreservation is a widely used technique which helps the male partner for the fertility insurance by preserving their sperms for a long time. Cancer patients suffer from low semen quality especially who undergo chemotherapy or radiation treatments may face complete loss of their sperm production. This study aimed to investigate the effects of different thawing temperature (37 °C & 40 °C) on sperm motility recovery after cryopreservation-involving four types of cancer tumors at variable ages between 16 and 42 years- to help cancer patient for storage their sperms. A detailed semen analysis was collected under guidelines for 30 samples. The samples were then analysed and frozen in liquid nitrogen. The semen samples were subsequently thawed at 37 °C & 40°Cfor 3 min. This was then followed by statistical analysis of the comparative motilities. Results: thawing of cryopreserved human sperm at 40 °C results in a statistically significant increase in motility recovery compared with thawing at 37 °C.

Inhibition of sperm motility in male macaques with EP055, a potential non-hormonal male contraceptive.

Men have two practical choices for contraception; the condom which has a high typical use failure rate or vasectomy. New male hormonal and non-hormonal contraceptives are under development that target either the production of sperm (spermatogenesis) or the delivery of sperm. One particular target is the sperm protein EPPIN, which is present on the surface of human spermatozoa. EP055 is a small organic compound that targets EPPIN on the surface of sperm and inhibits motility. EP055 was tested in cynomolgus (Macaca fascicularis) males to determine its plasma half-life after intravenous (i.v.) infusion of a single dose and for binding to its target tissues. Our initial study demonstrated a plasma half-life for EP055 of 10.6 minutes. In a second study examination of macaque testis, epididymis, and plasma after i.v. infusion of a single dose of compound EP055 (63.25 mg/kg) demonstrated that EP055 was detected in testis and epididymis two hours and six hours post-infusion. We initiated a trial in rhesus (Macaca mulatta) males to assess the availability of EP055 in semen and its effect on sperm motility as a measure of the drug's efficacy. Four macaques were infused with a low dose (75–80 mg/kg) followed by a recovery period and a subsequent high dose (125–130 mg/kg) of EP055. After high dose administration, sperm motility fell to approximately 20% of pretreatment levels within 6 hours post-infusion; no normal motility was observed at 30 hours post-infusion. Recovery of sperm motility was obvious by 78 hours post-infusion; with full recovery in all animals by 18 days post-infusion. EP055 has the potential to be a male contraceptive that would provide a reversible, short-lived pharmacological alternative.

Fibroblast growth factor 2 (FGF2) is present in human spermatozoa and is related with sperm motility. The use of recombinant FGF2 to improve motile sperm recovery.

Fibroblast growth factors (FGFs) and their receptors (FGFRs) regulate several functions of somatic cells. In a previous work, we reported FGFR expression in human spermatozoa and their involvement in motility. This study aimed to evaluate the presence and localization of fibroblast growth factor 2 (FGF2) in human spermatozoa, to determine the relationship of FGF2 levels with conventional semen parameters and to assess the effect of recombinant FGF2 (rFGF2) on sperm recovery in a selection procedure. Western immunoblotting analysis using an antibody against FGF2 revealed an 18-kDa band in sperm protein extracts. The protein was immunolocalized in the sperm flagellum and acrosomal region, as well as in all germ cells. Sperm FGF2 levels, assessed by flow cytometry, showed a positive (p<0.05) correlation with sperm concentration, motility, total sperm number and total motile cells per ejaculate. Moreover, samples with abnormal motility depicted diminished (p<0.01) FGF2 levels compared to those with normal motility. Spermatozoa exposed to rFGF2 bound the protein, exhibited higher (p<0.05) total and motile sperm recoveries, and increased (p<0.01) kinematic parameters after the swim-up. Findings herein presented lead to consider sperm FGF2 level as a potential marker of sperm quality, and rFGF2 as a supplement for improving sperm recovery in selection techniques.

Comparación de 2 métodos de selección espermática sobre la fragmentación del ADN y el potencial de membrana mitocondrial de los espermatozoides humanos: en busca del gameto de oro

IntroductionTo select good-quality sperm for assisted reproductive technology procedures, the best spermatozoa should be elicited. The aim of this study is to compare the impact of sperm selection on human sperm DNA fragmentation and mitochondrial membrane potential by means of density gradient and swim-up techniques. Materials and methodsConventional and functional semen parameters (mitochondrial membrane potential and plasma membrane integrity) in sperm selected by density gradient and swim-up techniques in 14 semen samples were evaluated. ResultsIn the selected neat semen samples, the number of sperm recovered was lower using swim-up as compared to density gradients. By contrast, an increase in progressive motility was observed compared with the initial sample, with no statistically significant difference between both techniques. On the other hand, the swim-up technique was shown to decrease the amount of non-progressive motile sperm recovered, whereas the density gradient technique allowed for a better recovery of the total motile sperm selected. Finally, an increase in the proportion of cells with higher mitochondrial membrane potential and lower DNA fragmentation index was observed. ConclusionThe results of this study demonstrate that sperm selection using the swim-up technique allows for better-quality sperm to be recovered, with a higher sperm cell membrane potential and lower DNA fragmentation. The use of this method is therefore recommended prior to artificial insemination or in vitro fertilisation procedures.

A comparative analysis of sperm selection procedures prior to cryopreservation for Nili-Ravi buffalo bull (Bubalus bubalis) semen-: Assessment of its impact on post-thaw sperm functional quality

Sperm selection techniques have been developed to get sperm suspensions enriched in motile and functional cells. Studies show that selection before cryopreservation improves post-thaw quality of cryopreserved sperm but information on buffalo bull sperm is scarce. The study was aimed to 1) perform a comparative analysis of sperm selection procedures; Swim-Up (SU), Sephadex™-G15 Filtration (S-G15) or Glass Wool Filtration (GWF) for total and motile cell recovery, 2) to assess the impact of sperm selection prior to cryopreservation on sperm quality (motility, morphology, cell membrane and normal apical ridge, viability and livability, chromatin integrity) and sperm functionality (Embryo Cleavage after IVF with selected sperm) in post-thawed suspensions of buffalo bull sperm. Semen was collected from 5 Nili Ravi buffalo bulls maintained at the Semen Production Unit Qadirabad, District Sahiwal, Pakistan. Ejaculates were divided into four aliquots for SU, S-G15 and GWF and control. After sperm selection, total and motile sperm recovery was highest in GWF samples (total sperm=84.08±8.39%; motile sperm=80.42±3.57%). An improvement (P<0.05) in all post-thaw parameters was observed in S-G15-selected sperm and, in some parameters in GWF-filtered sperm suspensions compared to control. The highest (P<0.05) embryo cleavage rate (%) was achieved with frozen-thawed sperm selected with S-G15 prior to cryopreservation (44.72±4.18) compared to control (21.98±3.00). In conclusion, post thaw sperm quality was improved after sperm selection from fresh buffalo bull semen through S-G15 and GWF procedures compared to SU and control while, the fertility rate (cleavage rate) was improved with sperm processed using the S-G15 procedure.

Human Sperm Quality and Metal Toxicants: Protective Effects of some Flavonoids on Male Reproductive Function.

Metals can cause male infertility through affection of spermatogenesis and sperm quality. Strong evidences confirm that male infertility in metal-exposed humans is mediated via various mechanisms such as production of reactive oxygen species (ROS). Flavonoids have antioxidant and metal chelating properties which make them suitable candidates for neutralizing adverse effects of metals on semen quality. In the current study, we have evaluated the effects of five types of flavonoids (rutin, naringin, kaempferol, quercetin, and catechin) on recovery of sperm motility and prevention of membrane oxidative damage from aluminum chloride (AlCl3), cadmium chloride (CdCl2), and lead chloride (PbCl4). In this experimental study, motility and lipid peroxidation of metalexposed sperm was investigated in the presence of different concentrations of five kinds of flavonoids. Malondialdehyde (MDA) production was assessed as a lipid peroxidation marker. Aluminum chloride (AlCl3), cadmium chloride (CdCl2), and lead chloride (PbCl4) diminished sperm motility. Treatment of metal-exposed sperm with rutin, naringin, and kaempferol attenuated the negative effects of the metals on sperm motility. Quercetin and catechin decreased the motility of metal-exposed sperm. Based on the MDA production results, only AlCl3 significantly induced lipid peroxidation. Treatment with rutin, naringin, and kaempferol significantly decreased MDA production.

Validation-verification of a highly effective, practical human testicular tissue in vitro culture-cryopreservation procedure aimed to optimize pre-freeze and post-thaw motility.

Purpose The aim of our paper was to validate a testicular biopsy procedure that simplifies handling, processing, and cryopreservation, while at the same time optimizes sperm motility before freezing and after thawing.MethodsTwo prospective studies were conducted to verify, optimize, and understand the virtues of pre-freeze testicular tissue IVC at different temperatures (21, 30, or 37 °C). Testicular tissue was obtained from clinical specimens designated for whole tissue cryopreservation (i.e., intact mass of tubules) and/or for fresh use in IVF-ICSI cycles. Whole testicular biopsy pieces (1–3 mm3) were diluted in glycerol containing freeze solutions, slow cooled to 4 °C and then rapidly frozen in LN2 vapor. Fresh and post-thaw testicular biopsy tissue were evaluated for changes in the quantity (%) and pattern of motility (I–IV: twitching to rapid progression, respectively) over a 1 week duration. The clinical effectiveness of IVC-cryopreserved whole testicular biopsy tissue was also validated analyzing fresh embryo transfers.ResultsMore reliable recovery of motile testicular sperm was achieved using whole tissue freeze preservation combined with IVC (24–96 h) post-acquisition at an incubation temperature of 30 °C compared to ambient temperature (21 °C) or 37 °C. Up to 85 % of the pre-freeze motility was conserved post-thaw (+3 h) for easy ICSI selection. Sperm longevity was optimized to fresh tissue levels by implementing testicular biopsy sucrose dilution post-thaw. Favorable clinical outcomes were proven using frozen-thawed testicular biopsy sperm for ICSI.ConclusionsBy employing minimal tissue manipulation, integrating pre-freeze IVC processing at 30 °C and the freezing of whole testicular biopsy tissue, we have reduced the labor and improved the efficacy of processing testicular tissue for freeze-preservation and subsequent ICSI use.

Standardisation of a novel sperm banking kit - NextGen(®) - to preserve sperm parameters during shipment.

Many male patients diagnosed with cancer are within their reproductive years. These men are advised to freeze their spermatozoa prior to the start of cancer treatment. Very often, sperm banking facilities may not be readily available and patients may be required to travel to distant sperm bank centres. Our objective was to design and standardise a remote home shipping sperm kit that allows patients to collect a semen sample at home and ship it overnight to a sperm bank. A total of 21 semen samples and two transport media (refrigeration media and human tubal fluid) and five different combinations of ice packs were tested for maintaining desired shipping temperature. Ten semen samples were assessed for pre- and post-shipment changes in sperm motility, membrane integrity, total motile spermatozoa and recovery of motile spermatozoa. Even though motility, membrane integrity and total motile spermatozoa declined both in samples examined under simulated shipped conditions and in overnight-shipped samples, the observed motility and total motile spermatozoa were adequate for use with assisted reproductive techniques. Using refrigeration media, cooling sleeve and ice packs, adequate sperm motility can be maintained utilising NextGen(®) kit and these spermatozoa can be used for procreation utilising ART techniques such as intracytoplasmic sperm injection.

Ultrastructural Features of Sperm Storage Tubules in the Oviduct of the Indian Garden Lizard, Calotes Versicolor.

This study provides the first description of the ultrastructural features of sperm storage tubules (SSTs) in the uterovaginal region of the oviduct of the Indian garden lizard, Calotes versicolor. Abundant spermatozoa along with copious secretory material were found in the lumen of the SSTs. These secretory granules appeared similar in electron density to those found in the epithelial cells lining the SSTs indicating their similar origin. The close physical proximity of sperm with these granules suggests an intimate association between the two. The present study is also the first report of recovery of motile sperm from the flushings of SSTs in C. versicolor. The density of sperm found in the flushings varied, being most abundant during the reproductive phase and minimum/absent during the regressive phase. Understanding the microenvironment of the SSTs, the nature of the secretory granules and their interaction with sperm can guide us in unraveling the biology of oviductal sperm storage.

The protective effects of melatonin against cryopreservation-induced oxidative stress in human sperm.

Reactive oxygen species (ROS) production and lipid peroxidation during cryopreservation harm sperm membrane and as a result reduce the recovery of motile sperm. The antioxidant effects of melatonin on different cells have been widely reported. This study was aimed to evaluate changes in post-thaw motility, viability, and intracellular ROS and malondialdehyde (MDA) in response to the addition of melatonin to human sperm freezing extender. Semen of 43 fertile men was collected and each sample was divided into eight equal aliquots. An aliquot was analyzed freshly for viability, motility, and intracellular ROS and MDA. Melatonin was added to the recommended human freezing extender to yield six different final concentrations: 0.001, 0.005, 0.01, 0.05, 0.1, and 1 mM. A control group without melatonin was also included. Two weeks after cryopreservation, samples were thawed and pre-freeze analyses repeated. Obtained results showed that cryopreservation significantly (P <0.05) reduces viability and motility, but increases intracellular ROS and MDA of human sperm. The semen extender supplemented with various doses of melatonin (except for 0.001 mM) significantly (P <0.05) increased motility and viability, but decreased intracellular ROS and MDA levels of cryopreserved sperm after the thawing process, as compared with the control group. We also found that the most effective concentration of melatonin in protecting human spermatozoa from cryopreservation injuries was 0.01 mM. These findings suggest that melatonin exerts its cryoprotective effects on spermatozoa possibly by counteracting intracellular ROS, and thereby reduces MDA generation. This finally leads to increase of post-thaw viability and motility of cryopreserved spermatozoa.

Sperm Selection Using Three Semen Processing Techniques.

This study aimed to assess the efficiency, in terms of recovered motile spermatozoa with normal morphology, of three sperm selection techniques: migration- sedimentation (SS), swim-up from fresh semen (SF), and swim-up from washed (SL) sperm. Samples from 20 normozoospermic men were divided into three equal aliquots and processed in parallel. SS was performed in a Jondet tube, using 1 ml of semen and 2.5 ml of Human Tubal Fluid medium (HTF+10% Synthetic Serum Supplement, Irvine, USA). For SF, 1 ml of HTF was layered over 1 ml of fresh semen (SF). For SL, 1 ml of sperm was first centrifuged (300 g, 10 min) and the pellet resuspended in 1 ml of HTF; a second layer of HTF was placed on top. Migration time was 1h (SF and SL) and 1h30' for SS at 37°C. After migration, 200 μl were removed from the top layer (SF, SL) and from the central cone (SS). Concentration, morphology and motility were determined. Recovery rates were 25% for SS, 10.1% for SF and 4.5% for SL. SS recovery rate was significantly higher (P<0.01) than the two swim-up techniques. Total motility was statistically different (P<0.001), with 93.6% for SS, 91.2% for SF, and 77% for SL. Sperm morphology was similar between the three techniques (P= 0.12). SS is an efficient technique for the recovery of motile spermatozoa from native semen preparations and yielded better results than SF and SL. Routine use for assisted reproduction remains to be evaluated.

Seminal plasma concentrations of Anti‐Müllerian hormone and inhibin B predict motile sperm recovery from cryopreserved semen in asthenozoospermic men: a prospective cohort study

The rate of motile sperm recovery after cryopreservation is very variable and difficult to predict. Anti-Müllerian hormone (AMH) and inhibin B are produced by Sertoli cells and released into the seminal plasma, where they could be functional markers of spermatogenesis and sperm resistance to thermal stress. The aim of this study was to evaluate whether seminal plasma levels of AMH and inhibin B predict sperm recovery after cryopreservation. The study included 153 men enrolled prospectively during a semen analysis. The cohort was stratified by the fresh semen characteristics into: normal (n=52), high sperm count (n=55), asthenozoospermia (n=23), and oligozoospermia (n=23). The main outcome measure was motile sperm recovery rate, defined as post-thaw total motile sperm count × 100/pre-freezing total motile sperm count. In men with asthenozoospermia there was a significant correlation between motile sperm recovery rate and the pre-freezing concentrations of AMH (r=0.522, p<0.05) and inhibin B (0.471, p<0.05). In this group, the areas under the receiver operating characteristic curves of AMH and inhibin B for prediction of ≥50% motile sperm recovery after cryopreservation were, respectively, 0.808 and 0.638. AMH was particularly useful, with sensitivity of 0.85, specificity of 0.80, positive predictive value of 0.84 and negative predictive value of 0.80. The sensitivity, specificity, positive, and negative predictive values of inhibin B for the same outcome were, respectively, 0.62, 0.60, 0.67, and 0.55. The median motile sperm recovery rate was 83% when seminal plasma AMH concentration was ≥0.84ng/mL, vs. 27% when AMH concentration was <0.84ng/mL (p<0.05). In other patient groups, there was no correlation between the two hormone levels in seminal plasma and the motile sperm recovery rate. In conclusion, seminal plasma AMH and inhibin B concentrations correlate with and can be used to predict motile sperm recovery after semen cryopreservation in asthenozoospermic men.

Effect of Swim-Up and Percoll Treatment on Sperm Quality and In vitro Embryo Development in Yak

This study was designed to determine the effect of different sperm preparation treatments on yak sperm quality and in vitro embryo development. Frozen-thawed semen samples were treated using swim-up or percoll gradient centrifugation methods. Sperm concentration, progressive motility, recovery of motile sperm, membrane integrity, acrosome and chromatin integrity were scored and compared in recovered samples and controls. In addition, the effects of two sperm separation treatments on embryos capable of cleavage and in vitro development to the blastocyst stage were evaluated. Swim-up separated sperm showed a higher motility, while the concentration of spermatozoa recovered and percent recovery of motile sperm were higher with percoll gradient centrifugation separation. According to the optical and electron microscopies, swim-up produced higher percentage of sperm with intact plasma membrane and acrosome than percoll gradient centrifugation separation. However, there was no difference in the percentage of sperm with intact chromatin between two treatment groups. Cell numbers in the blastocysts of two groups were not different. The blastocyst rate was similar in both groups, whereas cleavage rate was higher when swim-up was used.

Semen cryopreservation for men banking for oligospermia, cancers, and other pathologies: prediction of post-thaw outcome using basal semen quality

To evaluate post-thawing sperm parameters in a large series of men cryopreserving for different cancers and oligospermia. Retrospective observational study. Semen cryopreservation laboratory. Six hundred twenty-three patients undergoing semen cryopreservation for cancer or oligospermia who discontinued banking. None. Postcryopreservation sperm motility and viability. In oligospermic men, recovery of motile sperm after cryopreservation was possible in only a few out of the 219 samples cryopreserved for this problem. Similarly, independent of the reason for which cryopreservation was required, if one basal semen parameter fell below the 5th percentile of the World Health Organization reference values, recovery of motile and viable spermatozoa after thawing was low. Among samples cryopreserved for cancer, those with testicular cancer showed the lowest basal semen quality and recovery after thawing. In cases of hematological cancers or other types of cancers, motility recovery was similar to that of non-cancer-related samples. Receiver operating characteristic analyses demonstrate that basal progressive and total motility predict the recovery rate of motile sperm after thawing with high accuracy, sensibility and specificity. Our study demonstrates the ability of prefreeze semen parameters to predict cryosurvival in terms of sensitivity and precision. Using this information, the clinician could perform appropriate counseling about the future possibilities of fertility for the patient.

Semen hyperviscosity treatment: n-acetil cysteine reduces viscosity and increases motile sperm recovery without detriment on sperm DNA integrity

Semen hyperviscosity treatment: n-acetil cysteine reduces viscosity and increases motile sperm recovery without detriment on sperm DNA integrity

Development of a Sperm Cryopreservation Protocol for Redside Dace: Implications for Genome Resource Banking

AbstractPopulations of Redside Dace Clinostomus elongatus have declined in many areas across the species’ North American range. Therefore, the development of sperm cryopreservation technology would provide an invaluable means of preserving genetic diversity in populations that are in imminent danger of extirpation. We developed cryopreservation protocols by testing the effects of diluent (buffered sperm motility‐inhibiting saline solution [BSMIS]; BSMIS + glycine; sucrose; and Hanks’ balanced salt solution [HBSS]), cryoprotectant (dimethyl sulfoxide [DMSO]; propylene glycol [PG]; N,N‐dimethylacetamide [DMA]; and methanol), freezing rate (1, 5, and 10°C/min), and male‐to‐male variation on sperm quality. Incubating sperm in extenders affected motility; BSMIS + glycine + methanol, BSMIS + glycine + PG, and HBSS + methanol were the only treatments for which motility was not significantly different from that of fresh sperm. Sperm frozen with sucrose had higher motility than sperm frozen with BSMIS + glycine, and sperm frozen with DMSO had higher motility than sperm frozen with methanol. Freezing rates were evaluated for BSMIS + glycine, HBSS, and sucrose; all diluents were frozen with DMSO. The effect of freezing rate was not significant for BSMIS + glycine or for HBSS, but an effect was detected for sucrose, with sperm frozen at 5°C/min or 10°C/min having higher motility than sperm frozen at 1°C/min. The effect of extender was not significant at 1°C/min or 5°C/min, but an effect was detected at 10°C/min such that sperm frozen with sucrose had the highest motility. Male‐to‐male variability was evaluated by using sucrose + DMSO and a freezing rate of 10°C/min. For these males, the sperm motility recovery index ranged from 6.67% to 79.27%, and the sperm velocity recovery index ranged from 21.37% to 57.33%. Our findings demonstrate that cryopreservation of Redside Dace sperm in a sucrose + DMSO extender at a freezing rate of 10°C/min is adequate for preserving genetic diversity via sperm banks.

2045 SUSPENDED ANIMATION OF HUMAN SPERM: ULTRA-RAPID VITRIFICATION USING A NOVEL MICROCAPILLARY SYSTEM

You have accessJournal of UrologyInfertility: Basic Research, Physiology, Pathophysiology1 Apr 20132045 SUSPENDED ANIMATION OF HUMAN SPERM: ULTRA-RAPID VITRIFICATION USING A NOVEL MICROCAPILLARY SYSTEM Yahir Santiago-Lastra, Joseph W. McQuaid, Diane Wright, Mehmet Toner, Cigdem Tanrikut, and Thomas L. Toth Yahir Santiago-LastraYahir Santiago-Lastra Boston, MA More articles by this author , Joseph W. McQuaidJoseph W. McQuaid Boston, MA More articles by this author , Diane WrightDiane Wright Boston, MA More articles by this author , Mehmet TonerMehmet Toner Boston, MA More articles by this author , Cigdem TanrikutCigdem Tanrikut Boston, MA More articles by this author , and Thomas L. TothThomas L. Toth Boston, MA More articles by this author View All Author Informationhttps://doi.org/10.1016/j.juro.2013.02.2464AboutPDF ToolsAdd to favoritesDownload CitationsTrack CitationsPermissionsReprints ShareFacebookTwitterLinked InEmail INTRODUCTION AND OBJECTIVES Freezing very low concentrations of sperm remains a challenge. Conventional slow-freeze methods require dilution with relatively large volumes and high concentrations of traditional cryoprotectants with potentially lethal osmotic and cytotoxic effects. Development of ultra-rapid vitrification techniques allows for freezing of very low volumes using low concentrations of nontoxic cryoprotectants, such as sugars, in addition to very low doses of permeable cryoprotectant. Our investigation was undertaken to determine the feasibility of ultra-rapid freezing via vitrification of human sperm samples with 0.5M trehalose and 3.0M propanediol (PROH) using a novel microcapillary system. METHODS 84 fresh ejaculated samples were obtained from healthy men and prepared using the swim-up method in human tubal fluid (HTF) media supplemented with 5% human serum albumin (HSA). All samples had at least 20 million spermatozoa/ml and at least 50% motility prior to swim-up preparation. These samples were then diluted to varying concentrations and divided into equal volumes for vitrification into the following groups: (1) 0.25M trehalose (a natural sugar) to act as an impermeable cryoprotectant, (2) vitrification with 0.25M trehalose and propanediol (PROH), (3) vitrification without cryoprotectant, or (4) no freezing/vitrification (control). Each sample was loaded into a 100μ microcapillary and the ends were heat-sealed to create a closed system. The samples undergoing vitrification were plunged into a liquid nitrogen bath for 20 seconds and then warmed in a 37°C solution of phosphate-buffered saline. Each sample was expelled from the capillaries and motility was qualitatively assessed via microscopic visualization. All samples were further assessed by computer-assisted semen analysis. RESULTS Preliminary results demonstrate consistent and statistically significant (p<0.05) recovery of motile sperm in samples that were vitrified in 100μ microcapillaries with HTF media plus 5% HSA supplemented by 0.25M trehalose and PROH. CONCLUSIONS Ultra-rapid vitrification of human sperm samples in microcapillaries is feasible using low volumes of a nontoxic sugar, such as trehalose, as an alternative to traditional toxic cryoprotectants. This is a particularly relevant issue with men who produce very little sperm in the ejaculate and in men who undergo any variety of surgical sperm extraction efforts for either obstructive or nonobstructive azoospermia. © 2013 by American Urological Association Education and Research, Inc.FiguresReferencesRelatedDetails Volume 189Issue 4SApril 2013Page: e839 Advertisement Copyright & Permissions© 2013 by American Urological Association Education and Research, Inc.MetricsAuthor Information Yahir Santiago-Lastra Boston, MA More articles by this author Joseph W. McQuaid Boston, MA More articles by this author Diane Wright Boston, MA More articles by this author Mehmet Toner Boston, MA More articles by this author Cigdem Tanrikut Boston, MA More articles by this author Thomas L. Toth Boston, MA More articles by this author Expand All Advertisement Advertisement PDF DownloadLoading ...

Efficacy of four density gradient separation media to remove erythrocytes and nonviable sperm from canine semen

The objective was to compare four commercially available density gradient centrifugation (DGC) media (ISolate [Irvine Scientific; Santa Ana, CA, USA], Percoll [Pharmacia; Uppsala, Sweden], PureCeption [SAGE In-Vitro Fertilization, Inc.; Trumbull, CT, USA], PureSperm 100 [Nidacon International AB; Molndal, Sweden]) for their ability to separate viable, motile sperm from contaminant nonviable (immotile and/or dead) sperm and red blood cells (RBC). Pooled sperm-rich fractions from four healthy dogs were assessed using Spermvison SAR (Minitube of America). For this, 1 mL of the blood/sperm admixture was pipetted over 4 mL of DGC media: 50%/90% ISolate (Irvine Scientific), 45%/90% Percoll (Pharmacia), 40%/80% PureCeption (SAGE In-Vitro Fertilization, Inc.), and 40%/80% PureSperm 100 (Nidacon International AB). After centrifugation, five 1-mL fractions (A, B, C, D, and E) and the sperm pellet (bottom fraction F) were separated. Sperm morphology and red blood cell/sperm ratio (RBC/S) per fraction were determined on stained slides. All DGC media separated RBC from sperm; the highest red blood cell/sperm ratio was present in ISolate (Irvine Scientific) and Percoll (Pharmacia) fraction A (29.4 ± 29.7 and 28.2 ± 20.8, respectively), and in fractions A and B of both PureCeption (SAGE In-Vitro Fertilization, Inc.) (37.0 ± 22.8 and 39.6 ± 24.3, respectively) and PureSperm 100 (Nidacon International AB) (25.2 ± 5.9 and 23.0 ± 3.9, respectively). The fractions with the highest total sperm recovery, motile sperm recovery, as well as overall motility were ISolate (Irvine Scientific) and Percoll (Pharmacia) fraction D (33.9 ± 29.4%; 40.99 ± 27.9%; 71.2 ± 21.8% and 36.4 ± 14.5%; 39.3 ± 15.8%; 88.6 ± 2.3%, respectively), and for PureCeption (SAGE In-Vitro Fertilization, Inc.) and PureSperm 100 (Nidacon International AB), the sperm pellet, fraction F (78.8 ± 28.3%; 88.0 ± 17.4%; 70.2 ± 11.1% and 73.1 ± 21.0%; 75.4 ± 24.6%; 80.6 ± 17.1%, respectively). In the pellet for PureCeption (SAGE In-Vitro Fertilization, Inc.), more sperm and motile sperm were recovered than in ISolate (Irvine Scientific) and Percoll (Pharmacia) fractions D (P < 0.0163). Therefore, DGC media should be considered for canine semen purification when contaminated with blood or when separation of motile versus immotile sperm is needed.

Cryopreservation of Atlantic cod (Gadus morhua) sperm in large-volume straws: applications for commercial production and gene banking

In our study, we used a full factorial analysis of variance design to examine the effects of diluent [Mounib's sucrose-based diluent+hen's egg yolk (EY) and Hanks' balanced salt solution (HBSS)+EY], freezing rate (−2.5, −5.0 and −7.5 °C min−1) and thawing rate (2.5, 5.0 and 7.5 °C min−1) on motility and velocity of Atlantic cod sperm cryopreserved in 2.5 mL cryogenic straws. We found that post-thaw sperm performance was strongly influenced by the presence of higher-order interactions of the factors we tested. For all models broken down by diluent, the 2.5 °C min−1 thawing rate had the lowest sperm motility recovery index. Mounib's sucrose-based diluent+EY had the highest motility recovery index at all thawing rates. Mean per cent motility for fresh sperm (87.7±2.9%) was not significantly different than of sperm cryopreserved using Mounib's sucrose-based diluent+EY, frozen at −2.5 °C min−1 and thawed at 5.0 °C min−1 (77.1±2.9%). For Mounib's sucrose-based diluent+EY, velocity was significantly higher with sperm thawed at 7.5 °C min−1, than sperm thawed at 2.5 °C min−1, while thawing rate had no effect for HBSS+EY. Our findings have implications for cod mariculture and aiding in conservation efforts for a dominant marine fish species.