TES-Tris Research Articles

Efficacious long-term cooling and freezing of Sapajus apella semen in ACP-118®

The objectives of the present study were to test the effect of coconut water solution (CWS), TES–TRIS and ACP-118® on the seminal cooling and cryopreservation of semen from capuchin monkeys (Sapajus apella). Semen was collected from six males by electro-ejaculation, diluted in TES–TRIS, CWS or ACP-118®, and maintained at 4°C for 28h. Semen was subsequently evaluated (Experiment I) or cryopreserved in the presence of different glycerol concentrations (3%, 5% or 7%) (Experiment II). ACP-118® was the preferred extender to preserve sperm motility and viability after 28h incubation at 4°C. Cooled sperm were successfully frozen–thawed in a medium containing 3% glycerol. After thawing, sperm retained the capacity to fertilize oocytes and zygotes were obtained. In conclusion, ACP-118® can be effectively and efficiently used as extender for the cooling of S. apella semen. Furthermore, cryopreservation using ACP-118® by adding 3% glycerol is suitable to maintain sperm morphology and the capacity of these cells to fertilize in vitro.

The effects of cooling rates and type of freezing extenders on cryosurvival of rat sperm

Cryopreservation of rat sperm is very challenging due to its sensitivity to various stress factors. The objective of this study was to determine the optimal cooling rate and extender for epididymal sperm of outbred Sprague Dawley (SD) and inbred Fischer 344 (F344) rat strains. The epididymal sperm from 10 to 12weeks old sexually mature SD and F344 strains were suspended in five different freezing extenders, namely HEPES buffered Tyrode’s lactate (TL-HEPES), modified Kreb’s Ringer bicarbonate (mKRB), 3% dehydrated skim milk (SM), Salamon’s Tris-citrate (TRIS), and tes/tris (TES). All extenders contained 20% egg yolk, 0.75% Equex Paste and 0.1M raffinose or 0.1M sucrose. The sperm samples in each extender were cooled to 4°C and held for 45min for equilibration before freezing. The equilibrated sperm samples in each extender were placed onto a shallow quartz dish inserted into Linkam Cryostage (BCS 196). The samples were then cooled to a final temperature of −150°C by using various cooling rates (10, 40, 70, and 100°C/min). For thawing, the quartz dish containing the sperm samples were rapidly removed from the Linkam cryo-stage and placed on a 37°C slide warmer and held for 1min before motility analysis. Sperm membrane and acrosomal integrity and mitochondrial membrane potential (MMP) were assessed by SYBR-14/Propidium iodide, Alexa Fluor-488-PNA conjugate and JC-1, respectively. The total motility, acrosomal integrity, membrane integrity and MMP values were compared among cooling rates and extenders. Both cooling rate and type of extender had significant effect on cryosurvival (P<0.05). Sperm motility increased as cooling rate was increased for both strains (P<0.05). Highest cryosurvival was achieved when 100°C/min cooling rate was used in combination with TES extender containing 20% egg yolk, 0.75% Equex paste and either 0.1M sucrose or raffinose (P<0.05). This study showed that TES extender containing 0.1M raffinose or sucrose with 70°C/min and 100°C/min cooling rate improved post-thaw motility of rat sperm.

73 EFFECT OF THE BUFFER SYSTEM, CRYOPROTECTANT, AND ANTIOXIDANT ON SPERM FROM ENDANGERED CATALONIAN SHEEP XISQUETA AND ARANESA AND GOAT BLANCA DE RASQUERA BREEDS DURING PRESERVATION

In an attempt to increase the possibilities of a better sperm cryopreservation from these endangered small ruminant catalonian breeds, we are studying different strategies. One of them is to study the effect of the buffer system Tes-Tris (TEST) compared with the Tris and citric acid (TRIS) buffer system, testing simultaneously both systems in a 1% (w/v) soybean lecithin or in a 15% (v/v) powdered egg yolk–based media supplemented both with 5% glycerol and to assess also the inclusion of 5 mM of butylated hydroxytoluene (BHT) as an antioxidant on the cryopreservation media. However, the main objective of the present study was to test first the effect of the different media when the sperm were cultured 4 h at 5°C. Briefly, fresh ejaculates from 6 young bucks of Blanca de Rasquera breed (1 year old) were collected by an artificial vagina in favourable reproductive period and immediately mixed in equal quantities. Pooled ejaculates were split into 2 equal aliquots and washed by centrifugation (twice for 10 min at 600g) in TRIS- or TEST-based media without cryoprotectants and antioxidant. Afterward each pellet was split into 4 equal aliquots, re-suspended in TRIS or TEST, depending on the experimental group, soybean lecithin, or powdered egg yolk-based media, and supplemented or not with BHT and kept for 4 h at 5°C. Likewise, fresh ejaculates from 8 young rams (4 rams of Xisqueta and 4 rams of Aranesa breed, 1 year old) were collected and processed as buck semen samples. Sperm survival before cryopreservation was determined by eosine-nigrosine stain, and sperm motion parameters were analysed by a computer-assisted sperm analysis system (ISAS®). Six replications were performed in both species, and General Lineal Model (SAS®, Cary, NC, USA) was used for the statistical analysis. The highest sperm viability percentage (mean ± SE) on goat sperm cultured 4 h at 5°C was observed in the extender with TRIS buffer system in powdered egg yolk-based media supplemented with BHT (81.1 ± 2.8), not showing significant differences with the others extenders, except with the viability of the samples in the extenders with TEST buffer system in soybean lecithin-based media supplemented (56.5 ± 2.9; P &lt; 0.001) or not with BHT (60.1 ± 5.1; P &lt; 0.001). On the other hand, no significant differences on sperm viability were observed on ram sperm between treatments. Nevertheless, the sperm quality motion characteristics (data not shown) were quite different between all the treatments in both species. Considering that the present results are still preliminary, we suggest that more analysis should be made to explain how the different composition of the extenders affect sperm quality during the cryopreservation process. Supported by INIA (RZ2009-00008-00-00), Generalitat de Catalunya (2009SGR0621 and CUR-DIUE), and FSE and Fundacion Carolina.

Semen coagulum liquefaction, sperm activation and cryopreservation of capuchin monkey ( Cebus apella) semen in coconut water solution (CWS) and TES–TRIS

The objectives of the present study were to test the effect of coconut water solution and TES–TRIS on the seminal coagulum liquefaction, sperm activation in fresh diluted semen, and on the cryopreservation of semen from capuchin monkeys ( Cebus apella). Semen was collected from six males by electro-ejaculation, diluted in TES–TRIS or coconut water solution (CWS), and incubated at 35 °C until the coagulated fraction of the semen was completely liquefied. In the experiment I, after liquefaction, samples were diluted in TES–TRIS or CWS, plus 6 and 10 mM/mL of caffeine. Sperm motility and vigor were evaluated during 5 h. For experiment II, after liquefaction, semen samples were extended in TES–TRIS (3.5% glycerol in the final solution) or CWS (2.5% glycerol in the final solution), cryopreserved and stored in liquid nitrogen for 1 week. The seminal coagulum was liquefied in (mean ± SDM) 4.5 ± 1.7 and 2.8 ± 1.1 h in TES–TRIS and CWS, respectively. Sperm were motile in TES–TRIS and CWS for 5.0 ± 1.4 and 1.0 ± 0.5 h, respectively. The mean motility in this period was 38 ± 22% (TES–TRIS) and 22.0 ± 16.0 (CWS). Motility increased after caffeine addition only in samples diluted in CWS containing 6 mM (22.5 ± 16.0) or 10 mM (28.0 ± 19.0) caffeine. Post-thaw live sperm percentage was 26.2% in TES–TRIS and 13.2% in CWS. For cryopreservation of semen from C. apella TES–TRIS (3.5% glycerol) was more appropriate than CWS (2.5% glycerol). CWS + caffeine potentially increase sperm motility and may be useful in artificial insemination of fresh diluted semen.

A pilot study on post-thawing quality of Iberian red deer spermatozoa (epididymal and electroejaculated) depending on glycerol concentration and extender osmolality

The optimization of cryopreservation extenders is a fundamental issue for adequately performing germplasm banking on wild species. We have tested two glycerol concentrations (4 and 8%), and three extender osmolalities (320, 380 and 430 mOsm/kg; before adding cryoprotectants), for cryopreservation of epididymal and ejaculated sperm samples from Iberian red deer. All the extenders were based on Tes–Tris and fructose (for osmolality adjustment), and complemented with 20% egg yolk. Epididymal and ejaculated sperm samples were obtained from the cauda epididymis (post-mortem) and using electroejaculation, respectively. Samples were diluted 1:1 with each extender and equilibrated for 2 h at 5 °C. Then, they were diluted down to 100 × 10 6 sperm/mL and frozen at −20 °C/min. Post-thawed samples were assessed for motility (CASA), HOS test, proportion of swollen (osmotically challenged) cells in the untreated sample, viability and acrosomal status. For epididymal samples, 8% glycerol rendered a slightly higher proportion of intact acrosomes on viable spermatozoa than 4%; regarding extender osmolality, 380 and 430 mOsm/kg rendered higher motility results, and the 430 mOsm/kg yielded the lowest proportion of swollen spermatozoa. For ejaculated samples, 4% glycerol yielded more viable spermatozoa than 8%; for extender osmolality, 320 mOsm/kg rendered the highest percentages of progressively motile and viable spermatozoa, although 380 mOsm/kg extender was not significantly different. These results show that sample source influences extender suitability, and that extenders should be isoosmotic or rather slightly hyperosmotic. Future studies should test multiple glycerol concentrations and extender osmolalities in order to adjust them to these kinds of sample.

Leucocytes in semen from spinal injury patients

The objectives of the present study were to test the effect of coconut water solution (CWS), TES–TRIS and ACP-118® on the seminal cooling and cryopreservation of semen from capuchin monkeys (Sapajus apella). Semen was collected from six males by electro-ejaculation, diluted in TES–TRIS, CWS or ACP-118®, and maintained at 4 °C for 28 h. Semen was subsequently evaluated (Experiment I) or cryopreserved in the presence of different glycerol concentrations (3%, 5% or 7%) (Experiment II). ACP-118® was the preferred extender to preserve sperm motility and viability after 28 h incubation at 4 °C. Cooled sperm were successfully frozen–thawed in a medium containing 3% glycerol. After thawing, sperm retained the capacity to fertilize oocytes and zygotes were obtained. In conclusion, ACP-118® can be effectively and efficiently used as extender for the cooling of S. apella semen. Furthermore, cryopreservation using ACP-118® by adding 3% glycerol is suitable to maintain sperm morphology and the capacity of these cells to fertilize in vitro.

* - Prediction of in vitro fertilization outcome by sperm penetration assay with TEST-yolk buffer preincubation

To evaluate sperm penetration assay (SPA) value as a screening tool before in vitro fertilization (IVF). Follow-up study comparing sperm variables and IVF outcome. Infertile couples in an academic research environment. Two hundred forty-one infertile couples scheduled for IVF. Sperm penetrating assay with cold Tes-TRIS (TEST) Yolk buffer semen preincubation and IVF. Percent of egg penetration recoded into poor and good category (0% to 20%, 21% to 100%) and compared with fertilization, embryo transfer, and pregnancy rate (PR) in IVF, as well as sperm count motility and morphology. Sperm penetrating assay predicted fertilization with a high negative (74%) and positive (82%) predictive rate and good specificity value (0.96). One of 31 patients in the poor SPA category (3%) fertilized less than 50% of eggs; no pregnancy occurred. In the good SPA category, 87 of 210 patients (41%) fertilized greater than or equal to 50% of eggs with 34.3% PR. Sperm penetrating assay correlated better with fertilization rate than did sperm count and motility but not morphology. Sperm penetrating assay is a useful screening assay before IVF together with sperm morphology.

Acrosome reaction of human spermatozoa in zona-free hamster egg penetration test

The acrosomal status of human sperm during preparation for the process of zona-free hamster egg penetration test (ZFHEPT) was determined. The incidence of acrosome reaction (AR), as assessed by triple-stain technique, was significantly increased after 24 hours of incubation at 4 degrees C in TES-Tris (TEST)-yolk buffer, but the absolute values were relatively low (20% or less). Sperm from fertile donors and infertile patients with normal or abnormal semen analysis displayed similar capacity to undergo the AR in vitro. Although a positive correlation was found between the incidence of AR and the score of ZFHEPT, a remarkable individual variation was noted. The incidence of AR in freely swimming human sperm does not accurately reflect the fertilizing ability of the sperm.

TEST-egg yolk buffer storage increases the capacity of human sperm to penetrate hamster eggs in vitro.

A total of 85 semen samples from infertility clinic patients were examined to study the effect of storage at 4 degrees C in TES-Tris (TEST)-egg yolk buffer for 24 h on the penetrating capacity of sperm in the zona-free hamster egg penetration assay (HEPA). The mean sperm penetration rate and the fertilization index increased significantly after storage in TEST-egg yolk buffer. Only five out of the 85 samples (5.9%) failed to show any improvement in sperm penetration rate after cold storage. The sperm penetration rate before cold storage showed no significant correlations with routine semen characteristics, semen ATP concentration or the functional integrity of sperm membranes as measured by the hypo-osmotic swelling technique. Significant but low correlations were observed between sperm penetration rate after cold storage and the following semen parameters: sperm count, % motility, total number of motile sperm, % normal sperm morphology, total number of normal sperm, semen ATP concentration and sperm penetration rate before cold storage. Partial correlation analysis revealed that the positive correlation between semen ATP concentration and sperm penetration rate after cold storage was not a direct relationship but was due to the correlation with sperm count. The combination of sperm penetration rate before cold storage, sperm count and % normal sperm morphology accounted for 26.2% of the variation in sperm penetration rate after cold storage by stepwise multiple regression analysis, while sperm penetration rate before cold storage alone explained 13.5% of the variation. The results indicate that TEST-egg yolk buffer treatment can enhance sperm penetration rate in vitro and may be useful in the treatment of impaired sperm fertility.(ABSTRACT TRUNCATED AT 250 WORDS)

Human sperm chromosomes obtained from hamster eggs after sperm capacitation in TEST‐yolk buffer

AbstractHuman sperm chromosomes were obtained after capacitation with TES‐Tris (TEST) yolk buffer and fusion with Syrian hamster eggs. Semen samples could be stored at 4°C for 3 days and remain functional in the assay system. The efficiency of TEST yoik buffer for obtaining karyotypes was as good as, or greater than, the efficiency of standard BWW medium containing human serum albumin.

Conditions influencing human sperm capacitation and penetration of zona-free hamster ova

The ability of human spermatozoa to penetrate zona-free hamster ova was examined following low-temperature capacitation (4 degrees C) in TES-Tris (TEST)-yolk buffer for periods of up to 66 hours. Results obtained from 66 individuals demonstrated that the number of penetrations per ova were increased by an average of 2.5-fold when spermatozoa were capacitated for 42 hours, as compared with 18 hours. Furthermore sperm with extremely poor penetration rates after 18-hour capacitation was often improved by longer capacitation periods. Patients from our infertility clinic with less than 20 X 10(6) spermatozoa/ml of ejaculate had significantly lower penetration rates when compared with patients and donors with greater than 20 X 10(6) spermatozoa/ml (P less than or equal to 0.001). The observed effects of TEST-yolk buffer appear to be related to its ability to preserve sperm motility over prolonged periods, during which increased capacitation of the total sperm population is achieved.

Human sperm penetration of zona-free hamster eggs after storage of the semen for 48 hours at 2° C to 5° C

The motility of human spermatozoa and their ability to penetrate zona-free hamster eggs were examined after dilution of the semen with TES-Tris (TEST) yolk buffer and storage for 48 hours at 2 degrees C to 5 degrees C. Semen samples from 10 fertile donors and 19 infertility patients were studied. More than 65% of the spermatozoa which were initially motile in the TEST yolk buffer remained active after storage. During storage, the mean swimming speed of the sperm declined to approximately 60% of the prestorage value. The percentage of zona-free hamster eggs that were penetrated by spermatozoa from patients and donors increased significantly following 48 hours of storage at 2 degrees C to 5 degrees C. Normal semen and abnormal semen were equally preserved by this storage method. This procedure may be used to ship semen samples by commercial transportation to specialized laboratories for testing. Low temperature storage in the TEST yolk buffer appears to enhance the fertilizing capacity of human spermatozoa in vitro.