Efecto de la borra de café sobre la biológica espermática: aproximación in vitro / Effect of spent coffee grounds on sperm biology: in vitro approach

OBJETIVO: El objetivo de este trabajo fue establecer el efecto de la borra de café sobre la movilidad y los parámetros funcionales de los espermatozoides humanos in vitro. MATERIALES Y MÉTODOS: La borra de café, un subproducto obtenido en establecimientos especializados en la preparación de café soluble a base de grano, se diluyo en tampón fosfato salino y se mezcló en proporciones iguales con las muestras de semen de 16 voluntarios aparentemente sanos. A cada muestra se le determinó el efecto sobre la movilidad espermática en función del tiempo (30, 60, 90 y 120 minutos, n=16) y sobre los parámetros funcionales (n=6) por medio de citometría de flujo: potencial de membrana mitocondrial, producción de especies reactivas de oxígeno y lipoperoxidación de la membrana espermática. RESULTADOS: La incubación de los espermatozoides con la borra de café evidencio un cambio positivo en la movilidad espermática. Adicionalmente, la incubación con la borra de café incremento significativamente el potencial de membrana mitocondrial en los espermatozoides. CONCLUSIÓN: La borra de café, seguramente debido a los compuestos antioxidantes, afecta positivamente la movilidad espermática aumentando el potencial de membrana mitocondrial. Por lo tanto, esto es un paso inicial en la búsqueda de un suplemento de origen natural que aumente la calidad seminal.

OBJECTIVE: The objective of this work is to establish the effect of spent coffee grounds on the motility and functional parameters of human spermatozoa, in vitro. MATERIALS AND METHODS: Spent coffee grounds, a by-product obtained in specialized establishments in the preparation of soluble coffee based on grain, was diluted in saline phosphate buffer and mixed in equal proportions with semen samples from 16 apparently healthy volunteers. Each sample was determined the effect on sperm motility as a function of time (30, 60, 90 and 120 minutes, n=16) and on functional parameters (n=6) by means of flow cytometry: mitochondrial membrane potential, reactive oxygen species production and membrane lipoperoxidation. RESULTS: The incubation of the spermatozoa with the spent coffee grounds showed a positive change in sperm motility. Additionally, incubation with spent coffee grounds significantly increased the mitochondrial membrane potential in human sperm cells. CONCLUSION: Spent coffee grounds, probably due to antioxidant compounds, positively affects sperm motility by increasing mitochondrial membrane potential. Therefore, this is an initial step in the search for a supplement of natural origin that increases seminal quality.

Participation of the inositol 1,4,5-trisphosphate-gated calcium channel in the zona pellucida- and progesterone-induced acrosome reaction and calcium influx in human spermatozoa / 亚洲男科学杂志(英文版)

The acrosome reaction is a prerequisite for fertilization, and its signaling pathway has been investigated for decades. Regardless of the type of inducers present, the acrosome reaction is ultimately mediated by the elevation of cytosolic calcium. Inositol 1,4,5-trisphosphate-gated calcium channels are important components of the acrosome reaction signaling pathway and have been confirmed by several researchers. In this study, we used a novel permeabilization tool BioPORTER® and first demonstrated its effectiveness in spermatozoa. The inositol 1,4,5-trisphosphate type-1 receptor antibody was introduced into spermatozoa by BioPORTER® and significantly reduced the calcium influx and acrosome reaction induced by progesterone, solubilized zona pellucida, and the calcium ionophore A23187. This finding indicates that the inositol 1,4,5-trisphosphate type-1 receptor antibody is a valid inositol 1,4,5-trisphosphate receptor inhibitor and provides evidence of inositol 1,4,5-trisphosphate-gated calcium channel involvement in the acrosome reaction in human spermatozoa. Moreover, we demonstrated that the transfer of 1,4,5-trisphosphate into spermatozoa induced acrosome reactions, which provides more reliable evidence for this process. In addition, by treating the spermatozoa with inositol 1,4,5-trisphosphate/BioPORTER® in the presence or absence of calcium in the culture medium, we showed that the opening of inositol 1,4,5-trisphosphate-gated calcium channels led to extracellular calcium influx. This particular extracellular calcium influx may be the major process of the final step of the acrosome reaction signaling pathway.

Fluorescent probes for the detection of reactive oxygen species in human spermatozoa / 亚洲男科学杂志(英文版)

Reactive oxygen species (ROS) production is a by-product of mitochondrial activity and is necessary for the acquisition of the capacitated state, a requirement for functional spermatozoa. However, an increase in oxidative stress, due to an abnormal production of ROS, has been shown to be related to loss of sperm function, highlighting the importance of an accurate detection of sperm ROS, given the specific nature of this cell. In this work, we tested a variety of commercially available fluorescent probes to detect ROS and reactive nitrogen species (RNS) in human sperm, to define their specificity. Using both flow cytometry (FC) and fluorescence microscopy (FM), we confirmed that MitoSOX™ Red and dihydroethidium (DHE) detect superoxide anion (as determined using antimycin A as a positive control), while DAF-2A detects reactive nitrogen species (namely, nitric oxide). For the first time, we also report that RedoxSensor™ Red CC-1, CellROX®Orange Reagent, and MitoPY1 seem to be mostly sensitive to hydrogen peroxide, but not superoxide. Furthermore, mean fluorescence intensity (and not percentage of labeled cells) is the main parameter that can be reproducibly monitored using this type of methodology.

Participation of the inositol 1,4,5-trisphosphate-gated calcium channel in the zona pellucida- and progesterone-induced acrosome reaction and calcium influx in human spermatozoa / 亚洲男科学杂志(英文版)

The acrosome reaction is a prerequisite for fertilization, and its signaling pathway has been investigated for decades. Regardless of the type of inducers present, the acrosome reaction is ultimately mediated by the elevation of cytosolic calcium. Inositol 1,4,5-trisphosphate-gated calcium channels are important components of the acrosome reaction signaling pathway and have been confirmed by several researchers. In this study, we used a novel permeabilization tool BioPORTER® and first demonstrated its effectiveness in spermatozoa. The inositol 1,4,5-trisphosphate type-1 receptor antibody was introduced into spermatozoa by BioPORTER® and significantly reduced the calcium influx and acrosome reaction induced by progesterone, solubilized zona pellucida, and the calcium ionophore A23187. This finding indicates that the inositol 1,4,5-trisphosphate type-1 receptor antibody is a valid inositol 1,4,5-trisphosphate receptor inhibitor and provides evidence of inositol 1,4,5-trisphosphate-gated calcium channel involvement in the acrosome reaction in human spermatozoa. Moreover, we demonstrated that the transfer of 1,4,5-trisphosphate into spermatozoa induced acrosome reactions, which provides more reliable evidence for this process. In addition, by treating the spermatozoa with inositol 1,4,5-trisphosphate/BioPORTER® in the presence or absence of calcium in the culture medium, we showed that the opening of inositol 1,4,5-trisphosphate-gated calcium channels led to extracellular calcium influx. This particular extracellular calcium influx may be the major process of the final step of the acrosome reaction signaling pathway.

Polymorphism of the testis-specific serine/threonine kinase 2 gene and risk of asthenozoospermia in Côte d'Ivoire

Background: The testis-specific serine/threonine protein kinase (TSSK2) is an indispensable protein responsible for the mobility of spermatozoa expressed specifically in the germ cells during spermatogenesis and present in the mature spermatozoa. Its gene mutation could constitute a risk of infertility. The aim of this study is to investigate the polymorphism of this TSSK2 gene in men with asthenozoospermia.Methods: The ejaculates were obtained from patients attending the reproductive biology unit of Institut Pasteur of Côte d’Ivoire for their spermiological evaluations. The semen analyses are performed with the automatic sperm analyzer SQA-Vision. 30 sperms, including 20 asthenozoosperms and 10 normosperms, were selected from their spermiological results and the spermatozoa DNA was extracted by the phenol/chloroform method. Direct Sequencing of the spermatozoa DNA fragments was done using the Sanger method. The frequencies of mutation were analysis with the Fisher and Mann-Whitney tests.   Results: It was revealed 17 mutations in 22 ejaculates. The frequent mutations are c.839C>T (T280M), c.816G>C (L372L), c.1026G>A (R342R), c.785A>C (H262P) and c.80A>G (K27R) with respectively frequencies of 50.0%, 26.67%, 16.67%, 13.33% and 10.0%. The analysis of these mutations indicated a significant difference in the frequency of occurrence of mutations between normosperms and asthenozoosperms (p-value = 0.01).Conclusions: This study shows that mutations in the TSSK2 gene are more common in asthenozoosperm ejaculates than normosperm ejaculates. This fact suggests the probable association of mutations in the TSSK2 gene with asthenozoospermia.

Acetyl-L-carnitine: An Effective Antioxidant against Cryo-damage on Human Spermatozoa with Asthenospermia / 华中科技大学学报（医学）（英德文版）

A variety of natural and artificial cryoprotectant extenders have been explored to enhance sperm recovery following cryopreservation-thawing process.The current investigation is aimed at evaluating the effect of acetyl-L-carnitine on human spermatozoa and reactive species oxygen (ROS) level after freezing-thawing process.The spermatozoa were collected from 35 male patients diagnosed as having asthenospermia.The cryopreservation of human spermatozoa treated with acetyl-L-camitine at different concentrations (group B:2.5 mmol/L,group C:7.5 mmol/L,group D:15 mmol/L) was compared with control (group A:no acetyl-L-carnitine given).For the frozen-thawed spermatozoa,the viability,motility and DNA integrity were measured by comet assay,acrosome integrity by FITC-PNA staining and ROS level was determined in each group.The results showed that there were no significant differences in motility and viability between group A and group B,while the motility and viability of spermatozoa in group C and group D were significantly increased as compared with those in group A.As compared with group A,the values for DNA integrity parameters including comet rate (CR),tail DNA percentage (TD),tail length (TL) and Oliver tail moment (OTM) were significantly reduced in group C and group D.Group C and group D also displayed a higher proportion of intact acrosome than group A.No significant difference in ROS level was found between group A and group B,while with the increase in acetyl-L-camitine concentration,the ROS level in groups C and D was significantly reduced as compared with that in group A.In conclusion,acetyl-L-camitine at a concentration of 7.5 mmol/L is an effective antioxidant against cryo-damage on post-thawed human spermatozoa.

Efecto in vitro del pentóxido de vanadio (V2O5) sobre la calidad espermática / In vitro effect of vanadium pentoxide (V2O5) on sperm quality

El vanadio es un metal pesado considerado como un contaminante ambiental, producto de la manipulación antropogénica, que incide sobre el potencial reproductivo masculino, especialmente sobre la movilidad espermática. Esta investigación tuvo como objetivo determinar el efecto in vitro del pentóxido de vanadio sobre la calidad espermática. Se incubaron suspensiones de espermatozoides humanos por 24 horas bajo concentraciones de 0, 1 y 2 ppm V2O5, se realizó la valoración de la movilidad y vitalidad espermática, la integridad de membrana, la reacción acrosómica y la integridad de la cromatina espermática. El V2O5 alteró de manera significativa la movilidad espermática, específicamente los patrones de movilidad espermática moderado (p < 0.01), lento (p = 0.01) e inmóvil (p < 0.01). Los grupos tratados con V2O5 presentaron un porcentaje de espermatozoides vivos significativamente mayor al grupo control (p < 0.01). La integridad de membrana y reacción acrosómica no resultaron afectadas por la exposición de espermatozoides humanos a V2O5 (p > 0.05). De igual modo, no se observaron alteraciones del material nuclear (p > 0.05). El vanadio es capaz de alterar la movilidad y vitalidad espermática sin inducir cambios sobre la integridad de membrana y la cromatina espermática.

Vanadium is a heavy metal considered an environmental pollutant product of anthropogenic manipulation; it influences the masculine reproductive potential, especially the sperm motility. This research had the objective of determining the effect of vanadium pentoxide on sperm quality in vitro. Spermatozoa were incubated for 24 hours under 0, 1 and 2 ppm V2O5 concentrations, and sperm motility and vitality, membrane integrity, acrosome reaction and sperm chromatin integrity were assessed. V2O5 altered sperm motility in a significant way, specifically moderated (p < 0.01), slow (p = 0.01), and non motile (p < 0.01) sperm motility patterns. The groups treated with V2O5 had a major percentage of live spermatozoa than the control group (p < 0.01). Membrane integrity and acrosome reaction were not affected by human sperm exposition to V2O5 (p > 0.05). Similarly, we did not observe nuclear material alterations. Vanadium is able to alter sperm motility and viability without inducing changes on membrane and chromatin integrity.

Laboratory processing and intracytoplasmic sperm injection using epididymal and testicular spermatozoa: what can be done to improve outcomes?

There are two main reasons why sperm may be absent from semen. Obstructive azoospermia is the result of a blockage in the male reproductive tract; in this case, sperm are produced in the testicle but are trapped in the epididymis. Non-obstructive azoospermia is the result of severely impaired or non-existent sperm production. There are three different sperm-harvesting procedures that obstructive azoospermic males can undergo, namely MESA (microsurgical epididymal sperm aspiration), PESA (percutaneous epididymal sperm aspiration), and TESA (testicular sperm aspiration). These three procedures are performed by fine-gauge needle aspiration of epididymal fluid that is examined by an embryologist. Additionally, one technique, called TESE (testicular sperm extraction), is offered for males with non-obstructive azoospermia. In this procedure, a urologist extracts a piece of tissue from the testis. Then, an embryologist minces the tissue and uses a microscope to locate sperm. Finding sperm in the testicular tissue can be a laborious 2- to 3-hour process depending on the degree of sperm production and the etiology of testicular failure. Sperm are freed from within the seminiferous tubules and then dissected from the surrounding testicular tissue. It is specifically these situations that require advanced reproductive techniques, such as ICSI, to establish a pregnancy. This review describes eight different lab processing techniques that an embryologist can use to harvest sperm. Additionally, sperm cryopreservation, which allows patients to undergo multiple ICSI cycles without the need for additional surgeries, will also be discussed.

Congelación ultra rápida de espermatozoides humanos: efecto sobre la función espermática y producción de especies reactiva de oxígeno / Ultra rapid freezing in human spermatozoa: effect on sperm function and reactive oxygen species production

El incremento del número de pacientes que desean mantener su fertilidad, ya sea por motivos oncológicos o de fertilidad, como son los pacientes con enfermedades infecciosas virales trasmitidas por vía sexual, o que se someten en forma voluntaria a la esterilización quirúrgica, requieren de métodos de congelación que preserven en forma adecuada la función de los espermatozoides. En el área de la criobiología, la utilización de técnicas de congelación ultrarrápida ha permitido preservar en forma exitosa ovocitos, embriones y tejido ovárico. Este método se ha incorporado recientemente para preservar el gameto masculino. El presente estudio evalúa el efecto de la congelación ultrarrápida (vitrificación) sobre la función espermática de 10 donantes normozoospérmicos. Los espermatozoides se seleccionaron por Swim-up y la solución espermática se dividió en dos subfracciones. Una fracción se vitrificó sumergiéndola directamente en nitrógeno líquido mientras que la segunda se utilizó como control. En ambas fracciones se determinaron viabilidad, movilidad, potencial de membrana mitocondrial (YMMit), integridad del ADN, reacción de acrosoma espontánea e inducida, y superóxido intracelular (O2.-). Se observó que la vitrificación preserva una adecuada función celular en un alto número de espermatozoides, siendo además un método simple, rápido y de menor costo, ya que no necesita equipo de congelación. No obstante, existe una significativa activación de la producción de especies reactivas de oxígeno, que conlleva a una prematura capacitación espermática, evento que es necesario de modular, especialmente si se utilizan estas células en técnicas de inseminación intrauterina. Futuros estudios con adición de antioxidantes a los medios de congelación parecen necesarios para optimizar esta técnica.

The number of patients who wish to maintain their fertility is ever increasing. This group of patients includes cancer patients, those with fertility problems or viral infectious diseases acquired through sexual contact and others submitting to voluntary surgical sterilization; all of the above requiring freezing methods to adequately preserve sperm function. In the field of cryobiology the use of ultra-rapid freezing techniques has successfully preserved oocytes, embryos and ovarian tissue. This method has recently been incorporated in preserving male gametes. This study evaluates the effect of ultra-rapid freezing (vitrification) on sperm function of 10 normozoospermic donors. The sperm were selected by swim-up technique and the solution divided into two fractions. One fraction is vitrified by dipping directly into liquid nitrogen and the second fraction is used as control. In both fractions, viability, motility, mitochondrial membrane potential (YMMit) DNA integrity, spontaneous and induced acrosome reaction and intracellular superoxide (O2.-) were determined. It was noted that vitrification preserves cell function in a great number of spermatozoon, and is also simple, rapid and cost effective as this method does not require freezing equipment. There is however, significant activation of the production of reactive oxygen species, which leads to premature sperm capacitation, an event necessary to modulate particularly when using these cells in intrauterine insemination techniques. Future studies with addition of antioxidants to freezing media are necessary to further improve this technique.

Molecular identification of Ca(2+)channels in human sperm

The acrosome reaction is a Ca(2+)-dependent exocytotic process that is a prerequisite step for fertilization. External calcium entry through voltage-activated Ca(2+)channels is known to be essential in inducing the acrosome reaction of mammalian spermatozoa. Due to their complex geometry, however, electrophysiological identification of sperm Ca(2+)channels has been limited. Here we identified Ca(2+)channel mRNAs expressed in motile human sperm using RT-PCR and their levels were compared using RNase protection assays. L-type, non- L-type, and T-type Ca(2+)channel mRNAs were detected by RT-PCR using degenerate primers. Cloning and sequencing of the PCR products revealed alpha1B, alpha1C, alpha1E, alpha1G, and alpha1H sequences. RT-PCR using specific primers repeatedly detected alpha1B, alpha1C, alpha1E, alpha1G, and alpha1H mRNAs, and additionally alpha1I mRNA. But alpha1A and alpha1D messages were not detected. Relative expression levels of the detected Ca(2+)channel subtypes were compared by RNase protection assays. The abundance of detected mRNA messages was in the following order: alpha1H> or =alpha1G> or =alpha1E> or =alpha1B>alpha1C>alpha1I. These findings indicated that human motile sperm express multiple voltage-activated Ca(2+)channel RNAs among which T-type and non-L-type channel messages are likely to be predominantly expressed. Based on their relative expression levels, we propose that not only T-type but also non-L-type calcium channels may be major gates for the external calcium influx, required for the acrosome reaction.

Cryopreservation of human spermatozoa: The effect of cryostorage time on the post – thaw sperm outcome.

A prospective study was reported comparing the effect of short-term (1 week) and long-term (6 months) cryostorage time on the post-thaw sperm outcome in four different freezing techniques. Eighty five semen samples were included in the study. Each sample was divided and subjected to four cryopreservative techniques: CEG-NCR, CEG-CR, TEST-EYG-NCR, and TEST-EYC-CR. The cryopreservation of each technique was performed in similar way by mixing the semen with the cryopreservative media (CEG or TEST-EYG) in ratio 1:1, then the mixture was aspirated into two straws. The temperature was then reduced (by NCR or CR freezing method) to -80 C, then the straws were transferred into liquid nitrogen. One straw of each technique was thawed after 1 week and the other straw after 6 months of cryostorage time for assessing the post-thaw sperm outcome. As the results, the post-thaw sperm motility and survival rate of the 1 week cryostorage group was slightly higher than the 6 months group in the CEG-NCR and the CER-CR freezing technique, but these did not have clinical significance because the differences were into small. There were no differences of post-thaw sperm outcome between 1 week and 6 months group in the TEST-EYG-NCR and TEST-EYG-CR freezing techniques. We concluded that the duration of cryostorage did not have clinical effect on the post-thaw sperm outcome.

Experimental Study on Reviability of Cryopreserved Human Spermatozoa / 대한비뇨기과학회지

In order to perform standardization in analytic methods of cryopreserved human semen, to investigate the differences of resistance to cryoinjury, to define the stage of critical cryoinjury during cryopreservation and to evaluate the quality change after thawing by time interval, the reviability of 30 normal and 30 abnormal semen were evaluated by the supravital stainings of spermatozoa using acridine orange and eosin yellow and the motility assay simultaneously according to the stages of freezing-thawing and the time interval after thawing. The results were summarized as follows: 1. Vitality was estimated higher than motility at all specimens and the gap between two became greater as motility decreased. 2. Reviability of abnormal semen was estimated lower than that of normal semen(p<0.05). 3. The critical cryoinjury to spermatozoa was noticed at the stage of freezing from 4 degrees C to -10 degrees C (p<0.05). 4. The significant decrease in quality of normal cryopreserved semen was noticed between 30 to 60 min. after thawing (p<0.05). These results suggest that the cryoinjury to human semen is different in nature, therefore it is advisable that the quality of cryopreserved human semen should be evaluated by vitality and motility assay simultaneously. And the resistance of abnormal semen to cryopreservation is so low that it would be difficult to be used clinically with satisfaction. Moreover the laboratory studies should be concentrated on freezing method to achieve better reviability and it is desirable in practice that post-thaw semen should be used within 30 min, after thawing.

ESTABLISHMENT OF HYBRIDOMA CELL LINES (G8,E10) SECRETING MONOCLONAL ANTIBODIES AGAINST HUMAN SPERMATOZOA / 中国法医学杂志

Two hybridoma cell lines (G8,E10) secreting monoclonal antibodies against human spermatozoa were established by fusing Sp2/0 mouse myeloma cells with splenocytes from Balb/c mice immunized with washed human spermatozoa. The results of the IIF, Elisa or peroxidase conjugated antibody im- munohistochemical staining demonstrated that both antibodies reacted with the spermatozoa in the human semen, testis, epididymus and vas deferens as well as the germ cells in the testis, cross reacted with the epithelial cells Of the thyroid gland, renal tubules, pancreatic ducts as well as the ilets of Langhans of the pancrease and not cross reacted with 33 normal human tissue cells, 9 different sources of body fluids and secretions as well as 8 different species of animal's spermatozoa. It is concluded that both monoclonal antibodies can be used for the species identification of semen stain in sex assault cases. The results of IIF demonstrated that each antibody bind to the localized region of the sperm cell surface. The antibody binding patterns were restricted to the acromosal cap, equatorial segment, postacromosal region, neck and the midpiece. The observed antibody binding patterns suggest that the human sperm surface is divided into a minimum of five domains. Both monoclonal antibodies were belong to IgG_1 subclass. The titre of Gs was 1∶512, while of E_(10) 1∶1024.

SPERMICIDAL STUDY ON MANDELIC ACID IN VITRO / 西安交通大学学报(医学版)

Mandelic acid(MA). the hydrolysate of a compound extracted from the leaves of prunus Persica B, possesses a strong spermicidal effect in vitro. In this experiment. the International Planned Parenthood Federation screening method for spermicides was used. Dilutions of MA, between 0.8mg/ml and 3.3mg/ml, totally immobilized human spermatozoa in less than 1 min. When the lowest spermicidal concentrotions of MA and boric acid against human spermatozoa were compared, it was found that MA was 40 times more potent than boric acid.

Study on the Fluorescent Body (F-body) of the Human Spermatozoa / 대한비뇨기과학회지

In this study seminal fluid samples were obtained from 20 normal adult males to know the percentage of the spermatozoa containing the fluorescent body (F-body), after staining the seminal fluid with 0.5% aqueous quinacrine HCI. In this study the preparations were observed with an AO fluorescent microscope. The following results were obtained. 1) In 20 men, the mean percentage of F-body containing spermatozoa was 31.3%. 2) There was no relationship between sperm density in semen analysis and F-body rate 3) There was no relationship between sperm motility in semen analysis and F-body rate. 4) In 5 men, methyl alcohol-acetic acid (3:1) preparations, as a fixation solution, showed better F-body rate than that of ethyl alcohol preparations. 5) In 4 men, the preparations which were fixed and stained within 2 hours after obtaining seminal fluid showed better F-body rate than that fixed and stained after 6 hours. 6) In 10 men, after low speed centrifuging of the seminal fluid, the deposit preparations, showed decreased mean percentage of F-body containing spermatozoa by 4.2%.

A STUDY OF THE EFFECTS OF PROTECTANTS WITH DIFFERENT CONCENTRATION OF GLYCEROL AND SUCROSE ON CRYOPRESERVATION OF HUMAN SPERMATOZOA / 解剖学报

The effects on frozen-thawed sperm survival rate of 16 different cryoprotective media containing different ratio of glycerol (0%, 2.5%, 5% and 7%) and sucrose (0,25,50 and 100 mmol/L) were compared. The results showed that glycerol-sucrose cryoprotective media had better effect on cryoprotection of spermatozoa than that of traditional glycerol protective medium, and appropriately increasing the concentration of sucrose and decreasing the concentration of glycerol could improve sperm motility, and especially benefit to preserve sperm linear motility at 12h postthawed. Using 5% glycerol combined with 50 mmol/L sucrose as cryoprotective medium, the sperm survival rate at 0, 6, 12h postthawed was 85.38%, 51.22%, 33.38%, respectively, the linear moving sperm survival rate was 83.74%, 33.33%, 18.38% respectively.