Effects of energy drinks on biochemical and sperm parameters in Wistar rats

BACKGROUND: The present study evaluates the effects of energy drinks on the reproductive and biochemical parameters of adult male rats. METHODS: A total of 40 male rats (Wistar) were exposed to an energy drink mixed with the drinking water for a period of 120 days. The animals were divided into four groups and exposed to increasing therapeutic doses (DT) of an energy drink, based on allometric extrapolation, resulting in values (mL/day) per animal of 250 g: DT1 2.36 mL, DT3 7.47 mL, and DT6 14.16 mL. The control group (CTRL) consumed water only. During the treatment, the rats were assessed for signs of toxicity. After treatment, the animals were sacrificed and their organs were weighed. Sperm parameters (motility, concentration, and morphology) were evaluated. The biochemical markers alanine eamino transferase, aspartate amino transferase, alkaline phosphatase, lactic dehydrogenase, urea, creatinine, creatine phosphokinase, and creatine kinase MB fraction were measured, in addition to total cholesterol and testosterone. RESULTS: There was a significant decrease (p< 0.05) in the concentration of sperm in the treated groups (DT18.5 ± 0.7; DT3 7.2 ± 0.9; DT6 8.4 ± 0.9) compared to the control group (12.3 ± 1.2). No difference was observed with respect to relative weights of the animals'organs, water consumption, signs of toxicity, behavioral changes, biochemical markers, and sperm motility and morphology. CONCLUSION: The long-term consumption of energy drinks interferes negatively with sperm concentration, without affecting sperm motility and morphology or altering the hepatic, cardiac, or renal functions

Evaluación in vitro del efecto de Neisseria gonorrhoeae y los factores solubles producto de su metabolismo sobre la calidad espermática / In vitro evaluation of the effect of Neisseria gonorrhoeae and the soluble factors produced by its metabolism on sperm quality

AANTEDENTES: N. gonorrhoeae es una de las principales causas de uretritis, prostatitis y epididimitis en hombres. En el tracto urogenital masculino, esta bacteria o los factores solubles producto de su metabolismo pueden interactuar con los espermatozoides y alterar su calidad. OBJETIVOS: Determinar el efecto de la incubación in vitro de N. gonorrhoeae y los factores solubles producto de su metabolismo sobre la calidad espermática y evaluar la interacción bacteria-espermatozoide. MÉTODO: Se realizó incubación de una concentración 0,5 McFarland de N. gonorrhoeae y de los factores solubles producto de su metabolismo bacteriano con muestras de semen de voluntarios aparentemente sanos. Se cuantificaron los parámetros espermáticos convencionales (movilidad y viabilidad) y funcionales (potencial de membrana mitocondrial, integridad y lipoperoxidación de la membrana espermática, detección de especies reactivas del oxígeno, integridad de la cromatina y expresión de Anexina V), empleando microscopía y citometría de flujo, respectivamente. Para evaluar la interacción de N. gonorrhoeae con los espermatozoides humanos se realizaron extendidos en placas para su observación al microscopio. RESULTADOS: N. gonorrhoeae puede unirse al espermatozoide y disminuir la viabilidad espermática luego de 1,5 horas de incubación con los espermatozoides humanos (84,5% vs 66,5%, p<0,05), sin afectar los parámetros espermáticos funcionales. CONCLUSIÓN: N. gonorrhoeae interactúa con los espermatozoides humanos afectando la viabilidad espermática.

BACKGROUND: N. gonorrhoeae is a major cause of urethritis, prostatitis and epididymitis in men. In the male urogenital tract, the bacteria or soluble products of their metabolism may interact with sperm, and alter their quality. AIMS: To determine the in vitro effect of incubation of N. gonorrhoeae and soluble products of their metabolism on sperm quality and assess the bacteria-sperm interaction. METHODS: Soluble products of bacterial metabolism and 0.5 McFarland concentration of N. gonorrhoeae were incubated with semen samples from healthy volunteers. Conventional (motility and viability) and functional sperm parameters (potential of mitochondrial membrane integrity and sperm membrane lipid peroxidation, detection of reactive oxygen species, chromatin integrity and expression of annexin V) were quantified using microscopy and cytometry flow, respectively. To assess the interaction of N. gonorrhoeae to human sperm microscopic observation was performed. RESULTS: N. gonorrhoeae can join the sperm and decreases sperm viability after 1.5 hours of incubation with human sperm (84.5% vs 66.5%, p<0.05), without affecting the functional sperm parameters. CONCLUSION: N. gonorrhoeae interacts with human sperm affecting sperm viability.

Effects of Fluid Resuscitation on Testicular Damage in Severely Burned Rats.

Background: Severe burns cause a wide range of complications that challenge care in the short term and often leaves long term sequelae in survivors. Research evidence suggests that burns can affect testosterone secretion, and impair spermatogenesis and fertility. This study was designed to investigate the effects of fluid resuscitation on spermatogenesis and histology of the testes after major burns. Methodology: Third degree burns was induced on dorsal skin of 3 groups of male Wistar rats. Intra-venous resuscitation fluid was administered, ½ h and 3 h post-burn in the first 2 groups. A fourth group had sham burn only and served as negative control. After 8 weeks the animals were sacrificed evaluated for sperm parameters, testicular histology and assays of oxidative status. Main Findings: Fluid administration did not cause significant difference in sperm parameters or in levels of markers of oxidative stress among the animals with burn injury. Mean sperm density in groups 1 and 2 which had fluid treatment were 57.00±11.99 and 56.33±9.49 respectively compared to 49.00±6.24 in the untreated group 3 (p=0.921 and 0.947). However total counts were significantly lower in all burned groups than in the sham burn group. Fluid treatment produced a time-dependent relief from the histological disruptions associated with the burns. Tubules with germ cell loss were fewer in the fluid treated groups than in the untreated one. Conclusion: Fluid treatment in burn patients may not protect them from suppression of testicular function. Fertility damage in severe burns may involve mechanisms that do not depend on intravascular volumes and pressures.

Potential role of punicalagin against oxidative stress induced testicular damage / 亚洲男科学杂志(英文版)

Punicalagin is isolated from pomegranate and widely used for the treatment of different diseases in Chinese traditional medicine. This study aimed to evaluate the effect of Punicalagin (purity ≥98%) on oxidative stress induced testicular damage and its effect on fertility. We detected the antioxidant potential of punicalagin in lipopolysaccharide (LPS) induced oxidative stress damage in testes, also tried to uncover the boosting fertility effect of Punicalagin (PU) against oxidative stress-induced infertility. Results demonstrated that 9 mg kg-1 for 7 days treatment significantly decreases LPS induced oxidative damage in testes and nitric oxide production. The administration of oxidative stress resulted in a significant reduction in testes antioxidants GSH, T-SOD, and CAT raised LPO, but treatment with punicalagin for 7 days increased antioxidant defense GSH, T-SOD, and CAT by the end of the experiment and reduced LPO level as well. PU also significantly activates Nrf2, which is involved in regulation of antioxidant defense systems. Hence, the present research categorically elucidates the protective effect of punicalagin against LPS induced oxidative stress induced perturbation in the process of spermatogenesis and significantly increased sperm health and number. Moreover, fertility success significantly decreased in LPS-injected mice compared to controls. Mice injected with LPS had fertility indices of 12.5%, while others treated with a combination of PU + LPS exhibited 75% indices. By promoting fertility and eliminating oxidative stress and inflammation, PU may be a useful nutrient for the treatment of infertility.

The effects of curcumin on sperm parameters and nitric oxide production in varicocelized rats / Efectos de la cúrcuma sobre los parámetros espermáticos y producción de óxido nítrico en ratas varicocelizadas

One of the proposed mechanism by which varicocele induces its damage is excessive release of nitric oxide (NO). Several studies have shown the role of NO in poor sperm quality in infertile patients with varicocele. Scientific studies have demonstrated the beneficial effects of curcumin on the sperm parameters. Curcumin as an atoxic antioxidant can reduce production of NO. The aim of this study was to determine the effect of curcumin on NO levels and investigate if curcumin can improve sperm parameters in varicocelized male rats. Thirty male Wistar rats were randomly divided into 5 groups (V1 and V2 (varicocele), T (treatment), Sh (sham) and C was control). In groups V1, V2, T and Sh, the left renal vein was partially ligated to induce varicocele. In groups V1 and V2, sperm parameters and NO level were evaluated 8 and 16 weeks respectively after varicocele induction. Groups T and Sh received 100 mg/kg curcumin and placebo respectively, daily for 8 weeks after 2 months of induced varicocele. Sperm parameters (count, motility, viability and morphology), epididymis and testis weight and also NO concentration were measured. Sperm parameters (count, motility and viability) in groups V1, V2 and Sh were significantly low in comparison with control and treatment groups. The level of NO was significantly increased in serum of rats in groups V1 and V2, whereas group T rat serum in which curcumin was administered, showed decreased NO levels. The values of the epididymis and testis weight had no significant changes (P 0.05) in all groups. Administration of curcumin as a free radical scavenger, can decrease NO level and improve sperm parameters in varicocelized male rats.

Uno de los mecanismos propuestos por el cual los varicoceles inducen daño es la excesiva liberación de óxido nítrico (ON). Varios estudios han demostrado el efecto del ON en la mala calidad del semen en pacientes infértiles con varicocele. Investigaciones han demostrado los efectos beneficiosos de la cúrcuma sobre los parámetros de esperma. La cúrcuma como un antioxidanteatóxico puede reducir la producción de ON. El objetivo de este estudio fue determinar el efecto de la cúrcuma en el nivel de ON e investigar si la cúrcuma puede mejorar los parámetros del semen en ratas macho. Treinta ratas macho Wistar fueron divididas aleatoriamente en 5 grupos (V1y V2 (varicocele), T (tratamiento), Sh (simulado) y C (control)). En los grupos V1, V2, T y Sh, la vena renal izquierda fue parcialmente ligada para inducir varicocele. En los grupos de V1 y V2, los parámetros de semen y nivel de ON se evaluaron a las 8 y 16 semanas respectivamente, después de la inducción de varicocele. Los grupos T y Sh recibieron diariamente 100 mg/kg de cúrcuma y placebo durante 8 semanas, después de 2 meses de inducir el varicocele. Fueron medidos los parámetros del semen (recuento, motilidad, viabilidad y morfología espemática), peso del epidídimo y testículos, junto con la concentración del ON. El recuento, motilidad y viabilidad de los espermatozoides en los grupos V1, V2 y Sh fueron significativamente más bajos en comparación con los grupos C y T. El nivel de ON se incrementó significativamente en el suero de las ratas de los grupos V1 y V2, mientras que el suero del grupo T, en el que se administró cúrcuma, hubo una disminución de los niveles de ON. El peso del epidídimo y testículos tuvieron cambios significativos (P 0,05) en todos los grupos. La administración de cúrcuma como un eliminador de radicales libres, puede disminuir el nivel de ON y mejorar los parámetros espermáticos en ratas macho varicocelizadas.

Spermatozoa characterization in the one-sided livebearing Jenynsia multidentata (Cyprinodontiformes: Anablepidae) / Caracterización de parámetros espermáticos del pez vivíparo Jenynsia multidentata (Cyprinodontiformes: Anablepidae)

Several sperm parameters have been employed as useful tools to evaluate fish fertility. Within teleosts, approximately 3% of fish species are known to be viviparous. The Order Cyprinodontiformes includes several species with internal fertilization, and within this group most of the studies about sperm quality have been mainly focused on the Poeciliidae family. The livebearing fish Jenynsia multidentata (Anablepidae) inhabits an extensive area of the Neotropical region and it has been used as a useful fish laboratory model to evaluate the effects of xenobiotics through different biomarkers. The present work characterized the sperm of this species through a simple protocol of semen collection. Sperm population showed linearity greater than 89% and 70% of fish have a straight line and curvilinear velocity valued between 50 and 100µm/s. Although 85% of individuals showed a proportion of live sperm higher than 60%, the male population had a high degree of heterogeneity in its sperm count. Morphometry analyses showed a total sperm and head lengths of 46.66±2.06µm and 3.46±0.41mm, respectively. A rather long midpiece region (9.12±0.65µm) was registered, which may indicate high energy-producing capabilities of the spermatozoa. This study established basic parameter values which could be useful for evaluating reproductive potential of J. multidentata populations.

Diversos parámetros espermáticos han sido utilizados para evaluar la fertilidad de peces. Dentro de los peces teleósteos, aproximadamente el 3% de las especies son vivíparas. El orden Cyprinodontiformes incluye varias especies con fecundación interna. Dentro de este orden la mayor parte de los estudios sobre la calidad del esperma se han centrado principalmente en la familia Poeciliidae. El pez vivíparo Jenynsia multidentata (Anablepidae) habita una extensa área de la región Neotropical y ha sido utilizado como un exitoso modelo de laboratorio. El objetivo del presente trabajo fue caracterizar los espermatozoides de esta especie a través de un simple protocolo de recolección de esperma. La población de espermatozoides mostró una linealidad superior al 89% y el 70% de los peces tienen una velocidad lineal y curvilineal entre 50 y 100µm/s. Aunque el 85% de los individuos mostró una proporción de espermatozoides vivos de más del 60%, se observó una alta heterogeneidad en el recuento espermático. Los análisis morfométricos mostraron una longitud total de espermatozoides de 46.66±2.06µm y una longitud de la cabeza de 3.46±0.41µm. Los espermatozoides presentan una pieza media larga (9.12±0.65µm) lo que puede indicar una alta capacidad de producción de energía. El presente estudio establece valores básicos de parámetros que pueden ser útiles para evaluar el potencial reproductivo de las poblaciones de J. multidentata.

Protective effect of Tribulus terrestris hydroalcoholic extract against cisplatin-induced cytotoxicity on sperm parameters in male mice / Efecto protector del extracto hidroalcohólico de Tribulus terrestris contra la citotoxicidad inducida por cisplatino sobre parámetros espermáticos en ratones macho

Cisplatin is an anti-cancer drug used in chemotherapy. One of the limiting side effects of cisplatin is decreasing genital gland function, azoospermia and oligospermia. Tribulus terrestris has been used as an aphrodisiac. The present study amid to investigate protective effect of Tribulus terrestris hydroalcoholic extract against cisplatin-induced cytotoxicity on sperm parameters in mice. Male adult mice (n=30) were divided into control and 4 experimental groups (n=6). Control group received saline, first experimental group received cisplatin (5.5 mg/kg) and other three experimental groups received cisplatin (5.5 mg/kg) and different doses of hydroalcoholic extact of Tribulus terrestris (100, 300 and 500 mg/kg/i.p) respectively. On the day after the last injection, blood samples were collected for serum Nitric oxide (NO) assay. Also weights of body and testis, sperm parameters and seminiferous tubules diameter were assessed. Data analysis was performed using one-way ANOVA followed by Tukeys' test. The results showed that cisplatin lead to a reduction in the weight of body and testes, sperm parameters and increased level serum of NO significantly compared to the control group (P<0.05), while in treated groups with Tribulus terrestris, the weights of body and testes, sperm parameters, seminiferous tubules diameter were significantly higher compared with cisplatin group (P<0.05), but serum level of NO did not show significant differences. The study demonstrates that extract of Tribulus terrestris could protective effect against cisplatin- induced cytotoxicity on sperm parameters that may be related to the presence of antioxidant components acting via a multitude of central and peripheral mechanisms.

El cisplatino es un medicamento contra el cáncer utilizado en la quimioterapia. Uno de los efectos secundarios limitantes de cisplatino es la decreciente función glándular genital, azoospermia y oligospermia. Tribulus terrestris ha sido utilizado como un afrodisíaco. En el presente estudio se investiga el efecto protector del extracto hidroalcohólico de Tribulus terrestris (TT) contra la citotoxicidad inducida por cisplatino en los parámetros espermáticos en ratones. Ratones adultos machos (n = 30) fueron divididos en 4 grupos control y experimentales (n = 6). En el grupo control se administró solución salina, el primer grupo experimental recibió cisplatino (5,5 mg/kg) mientras que otros tres grupos experimentales recibieron cisplatino (5.5 mg/kg) además de diferentes dosis de extracto hidroalcohólico de TT (100, 300 y 500 mg/kg/IP) respectivamente. El día siguiente de la última inyección, se analizaron muestras de sangre para expresión de óxido nítrito (ON). Además, fueron evaluados el peso del cuerpo y testículos, los parámetros espermáticos y el diámetro de los túbulos seminíferos. Los datos fueron analizados mediante análisis ANOVA y la prueba de Tukey. El uso de cisplatino causó reducción del peso corporal y testicular, parámetros espermáticos y aumento significativo de los valores de ON en comparación con el grupo control (P<0,05), mientras que los grupos tratados con TT, fue significativamente mayor el peso corporal y testicular, parámetros espermáticos y diámetro de los túbulos seminíferos en comparación al grupo tratado con cisplatino (P<0,05). No se observaron diferencias significativas en los valores ON. El extracto de TT puede tener un efecto protector frente a la citotoxicidad inducida por el cisplatino sobre los parámetros espermáticos, al estar relacionado a la presencia de componentes antioxidantes que actúan mediante mecanismos centrales y periféricos.

The effect of cannabis sativa hydroalcoholic extract on sperm parameters and testis histology in rats / Efecto del extracto hidroalcohólico de cannabis sativa sobre los parámetros espermáticos e histología testicular en ratas

Cannabis Sativa is a multiuse herb in traditional medicine, its hydroalcoholic extract (10, 50, and 100 mg/kg) administered interaperitoneally for 14 consequent days to Wistar male rats resulted in significant decrease in progressive motility of sperm. Sperm count and seminiferous tubules diameter decreased significantly in comparison with control group. Also decrease in animal body weight in doses of 50 and 100 mg/kg was observed. Changes in testes weight and serum testosterone were not significant. Cannabis sativa extract has negative effect on sperm parameters such as motility, sperm count, and seminiferous tubules diameter.

La Cannabis Sativa es una hierba de múltiples usos en la medicina tradicional. Su extracto hidroalcohólico (10, 50, y 100 mg/kg) administrado intraperito-nealmente durante 14 días consecutivos a ratas Wistar macho produjo una disminución significativa en la motilidad progresiva de los espermatozoides. El recuento de espermatozoides y el diámetro de los túbulos seminíferos se redujo significativamente en comparación con el grupo control. También se observó disminución del peso corporal de los animales en dosis de 50 y 100 mg/kg. Cambios en el peso de los testículos y la testosterona sérica no fueron significativos. El extracto de Cannabis sativa tiene un efecto negativo sobre los parámetros seminales tales como la motilidad, conteo espermático, y el diámetro de los túbulos seminíferos.

Association of sperm-nucleoprotein transition with sperm parameters and embryo development / 中华泌尿外科杂志

Objective To investigate the correlation between sperm-nucleporotein transition and sperm parameters and embryo development,also to evaluate the influence of pregnancy out comes of assisted reproductive technology (ART).Methods Sperm-nucleoprotein transition assay of a total of 676 patients underwent ART treatment were detected by aniline blue staining,and the correlation analysis between spermnucleoprotein transition and sperm parameters,DNA damage,acrosin activity,fertilization rate,cleavage rate,quality of early embryo development as well as blastocyst formation rate was performed.Results The sperm concentration,(a+b) ％ sperm,sperm count and acrosin activity was (66.5±4.6) × 109/L,(149.2±9.9)×109/L,(51.2±1.3)％ and (72.2±3.3) mU/106 sperm in abnormal group,and (91.9±2.7) ×109/L,(240.0±8.0) ×109/L,(57.3±0.8)％ and (85.7±1.9) mU/106 sperm in normal group,which reached significant difference (P＜0.01).DNA fragmentation index (DFI) was (17.3± 1.0)％ in abnormal group,which was significantly higher than (14.6±0.5)％ in normal group.The cleavage rate of 95.0％,D3/D5 high quality embryo rates of 34.2％ and 1.28％,D5 blastocyst formation rate and the total rate of blastocyst formation rate of 22.4％ and 38.6％ in abnormal group,which were significantly lower than that in normal group (96.9％,38.2％,2.70％,27.9％ and 46.4％) (P＜0.01).The rate of spontaneous abortion was 12.3％ in abnormal group,which was significantly higher than that in normal group (4.7％) (P＜0.01).However,there was no significant difference in biochemical pregnancy rate and ectopic pregnancy rate between the 2 groups (P＞0.05).Conclusions Sperm-nucleoprotein transition was positively related with sperm parameters,DNA damage,acrosin activity,and also has an adverse effect on embryo development and the outcomes of ART.It is suggested that the sperm-nucleoprotein transition should be detected before ART.

Antioxidant levels in blood and seminal plasma and their impact on sperm parameters in infertile men.

Excess reactive oxygen species (ROS) beyond the scavenging capacity of antioxidants leads to DNA damage and oxidation of lipoprotein components at the cellular and subcellular level. The oxidative stress (OS) adversely affects sperm function by altering membrane fluidity, permeability and impairs sperm functional competence. In the present study, the OS status in seminal plasma and blood serum in infertile men and its relationship with spermatozoa parameters have been investigated. Four groups of infertile men viz., oligozoospermic (n = 15), asthenozoospermic (n = 17), teratozoospermic (n = 19), and oligoasthenoteratozoospermic (n = 9), and healthy fertile controls (n = 40) have been analyzed for superoxide dismutase (SOD), catalase (CAT), glutathione (GSH) and malondialdehyde (MDA) in seminal plasma and blood serum. Significant correlation between blood serum SOD and sperm count has been observed in patients (p = 0.018) and controls (p = 0.021). Similarly, significant correlation between blood serum GSH and sperm progressive motility in patients (p = 0.036) and controls (p = 0.029) is observed. The low seminal MDA is associated with increase in sperm progressive motility in patients (p = 0.039) and controls (p = 0.028). Positive correlation is found between increased seminal MDA levels and abnormal sperm morphology in both patients and controls (r = 0.523, p = 0.029; r = 0.612, p = 0.034 respectively). Correlations between blood SOD and sperm count and between blood GSH levels and progressive motility suggest that these can be important biochemical markers in assaying the sperm count and motility. A negative correlation of motility with seminal MDA indicates that sperm membrane lipid peroxidation affects the fluidity and thus mobility of sperm axoneme. This affects functional competence of the sperm and acts like a biological safeguard. The results of the present study suggest the prospects of using the blood serum and seminal plasma antioxidants as a valuable tool to evaluate the sperm reproductive capacity and functional competence.

Effect of Semen Quality on the Embryo Development / 华中科技大学学报（医学）（英德文版）

To investigate the influences of sperm quality on the zygotes and embryos development,as the role of the paternal factor in early human embryogenesis is gaining more attention because of the application of techniques such as intracytoplasmic sperm injection (ICSI) for the treatment of men infertility, 136 infertility couples with men factors (Group Ⅰ ) were included from May 2002 to January 2004. One hundred and seventy two infertility couples with tube factors (Group Ⅱ ) served as controls. The sperm parameters, gemmates and embryos quality, implantation rate and pregnant rate in both groups were analyzed. It was found that there was no significant differences in the number of oocytes retrieved, the fertilization rate and number of embryos transferred between two groups. Sperm concentration, percentage of motile sperm and percentage of sperm with normal morphology were significantly lower in group Ⅰ than in group Ⅱ (P＜0.01). The proportion of good quality zygotes and good quality embryos were significantly lower in the male infertility group than in the tubal disease group (P＜0.05). Implantation rate and pregnancy rate were similar in two groups. It was concluded that spermatozoa is involved in the embryo quality, even in the early stages of development, which limited the treatment potency of IVF procedure.

The Effects on Sperm Parameters and Membrane after Treatment with Progesteroneand/or Acetyl-L-Carnitine; Cryopreservation-Thawing / 대한불임학회지

OBJETIVE: To assess the effects of progesterone and acetyl-L-carnitine used after treated with IsolateR gradient before semen cryopreservation-thawing on sperm parameters and membrane integrity. MATERIALS AND METHODS: From April 2001 to July 2001, ten normal male partner of couples who were visited in vitro fertilization (IVF) clinics. the semens were treated with IsolateR gradient before cryopreservation, spermatozoa was incubated with progesterone (1, 5 and 10 micrometer), acetyl-L-carnitine (2.5, 5 and 10 micrometer), or both (progesterone, 1 micrometer; and acetyl-L-carnitine, 5 micrometer) for 30 min. RESULTS: There were no differences in sperm parameters and vital stain among isolate only treated group, progesterone (1, 5 and 10 micrometer), acetyl-L-carnitine (2.5, 5 and 10 micrometer) and both (progesterone, 1 micrometer; and acetyl-L-carnitine, 5 micrometer). But, in high concentration of acetyl-L-carnitine (10 micrometer) treated group, sperm parameters and vital stain were decreased. The statistical method was used ANOVA (Kruskal-Wallis test) and p value was <0.01. CONCLUSIONS: Neither progesterone nor acetyl-L-carnitine show to be protective effect on the cryodamage assessed by sperm parameters and vital stain (eosin-Y stain) in normal sperm. High concentration of acetyl-L-carnitine (10 micrometer), however, was harmful effect on cryoprevention.