Addition of synthetic amino acids in sheep semen improves its quality after cryopreservation / Adição de aminoácidos sintéticos no sêmen ovino melhora a qualidade espermática após a criopreservação do sêmen

Some amino acids can protect mammalian sperm cells against oxidation during thermal stress caused by freezing/thawing. Thus, the objective was to evaluate the protective action of the association of the amino acids L-proline (Pro) and L-glutamine (Glu) against the cryoinjury caused to sheep sperm after cryopreservation. Eight ejaculates were collected from four sheep (n=32) and diluted in Tris-Egg Yolk-Glycerol until the final concentration of 200 x106 sptz/mL and kept in a water bath at 32 °C. The amino acids were added as follows: control (without adding amino acids), Pro+Glu 1 (100 µM Pro + 500 µM Glu), Pro+Glu 2 (300 µMPro + 1000 µM Glu), Pro+Glu 3 (500 µM Pro + 1500 µM Glu) and Pro+Glu 4 (700 µM Pro + 2000 µM Glu). Afterwards, the semen was cooled to 5 °C for 2 h, after that period, filled in 0.5 mL straws and then placed under liquid nitrogen vapor (N2L), 8 cm from the liquid sheet for 15min, and then immersed on the N2L. The samples were analyzed for sperm motility, plasma membrane and acrosomal membrane integrity, mitochondrial activity and binding test. The variables were subjected to the normality tests (Lilliefors test) and homoscedasticity tests (Cochran and Bartlett test), afterwards the variables of normal distribution were subjected to analysis of variance and the means compared by the Tukey test with a significance level of 5%. The Pro+Glu 3 group exhibited sperm with a greater (P<0.05) motility after thawing. In addition, the highest percentage of plasma and acrosomal membrane integrity were obtained using Pro+Glu 1, Pro+Glu 2 and Pro+Glu 3; and Pro+Glu 2 and Pro+Glu 3, respectively. Amino acids also kept mitochondrial activity high compared to the control, with Pro+Glu 3 resulting in greater activity (P<0.05). Sperm viability was higher (P<0.05) with the use of Pro+Glu 2 and Pro+Glu 3 than in the control. The number of sperm that showed the ability to bind to the egg yolk perivitelline membrane was higher (P<0.05) in semen treated with amino acids. It is concluded that the addition of synthetic amino acids in the semen of sheep before cryopreservation improves sperm quality and fertilization potential and can thus be added in cryopreservation protocols.

Alguns aminoácidos podem proteger as células espermáticas de mamíferos contra a oxidação durante o estresse térmico causado na congelação/descongelação. Dessa forma, objetivou-se avaliar a ação protetora da associação dos aminoácidos L-prolina (Pro) e L-glutamina (Glu) contra as crioinjúrias causadas aos espermatozoides de ovino após a criopreservação. Foram coletados oito ejaculados de quatro carneiros (n=32) e diluídos em Tris-Gema de ovo-Glicerol até a concentração final de 200 x106 sptz/mL e, mantidos em banho maria a 32 °C. Os aminoácidos foram adicionados da seguinte forma: controle (sem adição de aminoácidos), Pro+Glu 1 (100 µM Pro + 500 µM Glu), Pro+Glu 2 (300 µM Pro + 1000 µM Glu), Pro+Glu 3 (500 µM Pro + 1500 µM Glu) e Pro+Glu 4 (700 µM Pro + 2000 µM Glu). Depois, o sêmen foi resfriado a 5 °C por 2 h, após esse período, envasado em palhetas de 0,5 mL e então acondicionado sob vapor de nitrogênio líquido (N2L), a 8 cm da lâmina líquida por 15 min, e depois imersos no N2L. As amostras foram analisadas quanto à motilidade espermática, integridade da membrana plasmática e da membrana acrossomal, atividade mitocondrial e teste de ligação. As variáveis foram submetidas aos testes de normalidade (Teste de Lilliefors) e homocedacidade (Teste de Cochran e Bartlett), posteriormente as variáveis de distribuição normal foram submetidas à análise de variância e as médias comparadas pelo teste de Tukey com nível de significância de 5%. O grupo Pro+Glu 3 exibiu espermatozoides com uma maior (P<0,05) motilidade após o descongelamento. Além disso o maior percentual de integridade da membrana plasmatica e acrossomal foram obtidos utilizando Pro+Glu 1, Pro+Glu 2 e Pro+Glu 3; e Pro+Glu 2 e Pro+Glu 3, respectivamente. Os aminoácidos também mantiveram alta a atividade mitocondrial em comparação com o controle, com Pro+Glu 3 resultando numa maior atividade (P<0,05). A viabilidade dos espermatozoides foi maior (P<0,05) com o uso de Pro+Glu 2 e Pro+Glu 3 do que no controle. O número de espermatozoides que apresentaram à capacidade de ligação a membrana perivitelina da gema de ovo foi maior (P<0,05) no sêmen tratado com aminoácidos. Conclui-se que, a adição dos aminoácidos sintéticos no sêmen de ovinos antes da criopreservação melhora a qualidade espermática e o potencial fecundante, podendo assim serem adicionados em protocolos de criopreservação.

Relationship between sperm quality and male reproductive hormones among male partners with fertility complications: attending CHUB

Background Infertility remains a highly prevalent global condition in the second decade of the new millennium. Reproductive hormones determine sperm quality as they initiate and maintain spermatogenesis. Hormonal imbalance can cause abnormal sperm quality that can be treated by hormonal replacement therapy. Objective To assess the relationship between sperm quality and male reproductive hormones among male partners with fertility complications attending CHUB. Methods The study was a descriptive cross-sectional, and a convenient sampling strategy was used to recruit subjects at CHUB. Sixty-two male subjects with fertility complications provided both semen and blood sample to analyze sperm quality and reproductive hormones. Descriptive statistics were used to analyze data. Results Both FSH and LH showed a strong negative correlation with sperm count which is more profound with FSH (r= -0.722) than LH (r= -0.545). Testosterone showed a strong positive correlation with sperm count (r= 0.712). FSH and LH showed a negative correlation with sperm motility which is more profound in FSH (r= - 0.312) than LH (r= -0.302). Testosterone also showed a positive correlation with sperm motility (r= 0.360). Conclusion Our study found a correlation between sperm quality and male reproductive hormones. We further suggest other studies to investigate predictive power of male reproductive hormones.

Actiflagelin, a new sperm activator isolated from Walterinnesia aegyptia venom using phenotypic screening

Background Sperm contains a wealth of cell surface receptors and ion channels that are required for most of its basic functions such as motility and acrosome reaction. Conversely, animal venoms are enriched in bioactive compounds that primarily target those ion channels and cell surface receptors. We hypothesized, therefore, that animal venoms should be rich enough in sperm-modulating compounds for a drug discovery program. Our objective was to demonstrate this fact by using a sperm-based phenotypic screening to identify positive modulators from the venom of Walterinnesia aegyptia. Methods Herein, as proof of concept that venoms contain interesting compounds for sperm physiology, we fractionated Walterinnesia aegyptia snake venom by RP-HPLC and screened for bioactive fractions capable of accelerating mouse sperm motility (primary screening). Next, we purified each compound from the positive fraction by cation exchange and identified the bioactive peptide by secondary screening. The peptide sequence was established by Edman sequencing of the reduced/alkylated compound combined to LC-ESI-QTOF MS/MS analyses of reduced/alkylated fragment peptides following trypsin or V8 protease digestion. Results Using this two-step purification protocol combined to cell phenotypic screening, we identified a new toxin of 7329.38 Da (actiflagelin) that activates sperm motility in vitro from OF1 male mice. Actiflagelin is 63 amino acids in length and contains five disulfide bridges along the proposed pattern of disulfide connectivity C1-C5, C2-C3, C4- C6, C7-C8 and C9-C10. Modeling of its structure suggests that it belongs to the family of three finger toxins with a noticeable homology with bucandin, a peptide from Bungarus candidus venom. Conclusions This report demonstrates the feasibility of identifying profertility compounds that may be of therapeutic potential for infertility cases where motility is an issue.

Star anise extracts modulation of reproductive parameters, fertility potential and DNA fragmentation induced by growth promoter Equigan in rat testes

ABSTRACT Equigan is an anabolic steroid that has been developed for veterinary use and derived from endogenous sex hormone testosterone that plays a key role in the development of male reproductive tissue as well as in puberty and spermatogenesis. The current study is aimed to investigate the possible prophylactic effect of star anise extracts (SAE) on the toxicity of rat testes, sexual hormones alternations, sperm count, sperm abnormalities and testicular DNA damage by Equigan. Forty adult male rats were equally divided into four groups (1st Control group, 2nd SAE group, 3rd Equigan and 4th Equigan+SAE group). Food and fluid intakes, relative body weight, potassium, chloride, phosphorous, non-progressive and immotile sperms were significantly increased in Equigan group as compared to control group. In contrast; relative testes weight, sodium, magnesium, total calcium, testosterone, FSH, LH, PRL, sperm count, progressive motility, and viability showed a significant decrease in Equigan group as compared to control groups. The relative weight of epididymis, seminal vesicles, prostates and serum calcium ions didn't change significantly in different studied groups. Co-administration of SAE with Equigan improved the sexual toxicity, electrolyte alternations, sperm count, abnormalities and DNA damage induced by Equigan.

Short-term buserelin administration induces apoptosis and morphological changes in adult rat testes

Abstract Purpose: To investigate the effect of buserelin on gonadal structure and function in adult male rats. Methods: Twenty-four adult Wistar male rats were divided into three groups: two treated groups and controls. The first and second treated groups received 300 (low dose) and 500 (high dose) µg/kg buserelin, respectively, and the control group received normal saline. All groups were treated subcutaneously for five days. Results: The seminiferous tubular epithelial thickness was significant decreased in the treated groups compared with those in the control. There was a significant increase in apoptotic cell death in high dose treated group compared with low dose treated and control groups. No significant difference in serum testosterone level was observed after one month in the three groups. Conclusion: Buserelin induces apoptotic cell death and decreased diameter and epithelium thickness of seminiferous tubules in the adult rat testes.

Trigger effect of hMG and hCG in the treatment of unexplainable non-obstructive azoospermia / 中华男科学杂志

Objective@#To investigate whether the trigger effect of human menopausal gonadotropins (hMG) and human chorionic gonadotropins (hCG) attributes to the treatment of unexplainable non-obstructive azoospermia (NOA).@*METHODS@#We retrospectively analyzed the clinical data about 282 cases of unexplainable NOA treated in the Maternity and Child Health Hospital of Guizhou Province from January 2010 to May 2017. All the patients underwent trigger treatment by intramuscular injection of hMG at 75 IU 3 times a week for 2 weeks, followed by hCG at 2 000 IU twice a week for another 2 weeks, and meanwhile took vitamin E, Levocarnitine and Tamoxifen as an adjunctive therapy. The treatment lasted 3－12 months.@*RESULTS@#Fifty-eight of the 255 patients that completed the treatment were found with sperm in the semen after treatment, all with severe oligoasthenospermia. Forty-seven of the 58 cases received assisted reproductive technology (ART), of which 18 achieved clinical pregnancy. Semen centrifugation revealed no sperm in the other cases, of which 6 were found with epididymal sperm at epididymal and testicular biopsy after treatment and 3 of them achieved clinical pregnancy after ART. Sperm was found in the semen or at epididymal or testicular biopsy in 64 of the patients after treatment, with an effectiveness rate of 25.1%.@*CONCLUSIONS@#Trigger treatment by injection of hMG and hCG combined with adjunctive oral medication has a certain effect on unexplainable NOA.

Protective effect of royal jelly on fertility and biochemical parameters in bleomycin-induced male rats

Bleomycin [BL] is a glycopeptide antibiotic obtained from the bacterium Streptomyces verticillus which is routinely used for treatment of human cancers. Royal jelly [RJ] is a production from the hypo pharyngeal, mandibular and post cerebral glands of nurse bees. RJ consists of 66% water, 15% sugars, 5% lipids, and 13% proteins, essential amino acids and vitamins. The aim of present study was to evaluate protective effect of royal jelly on sperm parameters and malondialdehyde [MDA] production in rat. Forty adult male wistar rats [220 +/- 20gr] were randomly divided into 4 groups [n=10]. Control group [CG] received normal saline 10 ml/kg twice a week with Intraperitoneal [I.P] for 48 days [0.3 ml/rat]. Royal Jelly group [RJG] received jelly [100 mg/kg daily] for 48 days orally. Bleomycin group [BLG] received BL [10 mg/kg twice a week] with I.P for 48 days. Royal Jelly+ Bleomycin group [RJ+BLG] received royal Jelly [100 mg/kg /day] orally concomitant with BL administration. Sperm count, motility, and viability were investigated and chromatin quality and DNA integrity were also analyzed. Serum testosterone and MDA concentrations were measured as well. BL caused decline significantly [p<0.05] sperm count, sperm viability, motility as well as testosterone concentration compared to control group while significant [p<0.05] increases in immature sperm, sperm with damaged DNA and MDA concentration were announced in BL in comparison with CG and RJ+BLG. Royal jelly improved Bleomycin-induced toxicity on sperm parameters and testosterone and MDA concentrations. The present results support the idea that BL adversely affects sperm parameters and MDA and the RJ with antioxidant properties has positive effects on these parameters

Effects of Fumaria parviflora leaves extract on reproductive parameters in adult male rats

There is growing concern that occupational, environmental and lifestyle factors adversely affect male reproductive health. Fumaria parviflora Lam. is being used traditionally in Persian folk medicine to cure various ailments and has been supposed to have fertility-enhancing properties. A dose-response study was designed to assess effects of F. parviflora ethanolic leaves extract on reproductive parameters in adult male Wistar rats. In this experimental study, healthy adult male rats were treated with 100, 200 and 400 mg/kg/day of F. parviflora leaves extract via gavage for 70 days. Blood samples were collected for determination of testosterone, LH and FSH serum levels. Reproductive organs weight, motility, morphology and density of epididymal sperm, seminiferous tubules diameter and germinal epithelium height were evaluated in each experimental group. The body weight was not affected, while the weights of testis and epididymis were significantly enhanced in rats treated with 200 and 400 mg/kg/day F. parviflora extract. No significant changes were observed in seminal vesicle and ventral prostate weight between experiment groups. Significant increase was found in epididymal sperm density and percent of morphologically normal sperm in extract-treated rats. Serum testosterone levels were significantly higher in rats received 200 and 400 mg/kg/day. The results indicated that ethanolic extract of F. parviflora leaves have a potential to improve reproductive parameters and enhance male fertility

Investigation in vitro expression of catSper sub fragment followed by production of polyclonal antibody: potential candidate for the next generation of non hormonal contraceptive

CatSper is a voltage-sensitive calcium channel that is specifically expressed in the testis and it has a significant role in sperm performance. CatSper [1-4] ion channel subunit genes, causes sperm cell hyperactivation and male fertility. In this study, we have explored targeting of the extracellular loop as an approach for the generation of antibodies with the potential ability to block the ion channel and applicable method to the next generation of non-hormonal contraceptive. In this experimental study, a small extracellular fragment of Cat-Sper1 channel was cloned in pET-32a and pEGFP-N1 plasmids. Then, subsequent methods were performed to evaluate production of antibody: 1] pEGFP-N1/CatSper was used as a DNA vaccine to immunize Balb/c mice, 2] The purified protein of pET-32a/CatSper was used as an antigen in an enzyme-linked immunosorbent assay [ELISA] and western-blot, and 3] The serum of Balb/-c mice was used as an antibody in ELISA and western-blot. The statistical analysis was performed using the Mann Whitney test. The results showed that vaccination of the experimental group with DNA vaccine caused to produce antibody with [p< 0.05] unlike the control group. This antibody extracted from Balb/c serum could recognize the antigen, and it may be used potentially as a male contraception to prevent sperm motility. CatSpers are the promising targets to develop male contraceptive because they are designed highly specific for sperm; although, no antagonists of these channels have been reported in the literature to date. As results showed, this antibody can be used in male for blocking CatSper channel and it has the potential ability to use as a contraceptive

Effect of clomiphene citrate on sperm density in male partners of infertile couples.

Infertility is on the rise in today's world. A subnormal sperm count is frequently encountered in infertile couples. Clomiphene citrate, 1-[p-(beta-diethyl aminoethoxy) phenyl]-1,2-diphenyl chloroethylene, is an orally active nonsteroidal agent distantly related to diethylstilbestrol. It is thought to stimulate pituitary gonadotropin release by excluding estradiol from hypothalamic receptor sites. This interaction neutralizes the normal negative feedback control of estrogen and results in enhanced secretion of LH-RH, FSH-RH and gonadotropins. Testosterone is produced by the Leydig cells in response to LH secretion. The concentration of testosterone in the tubular environment is believed to maintain the gametogenic function of the testis. Clomiphene citrate in the dose of 25 mg daily for 25 days with five days rest was administered to 25 extreme oligozoospermic men (group I) and 40 moderate oligozoospermic men (group II) the cycle being continued for three months). Repeat semen analysis was done at the end of three months and all the routine seminal parameters were reevaluated. The data thus obtained was analyzed using Student's paired 't' test. The mean sperm count in Group I increased from 3.84 +/- 0.32 to 8.2 +/- 1.58 (P < 0.05) and in Group II from 13.05 +/- 0.48 to 24.55 +/- 1.73 (P < 0.001). The mean motile sperm count in Group I increased from 1.74 +/- 0.25 to 3.92 +/- 0.83 (P < 0.05) and in Group II from 8.27 +/- 0.40 to 10.05 +/- 0.56 (P < 0.01). Thus clomiphene citrate exerts its effect on spermatogenesis by raising the endogenous serum FSH, LH and testosterone levels to initiate and maintain gametogenesis (10). Researchers opined that this increase in endogenous gonadotrophins manifests itself in improving the sperm count, sperm motility and to certain extent morphology of the sperms, when there is no end-organ pathology.

Affection of Bushen Shengjing pill on NO and NOS in testicle and ante-oxidization of rat with spermatogenic cell injury / 中国中药杂志

<p><b>OBJECTIVE</b>Discuss the functional mechanism of treating male infertility with Bushen Shengjing pill (BS).</p><p><b>METHOD</b>Fill the SD rat with adenine so as to injure the germ cell which will produce the model rat with kidney yang deficiency. Measure the activity of SOD and NOS, the amount of NO and MDA in the testicle.</p><p><b>RESULT</b>BS can promote the activity of SOD, reduce the activity of NOS and the amount of NO and MDA.</p><p><b>CONCLUSION</b>BS has the function of protecting the germ cell which is closely related to the function that the pill can promote the activity of SOD, reduce the activity of NOS and the amount of NO and MDA.</p>

Interacción de los péptidos opiodes endógenos con la función del eje hipotálamo-hipófisis-testículo / Interaction of endogenous opoid peptides with hypothalamie-hypophisis-testis axis function

A partir del aislamiento e identificación de sustancias endógenas con actividad opoide (endorfinas y encefalinas) (1), y del descubrimiento de los receptores opoides en el cerebro de mamíferos (2), numerososo estudios se han realizado con el fin de conocer el papel que estos opoides endógenos tienen en la regulación neuroendocrina del eje hipotálamo-hipófisis-gónada. Tanto los opioides endógenos como los agentes químicos con actividad opoide (morfina, heroína, metadona, análogo sintéticos de encefalinas, etc.) interfieren con la producción de las hormonas secretadas por la hipófisis; el efecto de estos opoides es inhibitorio para algunas hormonas como la LH, FSH y la TSH, mientras que para otras como la PRL, GH y ACTH estimulan su producción (3), los opoides actúan a nivel hipotalámico y suprahipotalámico modulando la secreción de factores liberadores e inhibitorios. En varones adultos sanos los péptidos opioides endógenos (POE) produce una disminución de los niveles séricos de gonadotrofinas. La administración de antagonistas específicos de los receptores opiáceos aumentan la liberación de hormona luteinizante (LH) e incrementan la freuencia y amplitud de los pulsos de LH (6). Tanto los efectos de los POE como de los antagonistas específicos de sus receptores están alterados en la diferentes patologías del eje hipotálamo-hipófisis-testículo

Variations in zinc content of seminal plasma from fertile and sub-fertile men

Zinc concentration in the seminal plasma from 27 subfertile [less than 10 million sperms/ml] patients and 9 fertile [more than 60 millions sperms/ml] subjects was estimated by the atomic absorption spectrophotometry. Results showed a significant reduction in the zinc concentration in subfertile group as compared to the control group. Low levels of seminal plasma zinc concentration have been explained by a poor secretory function of the prostate gland, also nutritional causes may be considered to be responsible for reduced zinc content in semen