Low semen quality and adverse histological changes in testes of adult male mice treated with bee venom (Apis mellifera).

Background: Male infertility has been on the rise since the past seven decades. Recently, in Libya, bee venom therapy (BVT) has become a popular method among alternative healthcare practitioners for treating male infertility. However, a literature search did not find any published studies that investigated the use of BVT for infertility treatment. Aim: To investigate the effect of bee venom on the male reproductive status through measurements of semen quality parameters and testicular histological changes in adult male mice. Methods: A total of 48 male mice were randomly divided into three experimental groups (which were subdivided into two subgroups with eight mice each) as follows: control, bee venom sting (BVS), and bee venom injection (BVI). The normal control subgroup mice were not subjected to any treatment, while the vehicle control subgroup mice were injected (i.p.) with 200 µl of 0.9% saline solution. In the BVS-treated subgroups, each mouse was stung by one live bee for five times (BVS-5) or seven times (BVS-7) every third day for 2 or 3 weeks. While each mouse in the BVI-treated subgroups received 23 µg/kg in a dose volume of 200 µl BVIs (i.p.) for five times (BVI-5) or seven times (BVI-7) every third day for 15 or 21 days. Results: The findings of this study showed that repeated bee venom treatment by sting or injection to adult male mice resulted in a significant decline in testosterone levels, sperm count, sperm motility, and a very significant increase in the percentage of abnormal sperm morphology; also, there were harmful testicular histological changes in the structural organization of seminiferous tubules and degenerative changes in the germinal epithelium compared to control group. Conclusion: The results of this study provide evidence for the low semen quality and adverse testicular histological changes in male mice treated with bee venom. Hence, there is a desperate need for educating alternative healthcare practitioners and infertile couples about the harmful effects of BVT on reproductive status.

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.

A double-blinded, randomized placebo-controlled trial on the effect of traditional Chinese medicine formula Wuzi Yanzong pill on improving semen qualities in men with suboptimal parameters.

BACKGROUND: In Hong Kong, one of six couples is affected by subfertility problems. Male infertility contributes to half of the infertility cases. In male infertility, there is no effective treatment for patients with idiopathic infertility/poor semen parameters. Recent meta-analysis results suggest that a traditional Chinese medicine (TCM) formula - Wuzi Yanzong pill - showed a curative effect on male fertility. However, the heterogeneity of the studies could not draw a definitive conclusion on the therapeutic effect of this formula. The aim of this study is to conduct a well-designed randomized controlled trial to investigate the effect of TCM formula Wuzi Yanzong pill on improving semen qualities in men with suboptimal parameters. METHODS: This study is a double-blinded, randomized placebo-controlled trial conducted in a public hospital in Hong Kong. Participants will be randomized, using computer-generated random numbers, with a 1:1 ratio to either the Wuzi Yanzong pill formula group or the placebo group. Both groups will be administered the drugs for 12 weeks. Participants will have a total of four visits for their semen and blood assessments for a 6-month period, and we will follow up for another 6 months to record their conception outcome. The primary outcome is to compare the total motile sperm count, natural conception rate, and pregnancy outcome to those under placebo treatment. Secondary objectives are sperm functions and assisted reproductive technology outcome. DISCUSSION: To date, there are no studies using the disclosed Wuzi Yanzong formula or double-blinded, randomized trials. The Wuzi Yanzong TCM formula may provide a good clinical solution for subfertile males for which contemporary western medicine has no cure. Therefore, a well-designed randomized trial for evaluating the effect of Wuzi Yanzong TCM formula is urgently needed. TRIAL REGISTRATION: Chinese Clinical Trial Registry (ChiCTR), ChiCTR-INR-17010790 . Registered on 27 February 2017. Centre for Clinical Research and Biostatistics - Clinical Trials Registry, CUHK_CCRB00548 . Registered on 27 February 2017.

Sim1 Neurons Are Sufficient for MC4R-Mediated Sexual Function in Male Mice.

Sexual dysfunction is a poorly understood condition that affects up to one-third of men around the world. Existing treatments that target the periphery do not work for all men. Previous studies have shown that central melanocortins, which are released by pro-opiomelanocortin neurons in the arcuate nucleus of the hypothalamus, can lead to male erection and increased libido. Several studies specifically implicate the melanocortin 4 receptor (MC4R) in the central control of sexual function, but the specific neural circuitry involved is unknown. We hypothesized that single-minded homolog 1 (Sim1) neurons play an important role in the melanocortin-mediated regulation of male sexual behavior. To test this hypothesis, we examined the sexual behavior of mice expressing MC4R only on Sim1-positive neurons (tbMC4Rsim1 mice) in comparison with tbMC4R null mice and wild-type controls. In tbMC4Rsim1 mice, MC4R reexpression was found in the medial amygdala and paraventricular nucleus of the hypothalamus. These mice were paired with sexually experienced females, and their sexual function and behavior was scored based on mounting, intromission, and ejaculation. tbMC4R null mice showed a longer latency to mount, a reduced intromission efficiency, and an inability to reach ejaculation. Expression of MC4R only on Sim1 neurons reversed the sexual deficits seen in tbMC4R null mice. This study implicates melanocortin signaling via the MC4R on Sim1 neurons in the central control of male sexual behavior.

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

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.

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.

Safety assessment of powdered Cistanche deserticola Y. C. Ma by a 90-day feeding test in Sprague-Dawley rats.

Cistanche deserticola (C. deserticola), a holoparasitic plant widely distributed in arid or semi-arid areas in Eurasia and North Africa, has been used as an important tonic in traditional Eastern medicine for centuries. However, little information on the systemic toxicity and safety evaluation of it is available. The purpose of this study was to investigate the potential toxicity of powdered C. deserticola as a novel food ingredient by use of a subchronic toxicity study in Sprague-Dawley (SD) rats. A total of 80 male and female rats were fed with diets containing 8, 4, 2 and 0% (control) powdered C. deserticola for 90 days. A toxicological assessment was performed including mortality, body and organ weight, food consumption, blood biochemistry, hematology, gross necropsy and histopathological examinations. There were no signs of toxicity and treatment-related changes in rats treated with powdered C. deserticola. The no-observed-adverse-effect level (NOAEL) of powdered C. deserticola was 7.8 g kg-1 body weight for males and 8.0 g kg-1 body weight for females of rats under the experimental conditions of this study.

Mucuna pruriens and its major constituent L-DOPA recover spermatogenic loss by combating ROS, loss of mitochondrial membrane potential and apoptosis.

BACKGROUND: The Ayurvedic medicinal system claims Mucuna pruriens (MP) to possess pro-male fertility, aphrodisiac and adaptogenic properties. Some scientific evidence also supports its pro-male fertility properties; however, the mechanism of its action is not yet clear. The present study aimed at demonstrating spermatogenic restorative efficacy of MP and its major constituent L-DOPA (LD), and finding the possible mechanism of action thereof in a rat model. METHODOLOGY/FINDINGS: Ethinyl estradiol (EE) was administered at a rate of 3 mg/kg body weight (BW)/day for a period of 14 days to generate a rat model with compromised spermatogenesis. MP and LD were administered in two separate groups of these animals starting 15(th) day for a period of 56 days, and the results were compared with an auto-recovery (AR) group. Sperm count and motility, testis histo-architecture, level of reactive oxygen species (ROS), mitochondrial membrane potential (MMP), apoptosis, peripheral hormone levels and testicular germ cell populations were analysed, in all experimental groups. We observed efficient and quick recovery of spermatogenesis in MP and LD groups in comparison to the auto-recovery group. The treatment regulated ROS level, apoptosis, and mitochondrial membrane potential (MMP), recovered the hypothalamic-pituitary-gonadal axis and the number of testicular germ cells, ultimately leading to increased sperm count and motility. CONCLUSION/SIGNIFICANCE: M. pruriens efficiently recovers the spermatogenic loss induced due to EE administration. The recovery is mediated by reduction in ROS level, restoration of MMP, regulation of apoptosis and eventual increase in the number of germ cells and regulation of apoptosis. The present study simplified the complexity of mechanism involved and provided meaningful insights into MP/LD mediated correction of spermatogenic impairment caused by estrogens exposure. This is the first study demonstrating that L-DOPA largely accounts for pro-spermatogenic properties of M. pruriens. The manuscript bears CDRI communication number 8374.

Use of human chorionic gonadotropin in a male Pacific walrus (Odobenus rosmarus divergens) to induce rut and achieve a pregnancy in a nulliparous female.

Walrus in US zoos have a very low reproductive rate of 11 births in 80 years, and little is known about Pacific walrus (Odobenus rosmarus divergens) reproductive biology. To address this, we initiated a program in which detailed biological data were recorded on captive walrus. As part of a 7-year study, 1 male and 1 female 16-year-old captive Pacific walrus were carefully monitored with weekly serum hormone analysis, daily glans penis smears for spermatozoa, and abdominal ultrasound for pregnancy. The female ovulated once annually from late December through mid-January and then exhibited 9 months of sustained elevated progesterone. This nonconceptive estrous cycle profile is consistent with reports from wild walrus females. In contrast, the male's seasonal rut routinely occurred in late February through May with a serum testosterone peak in March. This profile differed from the reported adult male cycle in wild walrus of November through March. During the period of the female's ovulation, the male had nadir testosterone levels and was consistently azoospermic. Likewise, during the male's spermatogenic rut in the spring, the female was anovulatory with elevated progesterone. On this basis, the male was treated for 14 weeks with human chorionic gonadotropin (hCG) in an attempt to increase testosterone levels in synchrony with the female's annual ovulation. The treatment successfully induced rut characterized by sustained elevated serum testosterone levels and production of spermatozoa. The male and female successfully bred, and the female became pregnant. Upon discontinuation of hCG treatment, the male resumed baseline testosterone levels. We theorize that the lack of synchronization of rut and ovulatory cycles is a primary reason for reproductive failure in these captive walrus.

Successful pregnancy and childbirth after intracytoplasmic sperm injection with calcium ionophore oocyte activation in a globozoospermic patient.

OBJECTIVE: To check the effectiveness of intracytoplasmic sperm injection (ICSI) combined with assisted oocyte activation (AOA) in a globozoospermic patient. DESIGN: Case report. SETTING: Instituto Valenciano de Infertilidad, Valencia, Spain. PATIENT(S): A patient with globozoospermia. INTERVENTION(S): ICSI was administered in 14 oocytes. ICSI combined with AOA, in which a small amount of calcium was injected followed by calcium ionophore exposure, was done in 9 oocytes. MAIN OUTCOME MEASURE(S): Fertilization rate and embryo quality was assessed in both groups. RESULT(S): Chemical activation increased fertilization rate (55.6% vs. 35.7%) and the number of embryos with less multinucleation on day 2 (0 vs. 60%). Two embryos generated from AOA were transferred into the uterus (on day 3), resulting in a pregnancy and a healthy newborn. CONCLUSION(S): The AOA with calcium ionophore treatment improved fertilization rate and quality of the embryos, and was found to be an effective method for AOA in this patient with a low fertilization rate after previous ICSI treatment.

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.

Effects of Butea superba on reproductive systems of rats.

The effects of Butea superba on the reproductive system in male Wistar rats were investigated. The animals were fed daily with the powdered crude drug suspended in distilled water by a gastric tube at the dose of 2, 25, 250 and 1250 mg/kg body weight for 8 weeks. Rats fed with 1 ml of distilled water were used as a negative control. The weights of all vital organs in all treated groups were not different from the control. The percentage weight ratios of body weights of seminal vesicles and prostate glands were not different from the control, except that the testis of the group fed with 1250 mg/kg was significantly different from the control and the other treated groups. In addition, the sperm counts in this group showed about 16% more than the control group. Hematology as well as the liver and kidney function of all treated groups showed no difference from the control. B. superba, drug at 250 mg/kg which was 100 times more than the Thai FDA recommended dose for humans appeared to be safe in rats. The crude drug has demonstrated an increase tendency on testis weight and sperm counts in rat. The information from the present study can be used to explain the Thai folklore application of this plant in Thailand.

Infertility in 5,10-methylenetetrahydrofolate reductase (MTHFR)-deficient male mice is partially alleviated by lifetime dietary betaine supplementation.

Metabolism of folate is essential for proper cellular function. Within the folate pathway, methylenetetrahydrofolate reductase (MTHFR) reduces 5,10-methylenetetrahydrofolate to 5-methyltetrahydrofolate, a methyl donor for remethylation of homocysteine to methionine, the precursor of S-adenosylmethionine. S-adenosylmethionine is the methyl donor for numerous cellular reactions. In adult male mice, MTHFR levels are highest in the testis; this finding, in conjunction with recent clinical evidence, suggest an important role for MTHFR in spermatogenesis. Indeed, we show here that severe MTHFR deficiency in male mice results in abnormal spermatogenesis and infertility. Maternal oral administration of betaine, an alternative methyl donor, throughout pregnancy and nursing, resulted in improved testicular histology in Mthfr-/- offspring at Postnatal Day 6, but not at 8 mo of age. However, when betaine supplementation was maintained postweaning, testicular histology improved, and sperm numbers and fertility increased significantly. We postulate that the adverse effects of MTHFR deficiency on spermatogenesis, may, in part, be mediated by alterations in the transmethylation pathway and suggest that betaine supplementation may provide a means to bypass MTHFR deficiency and its adverse effects on spermatogenesis by maintaining normal methylation levels within male germ cells.

[New method of endocrine therapy for male infertility].

We undertook a prospective trial to compare the efficacy and adverse effects of a gonadotropin releasing-hormone analogue (GnRHa) and clomiphene citrate therapy for idiopathic normogonadotropic oligoasthenozoospermia (INOA). Fifty-nine newly-diagnosed INOA patients were randomly allocated to treatment with GnRHa or clomiphene citrate. Efficacy was assessed by measuring changes in semen parameters prior to and after 3 months of treatment. Thirty-six INOA patients underwent GnRHa therapy with 15 microg of diluted buserelin acetate given once a day intranasally, and 23 INOA patients were treated with 50 mg of clomiphene citrate daily by oral administration. The mean sperm density in the GnRHa group increased from 13.7 x 10(6)/ml to 22.1 x 10(6)/ml (p < 0.01), while the mean sperm density did not change significantly in the group treated with clomiphene. Similarly, the mean sperm motility increased from 35.9% to 43.9% in the GnRHa group (p < 0.05), but did not significantly change in the clomiphene group. No adverse effects were observed in either group. This GnRHa treatment protocol can be administered on an outpatient basis and is hoped to be of benefit to INOA patients.

Adult-onset idiopathic hypogonadotropic hypogonadism presented with erectile and ejaculatory disorder.

A 37-year-old man who had fathered a child five years previously presented with erectile and ejaculatory disorder. Endocrinological examinations revealed isolated luteinizing hormone-releasing hormone (LHRH) deficiency of hypothalamus, resulting in hypogonadotropic hypogonadism, and no causative abnormality was detected in imaging studies, including magnetic resonance imaging (MRI). Having a diagnosis of adult-onset hypogonadotropic hypogonadism, the patient received pulsatile subcutaneous administration of gonadotropin-releasing hormone (GnRH). Sperm analysis and serum level of testosterone improved to normal in a few months. His wife became pregnant using artificial insemination with her husband's semen 15 months after the beginning of the treatment.

[Current viewpoints on the evaluation of testicular biopsy]. / A herebiopsiák értékelésének új szempontjai.

The aim of this study was to analize clinical diagnoses and histopathological reports in 185 cases of male infertility, investigated by testicular biopsies in the period 1993-1997. The classical objective of such investigations has been to clarify either obstructive, or so-called secretory lesions resulting in azoospermia. Histologically normal spermatogenesis was reported in 8 patients (4.3%). Hypospermatogenesis proved to be the most frequent pathologic lesion (69 cases, 37.3%), followed by maturation arrest (61 cases, 33%). Germ cell aplasia (Sertoli-cell-only syndrome) was found in 28 cases (15.1%). Tubular atrophy was the dominant lesion in 5 cases (2.7%). Inflammatory change without other histologic abnormalities was seen only in 1 biopsy (0.5%). Further 13 specimens (7%) showed miscellaneous lesions, including one intratubular germ cell neoplasia. Retrospective histopathologic analysis revealed the coexistence of different basic lesions in 43 of our cases (21.7%). According to the novel requirements, pathologists should always report the presence of germ cell forms in the specimen, which are potentially suitable for fertilization. This is essencial because the new methods of testicular spermium extraction and intracytoplasmatic spermium injection offer the chance of assisted fertilization, even for some patients with smaller testicles and slightly elevated FSH levels.