Epidydimal Sperm Research Articles

Microplastics are detected in bull epididymal sperm and polystyrene microparticles impair sperm fertilization.

An increase in global infertility has coincided with the accumulation of microplastics (MPs) in the environment. This trend is particularly troubling because only 10% of male infertility cases can be attributed to identifiable causes, leaving a knowledge gap in our understanding of their underlying factors. To bridge this, it is important to explore the connection between the accumulation of MPs and the observed decline in male fertility. We assessed the presence of microplastics in epididymal sperm from bulls and used it as baseline concentrations for sperm exposure. MPs were detected in all epidydimal sperm (ES) samples, with a mean concentration of 0.37μgmL-1. Next, to investigate the effects of MPs on fertility, bovine sperm was exposed to three different concentrations of a mixture of 1.1, 0.5, and 0.3μm polystyrene (PS) beads: (1) 0.7μgmL-1, blood concentration of PS in cows (bPS); (2) 0.37μgmL-1, based on the concentration of total MPs found in ES (esMP); and (3) 0.026μgmL-1, based on the concentration of PS found in ES (esPS). All sperm samples incubated with PS exhibited reduced motility compared with the control at 0.5h. However, PS exposure did not affect acrosome integrity or induced oxidative stress. Embryos produced from sperm exposed to PS had reduced blastocyst rates, in addition to increased ROS formation and apoptosis. By employing physiological exposure, this research provided evidence of MPs in bovine epididymal sperm and demonstrated the detrimental effect of PS on sperm functionality.

Technology for Biobanking of Epididymal Spermatozoa from Patient with Obstructive Azoospermia: Case Report about Baby Born after Conventional Freezing Only with a Nonpermeable Cryoprotectant 360 kDa Polyvinylpyrrolidone.

This publication reports, for the first time, the birth of a healthy child after intracytoplasmic sperm injection (ICSI) of motile spermatozoa after conventional ("slow") freezing of epididymal spermatozoa using 5% polyvinylpyrrolidone (PVP) of high molecular weight (360 kDa). Cryopreservation solution with 10% PVP was added to 30 µL of spermatozoa suspension in a 1:1 ratio, with a final PVP concentration of 5%. Then, polycarbonate capillaries for oocyte denudation with a diameter of 170 µm were filled with 60 µL of the resulting sperm suspension. After that, the capillaries were placed for 10 minutes at a height of 15 cm above liquid nitrogen and immersed into liquid nitrogen. To warm the spermatozoa, the capillaries were immersed in a water bath at a temperature of 40°C for 30 seconds. Oocyte fertilization was performed by ICSI. Zygotes were cultured in vitro for 5 days to the blastocyst stage. More than 100 spermatozoa were obtained after percutaneous epidydimal sperm aspiration, of which 80% were motile. After cryopreservation, storage for 3 months in liquid nitrogen, and thawing, 72% of the total sperm cells remained motile. Ten oocyte-cumulus complexes were found after follicle puncture, and eight metaphase II stage oocytes were fertilized using ICSI. After 18 hours, two pronuclei were found in seven (88%) of the oocytes. An analysis of the morphological characteristics of 5-day-old embryos showed that four (57%) of them reached the blastocyst stage. One embryo was transferred, and the remaining embryos were cryopreserved (vitrified). The onset of pregnancy was detected on the 14th day after embryo transfer, and one healthy girl (3300 g) was born at term.

P-558 Childhood exposure to high-fat diets changes sperm small RNA content up to two unexposed generations of mice

Abstract Study question Is the adoption of high-fat diet from childhood associated with sperm small RNA content in mice? Are those changes inherited by the offspring and grand-offspring? Summary answer The adoption of high-fat diet during childhood changes sperm small RNA content up to the grand-offspring even after dietary correction. What is known already The prevalence of childhood obesity is increasing worldwide. The adoption of high-fat diet (HFD) from early age is a major factor for this trend. However, the long-lasting health effects of childhood obesity associated to HFD is unknown, particularly after dietary reversion. Multiple studies demonstrated that sperm small non-coding RNA (sncRNA) content changes in response to acute and chronic dietary factors. Also, sperm sncRNAs can transmit acquired traits such as metabolic syndrome. However, it is not known how the HFD-related sperm sncRNA fingerprints varies across generations, and how it relates with the phenotypes of the progeny. Study design, size, duration At weaning, 36 C57BL/6 mice were randomly divided into three groups (n = 12) feeding on 1) standard chow (CTRL) for 200 days, 2) HFD for 200 days, or 3) HFD for 60 days reverted to chow for 140 days (childhood HFD, CHFD). Subsequent generations (36 mice/generation, all fed on chow) were obtained by mating F0 and F1 males with same-age normoponderal females 120 days post-weaning and were assigned to the same experimental group as the progenitor. Participants/materials, setting, methods Mice were euthanized 200 days after weaning and tissues collected. Epidydimal sperm was collected, and concentration, viability, motility, and morphology were evaluated. Sperm RNA were extracted using the tri-reagent protocol, and sncRNAs sequenced by NGS RNA-seq. Sequences were annotated using SPORTS 1.1 and differential expression analysis performed using DESeq2. Targets of differently expressed sequences were estimated and Gene Ontology (GO) analysis of biological function performed using topGO. Main results and the role of chance HFD consumption during childhood is associated with poorer sperm parameters, even after dietary correction. No changes in sperm parameters were found between the offspring of HFD, CHFD and CTRL but, the grand-offspring of HFD and HFDt mice had lower sperm counts than the grand-offspring of CTRL. Regarding sperm sncRNA content, no changes were found between HFD and CTRL, but several mitochondrial transcription initiation RNAs (tiRNA) and repeat-derived RNAs (repRNAs) were differently expressed comparing to CTRL. Sperm sncRNA content of the offspring of HFD (piwi-interacting RNAs and repRNA) and CHFD (tRNA-derived fragments and repRNAs) displayed several differently expressed sequences comparing to CTRL. Only microRNAs (miRNAs) were differently expressed in the grandoffspring (CTRL vs. CHFD). GO analysis of biological function based on the targets of differently expressed sncRNAs showed a potential impact of childhood obesity in pathways related to sperm motility, chemotaxis and membrane integrity. These results suggest that sperm sncRNA content is altered due to childhood obesity even after two generations. This signature is not preserved across the male germline, but may influence the sperm sncRNA content of the progeny. This long-lasting, transgenerational signature of childhood obesity may explain the inheritance of the poor sperm quality phenotype from grand-sire to grand-offspring. Limitations, reasons for caution This model does not evaluate the impact of earlier dietary intervention, before sexual maturation. Also, it is not clear if the reported effects are exclusively due to childhood obesity or to excessive caloric intake. The estimates of biological impact using GO analysis of sperm sncRNA targets require further experimental evidence. Wider implications of the findings Our findings evidence the long-lasting effects of childhood obesity to sperm parameters, even after dietary reversion, and the potential transgenerational effects to the health of the progeny. Our results raise awareness about the impact of childhood obesity to reproductive health of future fathers and their progeny. Trial registration number 0421/000/000/2016

Effects of epididymal sperm recovery methods on fresh and frozen-thawed sperm characteristics in dogs.

The cauda epididymis holds a collectible source of fertile spermatozoa in cases of obstructive azoospermia, sudden death, and after elective or emergency castration. The current study was conducted to compare three different epidydimal sperm collection methods (Percutaneous epididymal sperm aspiration (PESA), microsurgical epididymal sperm aspiration (MESA) and retrograde epididymal wash (EW)) in the dog. Fifteen large-breed adult dogs were applied for comparing the PESA (left testicles) with MESA (right testicles) techniques, while five dogs were used for evaluation of MESA (left testicles) versus EW (right testicles). The recovered sperm cells from MESA and EW were subjected to cryopreservation. Total sperm recovery, level of blood contamination and sperm quality markers (viability, morphology, plasma and acrosome membrane integrity, DNA fragmentation, and metabolic activity) were evaluated for fresh and frozen-thawed spermatozoa. We showed that the collection of epididymal sperm cells through the PESA method resulted in lower total sperm recovery and significantly reduced fresh sperm kinematic and quality measures. While, both MESA and EW procedures resulted in a high number of intact epididymal spermatozoa with appropriate cryo-tolerance potential. In conclusion, EW and MESA methods provide high-quality epidydimal spermatozoa with high cryopreservation potential in domestic dogs.

Intra-Cytosplamic sperm injection outcomes with fresh and cryopreserved human epidydimal sperm from patients with obstructive azoospermia

Techniques for the cryopreservation of epididymal sperm was are widely used in clinical practice. However, given the unique characteristics of sperm from patients with obstructive azoospermia, epididymal sperm cryopreservation is more difficult because of low count and weak motility; therefore, conventional methods of sperm cryopreservation may not result in the best outcomes. We used the micro-straw method to store small quantities of sperm obtained from patients with severe oligozoospermia or azoospermia and achieved successful deliveries in the previous study. This retrospective study of ICSI cycles included the first ICSI cycles of fresh or frozen/thawed epididymal sperm that were performed in patients suffering from obstructive azoospermia who were admitted to the CITIC-Xiangya Hospital of Reproduction and Genetics of China from June 1, 2015 to June 31, 2019. A total of 2441 patients with obstructive azoospermia were divided according to the use of fresh (n = 2342) or frozen/thawed (n = 99) epididymal sperm. The results showed that the fertilisation rate was higher with fresh epididymal sperm than that with frozen/thawed epididymal sperm (85.14% vs. 79.26%, respectively; p = 0.000). However, the rates of embryo cleavage, high-quality embryos, clinical pregnancy, miscarriage, singletons and birth defect were similar between fresh and frozen/thawed epididymal sperm (98.28% vs. 99.13%, 60.34% vs. 57.29%, 67.90% vs. 70.51%, 8.12% vs. 10.91%, 57.76% vs. 49.09%, 1.59% vs. 1.45%respectively; p = 0.088, 0.109, 0.628, 0.462,0.203 and 0.686). In addition, the short-term cryostorage of small quantities of epididymal sperm did not affect clinical outcomes. The results indicated that in cases of obstructive azoospermia, cryostorage of small quantities epididymal sperm is a reliable option.

Cryopreservation of Spix's yellow-toothed cavy epididymal sperm using Tris- and coconut water-based extenders supplemented with egg yolk or Aloe vera

Addressing the establishment of biobanks for the conservation of wild hystricomorph rodents' germplasm, we verified the effects of different extenders and distinct concentrations of non-permeant cryoprotectants on the sperm parameters of Spix's yellow-toothed cavies. Nine testis-epididymis complexes were used for sperm collection by retrograde washing using Tris or a powdered coconut water extender (ACP®-116c). Spermatozoa were diluted and frozen with the same extenders supplemented with egg yolk or Aloe vera at a 10% or 20% concentration. After recovery and cryopreservation, all samples were evaluated for sperm kinetic parameters, morphology, membrane integrity, osmotic response, and sperm-binding capability using an egg yolk perivitelline membrane assay. After recovery, no differences were observed between Tris and ACP®-116c that provided 515.4 × 106 sperm/mL and 561.6 × 106 sperm/mL, presenting >65% motile sperm, respectively. After cryopreservation, most effective preservation of sperm kinetic parameters (68.1 ± 5.9% motile sperm) and membrane integrity (48.2 ± 7.4%) was provided by Tris extender supplemented with 10% egg yolk. However, both extenders supplemented with any concentration of egg yolk or Aloe vera presented similar preservation of osmotic response and sperm-binding ability after cryopreservation. In summary, we suggest the use of a Tris extender supplemented of 10% egg yolk for cryopreservation of Spix's yellow-toothed cavy epidydimal sperm.

Cichorium intybus L. extract ameliorates testicular oxidative stress induced by lead acetate in male rats.

ObjectiveOxidative stress has been suggested as a possible mechanism for the adverse effects of heavy metal toxicity on male reproduction. Cichorium intybus L. is used in Iranian folk medicine as a hepatoprotective agent as well as for its supposed fertility-enhancing properties. The present study was performed to investigate whether the ethanolic extract of C. intybus leaves could protect male rats against lead-induced testicular oxidative stress.MethodsIn this experimental study, adult Wistar rats were treated with 0.1% lead acetate in drinking water alone or with 50, 100, or 200 mg/ kg body weight of C. intybus extract via gavage once daily for 70 days. The weight of their reproductive organs, levels of serum hormones, histometric parameters of the seminiferous tubules, epidydimal sperm quality, and oxidative stress status were evaluated.ResultsThe testis weight, seminiferous tubule diameter, epididymal sperm count, serum testosterone level, and testicular levels of superoxide dismutase and glutathione peroxidase were significantly reduced (p<0.05) in the lead-treated rats. Moreover, significantly (p<0.05) higher levels of malondialdehyde were observed in the lead-exposed group compared to the control. However, the co-administration of C. intybus ethanolic extract in lead-treated rats was associated with a significant improvement in reproductive parameters.ConclusionWe conclude that C. intybus leaf extract has the potential to prevent lead-induced testicular toxicity and to suppress the adverse effects of lead on male reproductive health.

Vitamin D3 Nanoemulsion Ameliorates Testicular Dysfunction in High-Fat Diet-Induced Obese Rat Model

Abstract Background: Obesity is a major health problem which may lead to infertility, vitamin D has anti-inflammatory, anti-oxidant, anti-apoptotic properties and metabolic activities Nano emulsions are very effective delivery system for medicines and nutrients. Nano vitamin D has a greater water solubility and stability compared to the conventional vitamin D. Aim of Study: The study aims to evaluate the potential protective effects of vitamin D3nanoemulsionintervention against some altered testicular, metabolic and hormonal changes in high-fat diet (HFD)-induced obese rats. Material and Methods: 32 adult male albino rats were equally and randomly divided into four groups: Group I (control group on standard diet), group II (untreated HFD), group III (HFD + conventional vitamin D) and group IV (HFD + Nano vitamin D3). Vitamin D3 administration was begun along with the start of introduction of the high fat diet. After eight weeks, body weight (BW) was measured and body mass index (BMI) was calculated then rats were euthanized. Serum testosterone, insulin and glucose levels were measured; home-ostasis model assessment of insulin resistance (HOMA-IR) was calculated. In addition, testicular levels of malondialdhyde (MDA), superoxide dismutase (SOD), reduced glutathione (GSH), TNF-a and IL-6 were measured. The changes in the histopathological aspects of the testis were examined. The testicular weight, testicular coefficient, epidydimal sperm count and motility all were investigated. Results: Nano vitamin D3 treatment showed significant reductions in BMI, fasting serum glucose, insulin resistance, total cholesterol, LDL-c, with significant elevations in serum HDL-c, final testicular weight, testicular coefficient, epidy-dimal sperm count, sperm motility, and serum testosterone. Moreover, Nano vitamin D3 significantly increased the tes-ticular SOD and GSH with significant decline in the lipid peroxidation contributed to MDA, testicular TNF-a and IL-6 content. Improvement of the metabolic and biochemical measurements induced by Nano vitamin D3 is linked toim-provement of testicular histology in obese rats. Interestingly, Nano vitamin D3 has more significant protective effects against HFD-induced testicular dysfunction compared to the conventional vitamin D3.Conclusions: Nano vitamin D3 has a potential protective role in improving the adverse effects of HFD induced obesity on testicular functions, withmore prominent protective effects than conventional vitamin D3.

The therapeutic potential of amifostine on cyclophosphamide-induced testicular dysfunction in rats: An experimental study.

BackgroundCyclophosphamide (CP) is a well-known alkylating anticancer agent used in the treatment of various malignant and non-malignant tumors. CP may also cause a variety of adverse effects, including reproductive toxicity. Amifostine is known as a cytoprotective drug having antioxidant properties.ObjectiveTo evaluate the possible beneficial effects of amifostine on testicular toxicity induced by CP in rats.Materials and MethodsA total of 35 Sprague-Dawley rats were used in this experimental study. The CP group animals received a single dose of 200 mg/kg CP on Day 8 by intraperitoneal injection and were left untreated for the following seven days. The two remaining groups of animals were treated with 200 mg/kg/day amifostine (AMF 200) and 400 mg/kg/day amifostine (AMF 400) for seven days prior to and following a single intraperitoneal injection of CP. Morphometrical analysis and histological examination of testicular tissue were performed. Serum testosterone, luteinizing hormone, and follicle-stimulating hormone levels were measured in serum using commercial ELISA kits. The epidydimal sperm count was determined.ResultsThe tubular epithelial height in the testis was significantly higher in the AMF400 group compared to other groups (p 0.001). Animals in the AMF400 group showed minimal debris in the tubules, no Sertoli cell damage, and the Johnsen scores were slightly higher in the AMF400 group. The epididymal sperm count was significantly lower in the CP-administered animals compared to the control animals and was significantly higher in the AMF200 and AMF400 groups compared to the CP group (p = 0.006, and p = 0.019 respectively).ConclusionAmifostine, at a dose of 400 mg/kg, may have a protective effect on testicular damage induced by CP in rats.

Effect of Glucagon Like Peptide-1 on Serum Kisspeptin Level in Adult Male Albino Rats Treated by Anabolic Androgenic Steroid

AbstractBackground: Anabolic Androgenic Steroids (AAS) are widely used among youth and athletes for improving their physical appearance and performance. However, its use has adverse effects on sexual health and metabolism. Glucagon Like Peptide-1 (GLP-1) is a gut hormone that participates in the neuroendocrine control of hypothalamic-pituitary axis (HPG), and may play an important role in the reproductive functions as a component of GIT-brain axis. Also, the hypoth-alamic neuropeptide kisspeptin stimulates the Hypothalamic Pituitary Gonadal (HPG) axis and controls gonadotropin secretion. Few researches were performed to study the rela-tionship between each of GLP-1 and kisspeptin with HPG axis.Aim: To evaluate the potential protective role of (GLP-1) against some altered sexual, metabolic, and histopathologic changes induced by the use of AAS in adult male albino rats.Material and Methods: 40 adult male albino rats were divided into 4 groups; Group I (control or vehicle treated), Group II (GLP-1 + vehicle treated), Group III (AAS-treated) and Group IV (GLP-1 + AAS). In all groups, Body Weight (BW) was measured and Body Mass Index (BMI) was calcu-lated. Serum testosterone, Luteinizing Hormone (LH), Follicle-Stimulating Hormone (FSH), kisspeptin, glucose and insulin levels were measured, homeostasis Model Assessment of Insulin Resistance (HOMA-IR) was calculated, and lipid profile was estimated. The changes in the histopathological aspects of testis were examined. The testicular weight, testicular coefficient, epidydimal sperm count and motility were inves-tigated.Results: The study revealed that peripheral injection of GLP-1 for 5 weeks to androgen treated-rats in Group IV (GLP-1 + AAS) significantly increased levels of serum kisspep-tin, LH, FSH, testosterone and consequently epidydimal sperm count and motility compared with rats in Group III (AAS-treated). In addition, GLP-1 treatment induced significant reductions in the values of fasting serum glucose and HOMA-IR, serum total cholesterol and LDL levels with no significant changes in serum insulin, HDL and TG levels in Group IV compared with Group III.Conclusion: The administered GLP-1 may attenuate some altered sexual, metabolic and histopathological adverse effects induced by AAS use in adult male rats. Therefore, it may be a novel approach for managing AAS abuse.

Fipronil-induced decrease in the epididymal sperm count: oxidative effect and protection by vitamin E.

The toxic effects of the insecticide fipronil on the sperm production and oxidative damage in the testis were evaluated, as well as the protective action of vitamin E. Male rats received vehicle or fipronil 5 mg/kg and fipronil 5 mg/kg + vitamin E 100 mg/kg for 14 days. Thereafter, the sperm concentration in the epididymis and parameters of oxidative damage in the homogenate of testicles were assessed. Fipronil reduced epidydimal sperm count. The activity of the glutathione peroxidase enzyme increased and that of catalase was reduced in the testis. Also, a reduction in GSH and an increase in the concentration of malondialdehyde were observed in the animals treated with fipronil. The vitamin E reestablished the analysed parameters to levels similar to those of the control group. We concluded that fipronil decreased sperm production in rats because of its oxidant activity and that this effect was reversed by vitamin E.

Changes in sperm banking rates within the first seven months of a formal male oncofertility program.

e21587 Background: Oncofertility counseling is an essential part of the care for cancer patients with risk of infertility. In June 2016, our institution formally initiated a standardized program for fertility preservation, including nursing and physician education. After the first 7 months of functioning, we here examine the sperm banking rates amongst male cancer patients, and its variation respect previous years. Methods: We retrospectively reviewed the charts of men newly diagnosed with cancer at our institution. We assessed the rates of sperm banking amongst this patient population before the implementation of a standardized fertility preservation program, and again after: from January 2011 to May 2016 and from June 2016 to December 2016, respectively. Results: Sperm banking rate amongst male cancer patients before the initiation of the program was 3.3% (30 out of 902 patients). Along the 7 months after initiation of the program, rate of fertility preservation increased 6.2 folds, up to 20.6% (21 out of 102). Patients undergoing sperm banking through the fertility preservation program were aged 14 to 62 years old; mean ± standard deviation = 25.9± 6.9 years. Two of the patients were minors under care of Pediatric Oncology department. 76.7% of the samples (17 patients) were obtained by masturbation. In the other 4 patients, viable sperm could not be preserved from ejaculation, but it was successfully obtained by testicular or epidydimal sperm extraction. Conclusions: The initiation of a formal fertility preservation program at our institution quickly increased the rate of sperm banking; in more than 6 folds within its first 7 months of functioning. Oncofertility is an essential component of the care of oncologic patients in reproductive age or younger, and our results manifest the benefit of a standardized program at academic institutions. Counseling in fertility preservation should be offered to all patients at risk prior to initiation of oncologic treatment.

Evaluación de la Integridad Acrosomal en Espermatozoides Epididimarios de Alpaca mediante Citometría de Flujo

El objetivo del presente trabajo fue determinar el porcentaje de integridad acrosomal en espermatozoides epididimarios de alpaca utilizando Arachis hypogaea (PNA) y Pisum sativum (PSA), conjugadas con isoticionato de fluoresceína (FITC). Se recolectaron 45 testículos de alpaca obtenidos del Camal Municipal de Ninacaca, provincia de Pasco (Perú) y se utilizaron 29 muestras recuperadas de la cola del epidídimo con motilidad mayor de 30% y concentración mayor de 50x106 espermatozoides/ml. Los espermatozoides fueron suspendidos con 1 ml de solución Tris base, se lavaron por centrifugación a 600 g por 8 min y los pellets fueron re-suspendidos en 300 μl de PBS. Cada muestra fue dividida en dos alícuotas e incubadas por 8 min a 38 °C con FITC-PNA (0.5 μg/ml) o FITC-PSA (2.5 μg/ml), junto con un marcador de vitalidad (ioduro de propidio [PI], 0.5 μg/ml). Las muestras fueron evaluadas mediante citometría de flujo, utilizando un láser de excitación de 488 nm y canales detectores de emisión de fluorescencia de 505-560 nm (Canal 02 para FITC) y de 642-740 nm (Canal 05 para PI). Los espermatozoides que emitieron fluorescencia verde en el Canal 02 fueron considerados con daño acrosomal, mientras que los que emitieron fluorescencia roja en el Canal 05 fueron considerados muertos. Se obtuvo 59.17 ± 4.84 y 61.13 ± 4.35% de espermatozoides vivos con integridad acrosomal para FITC-PNA y FITC-PSA, respectivamente. Los resultados presentan un indicador de la integridad acrosomal en espermatozoides epididimarios de alpaca, que podría constituir una base para el estudio de la fisiología espermática en esta especie.

Spermaurin, an La1-like peptide from the venom of the scorpion Scorpio maurus palmatus, improves sperm motility and fertilization in different mammalian species.

Is it possible to identify original compounds that are able to enhance sperm motility from the venom of the scorpion Scorpio maurus palmatus? We identified a potent disulfide-rich peptide (DRP) of 73 amino acids that significantly improved the motility of fresh and frozen-thawed sperm in different mammalian species, including human, and improved fertilization outcome in mouse IVF experiments. Any disturbance of sperm motility has a strong impact on fertilization and can lead to subfertility or infertility. Significant efforts have, therefore, been made to identify pharmacological drugs that might improve sperm motility. Such compounds are particularly useful in azoospermia to improve testicular sperm extraction and in the domain of cryopreservation because the motility of frozen-thawed sperm is reduced. This was a basic science/medical research study aimed at identifying original compounds from a library of venoms able to enhance mammalian sperm motility, including human. We first identified in the venom of a scorpion S. m. palmatus a fraction able to potently activate sperm motility. We next purified and characterized the compound by liquid chromatography, mass spectrometry and peptide synthesis. Finally, the potency and toxicity of both purified and synthetic versions of the identified compound on sperm motility were assessed using different in vitro tests in different mammalian species. For human sperm, biological samples were collected from normozoospermic donors and subfertile patients attending a reproduction department for diagnostic semen analysis. Testicular sperm was collected from cynomolgus monkeys (Macaca fascicularis) euthanized for the needs of specific authorized research projects. The peptide was also tested on bovine and mouse epidydimal sperm. We measured different sperm motility parameters with a computer-assisted sperm analysis system in the presence or absence of the peptide. Size exclusion chromatography enabled us to isolate a fraction of the venom of S. m. palmatus able to increase sperm motility. By liquid chromatography and mass spectrometry, a peptide comprising 73 amino acids with 4 disulfide bridges was identified as responsible for the biological activity and called 'spermaurin'. The identity of spermaurin was confirmed by chemical synthesis. We showed that the peptide increased the motility of fresh and frozen-thawed human sperm. We observed that the potency of the peptide was higher on fresh ejaculated spermatozoa with a low motility, achieving a 100% increase of curvilinear velocity in poorly performing sperm. We also demonstrated that peptide is effective on bovine and mouse fresh epididymal, bovine frozen-thawed ejaculated and fresh non-human primate testicular sperm. Finally, in mouse IVF, the production of 2-cell embryos was increased by 24% when sperm were treated with the peptide. This work is an in vitro evaluation of the ability of spermaurin to improve sperm motility parameters. Another limitation of this study is the small number of human sperm samples tested with the natural (n = 36) and synthetic (n = 12) peptides. Moreover, the effect of the peptide on IVF outcome was only tested in mouse and further tests with human and bovine gametes are required to confirm and extend this result in other mammalian species. This work confirms our initial study showing that venoms represent an interesting source of molecules that are able to modify sperm physiology. Moreover, this work presents the first demonstrated biological action of a venom peptide from the scorpion S. m. palmatus with sequence similarities to La1 peptide from Liocheles australasiae (Wood scorpion), a widespread family of DRPs. Not applicable. This work is part of the project 'LAB COM-14 LAB7 0004 01-LIPAV', funded by the program LabCom 2014 from the French Research Agency (ANR). Dr Arnoult reports grants from IMV Technologies during the conduct of the study. In addition, Drs Arnoult, Martinez, Ray and Schmitt have a patent EP16305642.7 pending containing some of the information presented in this manuscript.

Comprehensive Assessment of a Chlorinated Drinking Water Concentrate in a Rat Multigenerational Reproductive Toxicity Study

Some epidemiological studies report associations between drinking water disinfection byproducts (DBPs) and adverse reproductive/developmental effects, e.g., low birth weight, spontaneous abortion, stillbirth, and birth defects. Using a multigenerational rat bioassay, we evaluated an environmentally relevant "whole" mixture of DBPs representative of chlorinated drinking water, including unidentified DBPs as well as realistic proportions of known DBPs at low-toxicity concentrations. Source water from a water utility was concentrated 136-fold, chlorinated, and provided as drinking water to Sprague-Dawley rats. Timed-pregnant females (P0 generation) were exposed during gestation and lactation. Weanlings (F1 generation) continued exposures and were bred to produce an F2 generation. Large sample sizes enhanced statistical power, particularly for pup weight and prenatal loss. No adverse effects were observed for pup weight, prenatal loss, pregnancy rate, gestation length, puberty onset in males, growth, estrous cycles, hormone levels, immunological end points, and most neurobehavioral end points. Significant, albeit slight, effects included delayed puberty for F1 females, reduced caput epidydimal sperm counts in F1 adult males, and increased incidences of thyroid follicular cell hypertrophy in adult females. These results highlight areas for future research, while the largely negative findings, particularly for pup weight and prenatal loss, are notable.

Assessment of imidacloprid toxicity on reproductive organ system of adult male rats

In the current study it was aimed to investigate the toxicity of low doses of imidacloprid (IMI) on the reproductive organ systems of adult male rats. The treatment groups received 0.5 (IMI-0.5), 2 (IMI-2) or 8 mg IMI/kg body weight by oral gavage (IMI-8) for three months. The deterioration in sperm motility in IMI-8 group and epidydimal sperm concentration in IMI-2 and IMI-8 groups and abnormality in sperm morphology in IMI-8 were significant. The levels of testosterone (T) and GSH decreased significantly in group IMI-8 compared to the control group. Upon treatment with IMI, apoptotic index increased significantly only in germ cells of the seminiferous tubules of IMI-8 group when compared to control. Fragmentation was striking in the seminal DNA from the IMI-8 group, but it was much less obvious in the IMI-2 one. IMI exposure resulted in elevation of all fatty acids analyzed, but the increases were significant only in stearic, oleic, linoleic and arachidonic acids. The ratios of 20:4/20:3 and 20:4/18:2 were decreased and 16:1n-9/16:0 ratio was increased. In conclusion, the present animal experiments revealed that the treatment with IMI at NOAEL dose-levels caused deterioration in sperm parameters, decreased T level, increased apoptosis of germ cells, seminal DNA fragmentation, the depletion of antioxidants and change in disturbance of fatty acid composition. All these changes indicate the suppression of testicular function.

Sexual Behaviour of Hair Rams (Ovis aries) Subjected to Frequent Collection and Free from Habituation to the Female

Andrade, O., Vázquez, R. and Orihuela, A. 2010. Sexual behaviour of hair rams (Ovis aries) subjected to frequent collection and free from habituation to the female. J. Appl. Anim. Res., 37: 121–124. An experiment was designed to measure the relationship between repeated collection, semen characteristics and reaction time (RT) as an estimator of libido while isolating the effect of habituation to the female. Fourteen rams were equally and randomly assigned to two treatments. In TO (control) an ejaculation of each ram was collected daily with an artificial vagina (AV) for 10 consecutive days. In T1, semen was collected from the rams as in T0, but starting 60 min after this collection, electroejaculation was applied three times, with a 60 min interval to reduce epidydimal sperm reserves avoiding habituation to a female stimulus. No difference (P>0.05) was found in average RT and average sperm concentration between groups, while average semen volume was larger (P<0.01) in TO (0.82±0.04 ml) than in the T1 (0.64±0.04 ml). The slope for RT in the regression analysis was not different (P>0.05) from 0 in T0 and T1. However, a statistically significant (P<0.05) linear relationship was observed in ejaculate volumes and sperm concentration in the T1 as the experiment progressed. It was concluded that under no habituation to the female, rams RT was not affected by repeated collection, whereas, ejaculate volumes and sperm concentration declined proportionally as semen collection increased.

Nigerian Bonny Light Crude Oil Disrupts Antioxidant Systems in Testes and Sperm of Rats

Nigerian Bonny light crude oil (BLCO) is commonly used by the local population in folklore medicine for the management of various forms of gastrointestinal problems and male reproductive capacity. The study investigated the effects of BLCO on the antioxidant systems of the testes and epidydimal sperm in rats by oral exposure to 0, 200, 400 and 800 mg/kg BLCO for 7 days. In testes and sperm, BLCO treatment at all doses significantly (p<0.05) decreased superoxide dismutase (SOD), catalase (CAT), and glutathione S-transferase (GST) activities, whereas it markedly increased glucose 6-phosphate dehydrogenase (G6PD) and gamma glutamyl transferase (GGT) activities as well as increased glutathione (GSH), hydrogen peroxide, and malondialdehyde (MDA) levels in all treatment groups. Although epididymal sperm number (ESN), daily spermatozoa production (DSP), and sperm motility were significantly decreased, total sperm abnormalities were significantly increased without affecting sperm viability at all dose levels compared with controls. The adverse effect of BLCO on TSN was noted at the 800 mg/kg dose only. Histopathology results showed treatment-related lesions of the testes characterized by severe congestion of interstitial vessels, decreased germinal epithelium, and increased number of vacuolization. These results suggest that exposure to BLCO, such as its use in ailment management, may promote infertility by altering the function of the testes and sperm, particularly by way of induction of oxidative stress.

Artificial oocyte activation with calcium ionophore A23187 in intracytoplasmic sperm injection cycles using surgically retrieved spermatozoa

OBJECTIVE: Recent reports have suggested that fertilization failure after intracytoplasmic sperm injection (ICSI) may be due to the inability of the spermatozoa to trigger oocyte activation which, is characterized by a rise in intracellular calcium concentration. Treatment with calcium ionophore may increase the free intracellular calcium, thereby mimicking physiological cell-signaling mechanisms resulting in oocyte activation. However, to date, there have been few studies using AOA in ICSI cycles with testicular or epididymal sperm, retrieved from patients with either obstructive (OA) or non-obstructive azoospermia (NOA). We evaluated the effect of AOA with the calcium ionophore A23187 on ICSI cycles using sperm surgically retrieved. DESIGN: Case-control study. MATERIALS AND METHODS: The study was performed on 204 couples undergoing surgical sperm retrieval for ICSI. The cycles were divided into three groups according to the origin of the sperm and the type of azoospermia: (i) testicle sperm of NOA patients, TEST-NOA (n=58) (ii) testicle sperm of OA patients, TEST-OA group (n=48), and (iii) epididymis sperm of obstructive OA, EPID-OA (n=98). For each experimental group, cycles in which AOA was applied (subgroup activation) were compared with cycles in which AOA was not applied (subgroup control). RESULTS: The subgroups were not statistically different for fertilization, high-quality embryos, implantation, or pregnancy rates when testicular sperm were retrieved from both types of azoospermia (TEST-NOA and TEST-OA groups). When epidydimal sperm were injected, we observed a significant increase in high-quality embryo rate when AOA was performed (64.8% vs 48.0%, p=0.045). However, no significant difference was observed in the other evaluated parameters. CONCLUSIONS: We have demonstrated for the first time that, when epidydimal sperm are injected, AOA is able to increase the high-quality embryo rate. The reason why AOA did not improve the outcomes of ICSI cycles using testicular sperm may be explained by the sperm maturity status. We suggest that although the increase of intracellular calcium can be induced by extra-physiologic stimulus, testicular spermatozoon is unable to maintain the calcium oscillations, which is the key event leading to fertilization and further embryonic development. In summary, our results demonstrated that AOA might be useful in improving ICSI outcomes in azoospermic patients when epididymal, but not testicular, sperm are injected, independently on the type of azoospermia.

Epididymal sperm quality may influence intracytoplasmic sperm injection (ICSI) outcomes: role of testicular sperm extraction (TESE) in men with obstructive azoospermia

Epididymal sperm quality may influence intracytoplasmic sperm injection (ICSI) outcomes: role of testicular sperm extraction (TESE) in men with obstructive azoospermia