A comparison of the biological properties of European red deer (Cervus elaphus elaphus) spermatozoa stored in the epididymides and in a liquid state at 5 °C.

The biological properties of European red deer (Cervus elaphus elaphus) spermatozoa stored in the epididymides and in a liquid state were compared. Spermatozoa were collected from the epididymides harvested post-mortem. In the first variant, spermatozoa were diluted in two extenders (Bovidyl® and Salomon's), and were stored at 5 °C for up to 144 h. In the second variant, spermatozoa were stored in the epididymides at 5 °C for up to 144 h, and then diluted in the same extenders. Biological properties were evaluated after 0, 48, 96, and 144 h of storage. Sperm motility parameters were determined in the CASA system. Plasma and acrosomal membrane integrity, mitochondrial activity, apoptotic changes, and DNA integrity were assessed by the fluorescence method. Most variables were significantly influenced by the storage method and time. At 144 h, differences (P ≤ 0.05) in sperm parameters were observed between storage variants. Total motility (TMOT), plasma membrane integrity, and mitochondrial activity decreased below 50% of baseline values in the spermatozoa stored in the epididymides, but remained above 70% of baseline values in the spermatozoa stored in a liquid state. The compared storage variants did not differ in TMOT, mitochondrial activity, or the percentage of viable spermatozoa without apoptotic-like changes up to 96 h of storage, regardless of the applied extender. The earliest significant changes were noted in sperm motility parameters. In conclusion, European red deer spermatozoa can be stored in the epididymides at 5 °C for up to 96 h, but their biological parameters are more effectively preserved during liquid storage.

Nerve and Glial Cell Expressions in the Testes and Epididymides of Different Age Groups of Cane Rat (Thryonomys swinderianus).

PURPOSE: This study was conducted to examine the variations in the expressions of neuronal and glial cell markers in the testes and epididymides of different age groups of cane rat using histochemical and immunohistochemical techniques. METHOD: Thirty (32) healthy domesticated male cane rats were used for this investigation. The rats were divided into four groups (prepubertal [≤4 months], pubertal (>4 ≤12 months), adult (>12 ≤30 months), and aged (>30 months)] of 8 animals each. Subsequent to anesthesia and intracardiac perfusion of the rats with 10% buffered formalin, testes were harvested and preliminary assessment of nervous and glial structures was determined using the Golgi technique. Specific immunolocalization was done using the anti-neurofilament (NF-20) and anti-glial fibrillary acid protein (GFAP) for the expressions of neuronal and astrocyte-like cells, respectively. RESULT: Neuronal and astrocyte-like structures as revealed by the Golgi procedure were demonstrated in the tunica albuginea and interstitium of the testes as well as in the periductal muscle coat and epididymal interstitium of the caput down to the caudal segments. Golgi signal intensities of the expressions in both testes and epididymides increased with age advancement. Immunolocalization of the nerve structures and glial cells tallied with the Golgi results. However, NF signal intensity was significantly higher in the adult relative to others. Similarly, GFAP signal intensity increased with age increment. CONCLUSION: This study has shown that the variation in the expression of neuronal and glial cells in the testis and epididymis of the cane rat could be associated with increased reproductive reproductive activity.

Donkey Epididymal Transport for Semen Cooling and Freezing.

The objectives of this study were to assess the cooling and freezing of donkey epididymal semen harvested immediately after castration (Experiment 1, n = 4) or after the shipment (24 or 48 h) of epididymides attached to testicles (Experiment 2, n = 14) or dissected apart (Experiment 3, n = 36). In each experiment, semen was frozen immediately (Non-Centrif) in an egg yolk-based semen extender (EY) or after processing through cushion-centrifugation (Centrif) while extended in a skim milk-based extender (SC). In all three experiments, cooled, pre-freeze, and post-thaw epididymal semen was assessed for total motility (TM), progressive motility (PM), plasma membrane integrity (PMI), and high mitochondrial membrane potential (HMMP). Data were analyzed with R using mixed models and Tukey's test as posthoc. Results showed that the cooling of epididymal semen up to 24 h after harvesting did not affect motility parameters or plasma membrane integrity; furthermore, in Experiment 3, the post-thaw evaluation of both Centrif and Non-Centrif achieved similar TM and PM. Collectively, the post-thaw results revealed low motility parameters across groups; while, the PMI and HMMP did not reflect this trend, and the values remained high, suggesting that there was a lack of epididymal sperm activation with either centrifugation or extenders. In summary, freshly harvested and cooled-shipped and cooled semen had satisfactory semen parameters. Future studies need to address donkey epididymal semen fertility in mares and jennies.

Retrograde flushing collection and freezing of dromedary camel epididymal spermatozoa with seminal plasma.

The objectives of this study were to describe the parameters of dromedary camel epididymal spermatozoa collected by retrograde flushing (RF) technique and to evaluate the freezability of the collected sperm, diluted with and without the supplementation of seminal plasma (SP). Two experiments were conducted: in Experiment 1, ES were recovered within 6-8 h after castration; selected samples were diluted with a Tris-citrate egg-yolk glycerolated buffer and frozen. In Experiment 2, epididymides were stored for 24 h at 4 °C before RF and semen samples were frozen after dilution with a Tris-lactose egg-yolk glycerolated extender with and without 15% SP. In Experiment 1, eight semen samples were obtained from ten epididymides with a mean of 500 × 106 total spermatozoa recovered, per flushed epididymis. Mean post-thaw motility and progressive motility were 75 and 17%, respectively. In Experiment 2, 15 samples were collected, out of the 18 epididymides (mean number of collected spermatozoa: 700 × 106), and 13 of these samples were of excellent quality. Post-thaw parameters were not satisfactory but the supplementation of the freezing medium with 15% SP improved the progressive motility and kinematic parameters of the spermatozoa.

Zinc deficient diet increases the toxicity of bisphenol A in rat testis.

Zinc (Zn) plays an important role in maintaining the process of spermatogenesis and reproductive health. Bisphenol A (BPA), an endocrine disrupting chemical is known to be a reproductive toxicant in different animal models. The present study was designed to study the effect of two of the utmost determinative factors (Zn deficient condition and influence of toxicant BPA) on germ cell growth and overall male reproductive health in the testis, epididymis, and sperm using (a) biochemical, (b) antioxidant, (c) cellular damage, (d) apoptosis, and (e) protein expression measurements. Rats were divided into Control (normal feed and water), BPA (100 mg/kg/d), zinc deficient diet (ZDD; fed with ZDD), and BPA + ZDD for 8 weeks. Body and organ weights, sperm motility and counts, and sperm head morphology were evaluated. The histology of testes, epididymides, and prostate was investigated. Testicular deoxyribonucleic acid (DNA) damage was evaluated by Halo and Comet assay, apoptosis of sperm and testes were quantified by TUNEL assay. Serum protein electrophoretic patterns and testicular protein expressions such as Nrf-2, catalase, PCNA, and Keap1 were analyzed by Western blot analysis. The results showed that BPA significantly increased the testicular, epididymal, and prostrate toxicity in dietary Zn deficient condition due to testicular hypozincemia, hypogonadism, increased cellular and DNA damage, apoptosis, as well as perturbations in protein expression.

Prss55 but not Prss51 is required for male fertility in mice†.

Mammalian spermatozoa are produced in the testis through spermatogenesis and matured in the epididymis to acquire fertilizing ability. Spermatozoa are ejaculated and migrate from the uterus to the oviducts to fuse with oocytes. Although over 2000 genes are expressed abundantly in mouse testes, the genes responsible for male fertility are not yet fully clarified. Here, we focused on two testis-enriched serine protease genes, Serine protease (Prss) 51 and Prss55, which overlap their gene loci partially in both mice and humans. To characterize their functions in male fertility, we first generated Prss51 and Prss55 double knockout (DKO) mice by CRISPR/Cas9 system and found that the DKO mice were sterile. DKO spermatozoa exhibit impaired migration from the uterus to the oviduct and impaired ability to bind the zona pellucida (ZP) of oocytes. Moreover, a sperm membrane protein, ADAM3 (a disintegrin and metalloprotease 3), which plays a role in sperm migration through uterotubal junction (UTJ) and sperm-ZP binding, disappeared in the DKO spermatozoa from the epididymis. We next generated single knockout (KO) mice lacking Prss51 and found that Prss51 KO mice are fertile. We also generated single KO mice lacking Prss55 and found that Prss55 KO mice phenocopy the DKO mice, demonstrating impaired sperm migration and sperm-ZP binding and a severe defect in fertility. We conclude that Prss55, but not Prss51, is required for male fertility in mice, by stabilizing ADAM3 protein for efficient sperm-UTJ migration and sperm-ZP binding. Our findings have implications for understanding additional genetic causes of the idiopathic male infertility and for the development of male or female contraceptives.

Effect of storage time on the quality of cauda epididymal spermatozoa of West African dwarf (WAD) rams.

The objectives of the study were to develop a protocol for preserving Western African Dwarf (WAD) ram cauda epididymal semen that could be applied in preserving and storing the semen of endangered species/genetically valuable animals in case of death and to study the morphometric characteristic of epididymides after refrigerated storage. Thirty testes-epididymides were collected immediately after slaughter from mature WAD rams and transported in ice chest at (4.5-6 °C) to the laboratory. The samples were either processed immediately or stored in a refrigerator at 5 °C for 6, 12, 24, 48 h. The results indicate that the semen motility decreased (P < 0.05) when compared to the control [87.5 ± 2.1% (0 h), to 85.0 ± 1.8% (6 h), 73.3 ± 3.6% (12 h), 53.3 ± 2.5% (24 h) and 50.0 ± 2.9% (48 h)]. The sperm concentration also decreased (P < 0.05) as duration of storage increased (0 h) 157.5 ± 8.2, (6 h) 152.3 ± 5.8, (12 h) 125.3 ± 4.4, (24 h) 106.2 ± 2.9, (48 h) 98.5 ± 3.5. Semen viability decreased as duration of storage increased from the 0 h to 48 h [(P < 0.05; 84.0 ± 1.4%, 82.8 ± 2.2%, 77.3 ± 1.7%, 69.8 ± 1.5%, and 66.5 ± 1.2%, respectively]. Furthermore, there was a decrease (P < 0.05) in percentage of intact acrosome as duration of storage increased [(0 h) 90.7 ± 1.0%; (6 h) 89.3 ± 2.0%; (12 h) 85.5 ± 1.6%; (24 h) 70.0 ± 2.4%; and (48 h) 73.3 ± 2.1%]. The results from this study indicate that epididymal semen of WAD rams recovered and preserved at 5 °C for 48 h may be used for artificial insemination.

Evaluation of epididymis storage temperature and cryopreservation conditions for improved mitochondrial membrane potential, membrane integrity, sperm motility and in vitro fertilization in bovine epididymal sperm.

The maintaining of the epididymis at lower temperatures during storage and transport improves sperm quality. Our study aimed to test whether epididymis storage temperature (post-mortem) and sperm cryopreservation affect sperm kinetics, membrane integrity, mitochondrial potential and fertility capacity. Thirty-six epididymides were collected from 18 bulls after slaughter and divided into two groups: at 4 or 34°C for 2-3 hr. The sperm was collected from the epididymis cauda. The evaluation consisted of computer-assisted sperm analysis (CASA), SYBR14/PI/JC1 to evaluate membrane integrity, mitochondrial membrane potential (MMP) and measurement of lipid peroxidation (TBARS). The sperm was then frozen using an automatic device. After thawing, sperm samples were evaluated by the same variables and further in vitro fertilization rates. Cryopreservation negatively affected sperm motility in samples stored at 4 and 34°C. Nevertheless, the 4°C samples yielded higher rates of blastocyst formation. Pre-freeze sperm motility, progressive motility and velocity were higher in sperm from epididymis stored at 4°C while post-thaw sperm motility, progressive motility and velocity remained the same among samples from epididymis stored at 4 or 34°C. However, with regard to the kinetic patterns, samples collected from epididymis stored at 34°C had lower values when compared to those stored at 4°C prior the cryopreservation process. Our results indicate that epididymis handling conditions after cryopreservation may affect sperm quality after thawing, especially due to compromised MMP in sperm collected from epididymis stored at higher temperatures.

Resveratrol prevents oxidative damage and loss of sperm motility induced by long-term treatment with valproic acid in Wistar rats.

Valproic acid (VPA) is a drug widely use for the treatment of epilepsy in both children and adults. Evidence suggests that long-term use of VPA may lead to an impairment in the male reproductive function. Oxidative stress is considered to play a major role in VPA associated toxicity. In the present work, we demonstrated that the natural antioxidant compound resveratrol (RSV) can be use to prevent VPA oxidative damage. Wistar rats treated with VPA (400mgkg(-1)) by gavage for 28days showed decrease in sperm motility accompanied by increase in oxidative damage to lipids and proteins. Additionally, VPA administration leaded to depletion of reduced glutathione and decrease in total antioxidant potential in testes and epididymides of Wistar rats. The co-administration of RSV (10mgkg(-1)) efficiently prevented VPA pro-oxidant effects. In summary, RSV was shown to protect the reproductive system from the damage induced by VPA. Altogether, our data strongly suggests that RSV administration might be a valuable strategy to minimize reproductive impairment in patients requiring long-term VPA treatment.

Evaluation of sperm recovered after slaughter from cauda epididymides of red Sokoto bucks.

AIM: Viable spermatozoa could be recovered from the cauda epididymides for the purpose of preservation of genetic material of male animals with desirable traits and for use in reproductive biotechnology. The aim of this study was to determine the effect of storage time on testicular and epididymal biometry, sperm reserves and epididymal sperm characteristics of red Sokoto bucks post mortem. MATERIALS AND METHODS: Testes-epididymides were collected immediately after slaughter of mature red Sokoto bucks and transported in ice chest to the laboratory. The samples were either processed immediately or stored at 5°C in refrigerator for 24, 48 h and then processed. The testes and epididymides were measured and weighed. Sperm motility, concentration, livability, morphology, intact acrosome, and sperm reserves from different treatment groups including control were evaluated and means (±standard error of mean) were recorded. RESULTS: There was no significant difference (p>0.05) in the testicular and epididymal dimensions determined between the means of the groups. Percent sperm motility and viability decreased significantly (p<0.05) after 24 h from 69.00±0.46 and 71.27±0.50% to 50.60±0.48 and 60.47±0.70% at 48 h, respectively. Significant decreases (p<0.05) in epididymal sperm concentration and intact acrosome from 2.86±0.08 and 92.87±0.39 at 0 to 24 h of storage, respectively, were observed. CONCLUSION: The results of this study suggest that spermatozoa recovered from the epididymides of red Sokoto bucks were viable after storage for up to 48 h. Furthermore, this finding offers some hope that epididymal sperm recovered post-mortem can be used in assisted reproductive technologies.

Sperm of Galeorhinus galeus (Elasmobranchii, Triakidae) Traverse an Excurrent Duct System Characterized by Pronounced Regionalization: A Scanning Electron and Light Microscopy Study.

The transport and subsequent maturation of spermatozoa in the vertebrate excurrent duct require the creation of a series of biochemically defined luminal milieus along the length of the duct. Such specialization is accomplished, among others, by changes in the epididymal histoarchitecture. Here we show that the intratesticular and extratesticular genital ducts of mating Galeorhinus galeus exhibit pronounced regionalization both in terms of epithelial histology and lumen diameter size. Findings also reveal distinct differences in the manner in which the spermatozoa were found in each segment of the duct. Novel scanning electron microscopy evidence is presented showing that the wide lumen ductuli epididymides, which ultimately convey the spermatozoa to the proximal epididymis, show functional specialization as well. The wall of the former consisted of cuboidal ciliated and nonciliated cells whose spatial arrangement in the duct wall resulted in a luminal surface showing lengthy rows of cilia-free areas, with each row bordered on both sides by a single row of cilia. The proximal epididymis comprised several subregions whose epithelial histology varied widely. The distal epididymis and ampulla of the epididymis possessed many fingerlike projections and transverse septa, respectively. As the main storage site for spermatozoa, the ampulla completed the bundling of spermatozoa into spermatozeugmata. These were circular sperm masses in which the heads of the spermatozoa were aligned side by side and embedded in a seminal matrix, while their tails extended outward. These findings of pronounced regionalization differ greatly from the rather uniform epididymal histology seen in some rays.

Characteristics of spermatozoa and reproductive organs in relation to age and body weight in Swedish moose (Alces alces).

Knowledge of the reproductive biology of game species is vital for sustainable management. In moose (Alces alces), research in reproductive characteristics has focused on the female, whereas there are few studies in male moose. The aim of the present study was to investigate sperm morphology and chromatin integrity (SCSA), and their relationships with testicular and epididymal features, as well as temporal aspects with respect to the hunting season. In total, 143 male moose aged 1.5-11.5 years were sampled from 2008 to 2011. The proportion of normal spermatozoa (PNS) ranged from 1.5% to 82.0%, with a mean of 51%, and the %DFI (DNA fragmentation index) ranged from 2.5% to 36.7% (mean 9.5). PNS decreased temporally, and was positively associated with carcass and testes weight. Body weight and testes weight had positive effect on PNS regardless of age. No effect of any explanatory variables was observed on the DFI. The testis/body weight ratio of moose (0.033%) is among the lowest reported among mammals, indicating a less polygynous mating system than in roe deer and red deer. For reproduction success in moose, a high body weight in males is favorable, as is a balanced sex ratio. Thus, males should not be harvested prior to the time when the majority of females have passed their first oestrus of the season.

Investigations of motility and fertilization potential in thawed cryopreserved mouse sperm from cold-stored epididymides.

Cold transport of epididymides from genetically modified mice is an efficient alternative to the shipment of live animals between research facilities. Mouse sperm from epididymides cold-stored for short periods can maintain viability. We previously reported that cold storage of mouse epididymides in Lifor® perfusion medium prolonged sperm motility and fertilization potential and that the sperm efficiently fertilized oocytes when reduced glutathione was added to the fertilization medium. Cryopreservation usually results in decreased sperm viability; an optimized protocol for cold storage of epididymides plus sperm cryopreservation has yet to be established. Here, we examined the motility and fertilization potential of cryopreserved, thawed (frozen-thawed) sperm from previously cold-stored mouse epididymides. We also examined the protective effect of sphingosine-1-phosphate (S1P) on sperm viability when S1P was added to the preservation medium during cold storage. We assessed viability of frozen-thawed sperm from mouse epididymides that had been cold-transported domestically or internationally and investigated whether embryos fertilized in vitro with these sperm developed normally when implanted in pseudo-pregnant mice. Our results indicate that frozen-thawed sperm from epididymides cold-stored for up to 48 h maintained high fertilization potential. Fertilization potential was reduced after cold storage for 72 h, but not if S1P was included in the cold storage medium. Live pups were born normally to recipients after in vitro fertilization using frozen-thawed sperm from cold-transported epididymides. In summary, we demonstrate an improved protocol for cold-storage of epididymides that can facilitate transport of genetically engineered-mice and preserve sperm viability after cryopreservation.

Transporting mouse embryos and germplasm as frozen or unfrozen materials.

The 21st century has seen a huge proliferation in the availability of genetically altered mice. The availability of these resources has been accompanied by ever greater opportunities for international collaborations between laboratories involving the exchange of mouse strains. This exchange can involve significant costs in terms of animal welfare and transportation expenses. In an attempt to mitigate some of these costs, the mouse community has developed a battery of techniques that can be used to avoid transporting live mice. Transporting frozen embryos and sperm at liquid nitrogen (LN2 ) temperatures using dry shippers has been common practice for some time. However, current advances in this field have refined transportation procedures and introduced new techniques for disseminating embryos and sperm: for example, shipping frozen sperm on dry ice, exchanging unfrozen epididymides from which sperm can be extracted, and transporting frozen/thawed embryos in isotonic media. This article discusses some of the current practices used by laboratories to transport mouse strains around the world without having to exchange live mice.

Contemporary techniques for freezing mouse spermatozoa.

Each year, thousands of new mouse models are generated around the world to further biomedical research. Unfortunately, the cost of maintaining mouse colonies makes it uneconomical to keep strains on the shelf that are not part of active research programs. Ideally, these retired strains should be archived. If this is not done and the line is simply killed off, the genetics are lost to future generations of scientists. Traditionally, embryo freezing has been used to cryopreserve mice, but this is expensive, time consuming, requires large numbers of donor females, and usually involves invasive superovulation procedures. Sperm freezing circumvents all of these disadvantages and is rapidly becoming the technique of choice for many repositories. This has been made possible through the use of refined cryoprotective agents and the development of improved in vitro fertilization techniques. This article describes two popular sperm freezing techniques employed by mouse repositories to archive spermatozoa using cryoprotective agents supplemented with either L-glutamine or monothioglycerol.

Effects of Dibutyl Phthalate on Rat Sperm Production and Quality / 中国药师

Objective:To evaluate the effects of dibutyl phthalate on rat sperm production and quality. Methods:Totally 150 male rats were randomly divided into the low dose group (50 mg·kg-1), the middle dose group (200 mg·kg-1), the high dose group (1 000 mg·kg-1 ) , the blank control group and the solvent control group ( peanut oil as the control) with 30 ones in each. After continu-ous administration for every 30 days, 10 rats from each group were anatomized, the weight of testes and epididymides were determined, and one side of epididymis was used to carry out the sperm analysis including counting, survival rate and morphology. Results:After intragastric administration for 90 days, the sperm count and survival rate, the weight of testis and epididymis and organ coefficient in the middle dose group and high dose group were decreased significantly(P<0. 05 or 0. 01). Conclusion:The long-term administration of dibutyl phthalate at high dose exhibits notable toxicity on rat reproductive function.

The epididymal sperm viability, motility and DNA integrity in dead mice maintained at 4-6oC.

BACKGROUND: When male animals die, spermatozoa within the body of animal will be degenerated. Because of unique chromatin structure of sperm, maybe this degeneration is different from other cells. However there is not any research which considered directly the integrity of sperm DNA by keeping the cadaver in refrigerator. OBJECTIVE: The aim of this study was to assess viability, total motility and DNA integrity of sperm cells after death. MATERIALS AND METHODS: In this experimental study, 24 male Swiss white mice were killed by cervical dislocation and then kept in refrigerator (4-6(o)C) for up to 12 days. On the 0 (immediately after death as control group), 1(st), 2(nd), 3(rd), 5(th), 7(th), 10(th) and the 12(th) days after death cauda epididymides were removed and squeezed in Ham's F10 medium. The proportion of viable, motile and double stranded DNA spermatozoa was examined. Viability and DNA integrity of sperm cells were examined consecutively by eosin nigrosin and acridine orange stainings. RESULTS: The data obtained from this study showed that viability and total motility of sperm cells were significantly decreased during 12 days after death (p<0.001). In contrast with viability and motility, DNA integrity was without significant changes (even 12 days after death). CONCLUSION: This study suggests that integrity of sperm DNA would not change even after 12 days after death if the cadaver kept in refrigerator.

Effect of Triptolide on Epididymal Function and Sperm Motility Parameters in Male Rats / 环境与健康杂志

0.05).Compared with control group,contents of sialic acid in the high and moderate dose groups,epididymal coefficient and contents of carnitine in the high dose group reduced significantly(P