Epididymal Sperm Samples Research Articles

Horn size is linked to Sertoli cell efficiency and sperm size homogeneity during sexual development in common eland (Taurotragus oryx).

Background: In polygynous species, the development of secondary sexual characters is usually decisive for male reproductive success. However, our understanding about the links between the growth of these traits and reproductive efficiency is still elusive. Most research efforts in this topic have been also focused on adult males, although the development of some secondary sexual characters, like bovid horns, typically starts after birth, continues during the puberty and in some species, such as the common eland, slows or even stops during adulthood. In this study, we investigated the relationships between horn size and testicular function during sexual development in common elands using a comprehensive approach that considers both spermatogenic and sperm parameters. Methods: Twenty-two non-sexually mature common elands were used for the present study. Horn size, body mass, testes mass, and gonadosomatic index were assessed. Spermatogenic activity was determined by cytological and histological analyses. Sperm concentration, morphology, morphometry, and intramale variation in sperm size were evaluated on epididymal sperm samples. Cluster analysis was performed to explore the influence of age on relationships between horn size and reproductive function. Results: We found that bigger horns are associated with increased Sertoli cell efficiency and reduced intramale variation in sperm size. Both parameters were not related to one another while they have shown to be associated with enhanced sperm quality in ungulates. Moreover, horn size was positively linked to the testis mass, sperm concentration, and testicular investment in the seminiferous epithelium. Spiral length and basal circumference were the horn traits most strongly correlated with spermatogenic and sperm parameters as well as those responsible for the sexual dimorphism in this species. Cluster analysis rendered two groups: the first one including males ≤30months old, while the second one those >30months old. Horn development and reproductive function were still correlated within age groups, with the strongest relationship found between horn size and sperm size homogeneity in males >30months old. Conclusion: Taken together, our results indicate that horn size can be regarded as a good index of male reproductive potential during sexual development and provide insights into the role of secondary sexual characters in sexual selection dynamics.

Supplementation of extender with chamuangone-enriched Garcinia cowa leaf extract improves quality parameters and longevity of cold-stored cat semen

BACKGROUND: Semen preservation by cooling is less expensive, simpler and results in less sperm damage than freezing does. However, spermatozoa can only be preserved for a short period due to the excessive formation of reactive oxygen species (ROS). Although several antioxidants can protect sperms from ROS damage during storage at low temperatures, the use of natural antioxidants derived from plants would be a better alternative. OBJECTIVE: To assess the effects of chamuangone, which can reduce oxidation reactions in cells, on cat semen quality after preservation at 4°C for 15 days. MATERIALS AND METHODS: Epididymal sperm samples were collected before being diluted with tris-citric-fructose-egg yolk (TCFE) extender containing different concentrations of chamuangone (0, 50, 100, 150 and 200 μg/mL) and preserved at 4°C. Semen samples were evaluated before chilling and then every 3 days after chilling for up to 15 days. Each sample was assessed for sperm motility, viability, DNA integrity, plasma membrane integrity and percentage of spermatozoa with intact acrosomes. RESULTS: A significantly higher sperm motility was observed in the group supplemented with 100 μg/mL chamuangone compared to the control after 6 days of storage. However, the chamuangone concentration at 200 μg/mL did not significantly increase the sperm motility when compared to the control for the entire storage period. CONCLUSION: 100 μg/mL chamuangone can improve sperm characteristics during 15 days of preservation at 4°C, keeping sperm alive (49.3 ± 5.2%) and moving (7.1 ± 2.4%). These results can be used for the development of breeding programs using technologically advanced reproductive procedures in domestic and wild cats.

Sperm storage of wild cervid species: comparison of available cryoprotectants

We compared four diluents (Andromed, dimethylacetamide, dimethyl sulphoxide, Triladyl) used for semen cryopreservation in wild ungulates from the family Cervidae, i.e. roe deer (Capreolus capreolus), red deer (Cervus elaphus), and fallow deer (Dama dama). Epididymal sperm samples were collected and analysed immediately using Computer Assisted Sperm Analysis with the module for concentration and motility determination and, after equilibration, frozen and placed in liquid nitrogen for one month. After thawing, the samples were again subjected to the same examination procedure. Influence of cryopreservation and choice of the cryoprotectant was assessed by monitoring sperm concentration, average head area (HA), average velocity and progressivity (VAP), beat frequency (BF), total motility (MO), total progressive motility (PR), circular tracks (CT) and mucous penetration (MP). We observed no significant differences in sperm concentration between cryoprotective diluents or fresh and post-thaw samples in all species. All motility indicators (MO, PR, VAP, BF, CT) were influenced by the treatment but did not differ significantly between diluents used in red and roe deer. In fallow deer, commercial diluents (Andromed, Triladyl) resulted in better sperm survival than the alternatives (dimethyl sulphoxide, dimethylacetamide). Only HA showed significant differences (P < 0.001) in all species based on the diluent, with no effect of treatment. In contrast, MP was influenced by both the diluent and the cryopreservation process in roe deer and, partly, fallow deer. In future studies, we suggest expanding both the members of the Cervidae family examined and the sample size. Knowledge how to optimise cryopreservation protocols for different mammalian species has implications for conservation reproductive medicine of endangered wildlife.

Epididymal stallion sperm quality improves with single layer centrifugation prior to cryopreservation or post-thawing

Cryopreservation of epididymal spermatozoa is the last method for preserving gametes of valuable stallions in case of sudden death, severe injuries, or orchiectomy. Due to harvesting methods, sperm is unavoidably contaminated with epithelial and red blood cells, leukocytes, debris, and bacteria. These contaminants and dead spermatozoa can cause detrimental effects on sperm quality. Colloid centrifugation can reduce the load of these contaminants and select a subpopulation of motile spermatozoa. The objective of this experiment was to evaluate whether single layer centrifugation (SLC) prior to cryopreservation or post-thawing improves the quality of stallion epididymal sperm. Ten stallions were submitted to bilateral orchiectomy and harvesting of epididymal cauda spermatozoa (n=20) was performed by retrograde flushing with an extender. Epididymal sperm samples were subjected to conventional centrifugation (no colloid - 600xg for 20 minutes), and SLC prior to cryopreservation (SLC-PC) or SLC post-thaw (SLC+). SLC was performed at 300 x g for 10 minutes (Androcoll-Equine, Minitube, Tiefenbach, Germany). All samples were cryopreserved, thawed, and evaluated. The post-thaw group (SLC+) was thawed, centrifuged by SLC, and resuspended in freezing extender (SLC+F) or cooling extender (SLC+C). Total motility and progressive motility were evaluated with computer-assisted semen analyses (CASA). Sperm morphology was evaluated by examining a wetmount preparation of unstained samples fixed in formol saline. Mitochondrial functionality, membrane integrity, and DNA integrity were evaluated in a fluorescence microscope with specific fluorescent dyes. All variables were normally distributed according to the Shapiro–Wilk test. Data were evaluated by descriptive statistics, one-way analysis of variance, and Tukey's test. Significance was set at p <0.05. SLC-PC (24.6 ± 3.6%) and SLC+F (26.2 ± 4%) treatments yielded significantly higher total sperm motility than SLC+C (16.7 ± 1.7%) and conventional (16 ± 3.5%) treatment. SLC+F (17.5 ± 3.8%) yielded significantly higher progressive motility than the conventional (8.8 ± 2%), SLC+C (9.8 ± 1.3%), and SLC-PC (12.9 ± 4.1%) treatments. The mitochondrial functionality of all SLC groups was significantly higher (SLC-PC 72 ± 2%; SLC+C 74.6 ± 1.8%; SLC+F 82.5 ± 1.9%), than that of the conventional group (25.2 ± 2.1%). Plasma membrane integrity was higher in SLC-PC (48.6 ± 4%) than in SLC+F (30 ± 6.5%), SLC+C (36 ± 4.8%), and conventional (22.8 ± 4.5%) samples. DNA integrity and percentage of morphologically normal samples were unaffected by treatment. SLC is a simple procedure that can be performed prior to cryopreservation or post-thawing and that can improve stallion epididymal sperm quality. Epididymal spermatozoa submitted to SLC only after thawing is best extended using freezing extender than cooling extender.

P–121 Selection and separation of X- and Y- chromosome-bearing mammalian sperm using SPERMAN technology

Abstract Study question How can we select and separate X and Y-chromosome bearing sperm cells without causing cellular damage and reduced fertility? Summary answer AI powered SPERMAN technology can sort X and Y-chromosome bearing sperm populations without using harmful fluorescent dyes and lasers. What is known already Most common and reliable method to sex sorting (separation of X and Y- chromosome bearing sperm cells) is by using fluorescence activated cell sorting ( FACS) which takes advantage of the difference of amount of DNA in X and Y-chromosomes. Unfortunately this method causes reduced fertility and cellular damages due to the lasers and fluorescent dyes that are used. There are new and experimental developments in sex-sorting such as using immunological approaches to separate sperm cells. Current sorting method damages sperm cells. However, there is no automated and easy to use sperm sorting technology available. Study design, size, duration In this study, we have compared the quality parameters (motility, viability, AR status, DNA packaging) of sorted sperm cells to unsorted sperm cells. We have used epididymal sperm samples from 10 C57BL/6 mice (Charles River) and frozen semen samples from 6 Holstein bulls (Sexing Technologies) and frozen semen samples from 8 human sperm donors (Fairfax Cryobank). Samples were divided into two groups: sex-sorted and unsorted sperm. Participants/materials, setting, methods Sperm samples from 8-week-old C57BL/6 mice were collected from epididymal region, frozen bull semen samples and human semen samples were thawed at 37ºC then separated into two groups as sorted and unsorted sperm. Sorted samples in modified SP-TALP medium were loaded on the microfluidic chip of Sperman device, after the 30min long sorting process ended, all samples were centrifuged gently at 100g for 5 min, quality parameters were measured by using CASA and FACS. Main results and the role of chance Sperman device sorted X-bearing sperm cells at 81% purity and Y-bearing cells at 73% in mouse samples. Sperman device was able to sort X-bearing sperm cells at 78% purity and Y-bearing cells at 70% in bull samples.S perman device sorted X-bearing sperm cells at 85% purity and Y-bearing cells at 76% in human samples. Our study shows that in mouse, bull and human sperm samples, sperm DNA quality, sperm concentration, progressive motility and AR status results from the sorting with SPERMAN device are comparable with the unsorted samples. For sperm DNA quality, both the Spearman rank correlation coefficient and concordance correlation coefficient are above 0.97, indicating a high agreement between the unsorted samples and SPERMAN sorted samples. Limitations, reasons for caution In this study we only compared sperm quality parameters between unsorted sperm cells and Sperman-sorted sperm cells. Ideally a follow up study would be to look in detail at fertilization success rates and embryo growth. Also, genomic and proteomic profiles of unsorted and sorted sperm cells could be different. Wider implications of the findings: In livestock, sperm sex sorting has high value in terms of economic impact on the livestock management. Unfortunately, there is one expensive single technology dominates the sex-sorting market causing high fees poor outcomes. We believe that Sperman technology provides farmers more cheaper and better sex-sorting technology. Trial registration number Not applicable

Effect of Toxoplasma gondii on Ram Sperm Quality After Experimental Infection.

The aim of this study was to investigate the effect of experimental Toxoplasma gondii infection on ram sperm quality. Five months old, pre-pubertal, rams were divided into four groups (n = 8 per group). Group A was the control group; the remaining animals received per os (p.o.) 5000 oocysts per ram. Group B did not receive treatment post-infection (p.i.). Group C received sulphadimidine (intermuscular injection (i.m.) 33 mg/kg for eight days; every 48 h) two months p.i. and Group D received the same drug twice (24 h p.i. and two months later). Blood samples were collected every 15 days to detect serum immunoglobulin G (IgG). Epididymal sperm samples were analyzed for concentration, kinetics, morphology/viability, functional membrane integrity, DNA integrity, and the presence of parasite DNA. Histopathological examination was performed on the testes. The IgG titres in infected groups raised two weeks p.i. and remained high for four months. Higher values were noticed in viability and functional membrane integrity in positive spermatozoa in the control group compared to other groups, level of significance p < 0.05. Abnormal sperm was higher in groups C and D vs. A and C vs. B (p < 0.05). T. gondii DNA was detected in three sperm samples of the infected rams (12.5%). Histopathology revealed similar findings with little variation among all infected groups, characterized mostly by increased interstitial connective tissue, non-purulent inflammation, and presence of seminiferous tubules with spermatogenic cell depletion, which increased gradually from D to C and B groups. In conclusion Toxoplasmosis in pre-pubertal age negatively affected mature ram sperm quality, while sulphadimidine administration failed to alter this.

Characteristics of bull sperm acrosome associated 1 proteins

An atypical distribution of sperm acrosomal tyrosine-phosphorylated proteins [which include sperm acrosome associated 1 (SPACA1) proteins] may be related to the relatively lesser pregnancy rates when semen of some bulls are used for artificial insemination (AI). There may also be these associations with bull SPACA1 proteins that are translocated from the equatorial segment to the anterior part in the acrosomes during sperm maturation in the normally functioning epididymis. The aim of the present study, therefore, was assessment of the characteristics of bull SPACA1 proteins. Results from immunocytochemical evaluations indicate there were large variations in sperm percentages with typically distributed SPACA1 proteins in acrosomes of cauda epididymal sperm samples (7%–95%). These values were positively correlated with percentages of epididymal spermatozoa with typically distributed acrosomal tyrosine-phosphorylated proteins (r=0.8564, P<0.001). Results indicate there are individual differences in translocation of SPACA1 proteins in the epididymis during sperm maturation, and that SPACA1 protein is one of the main determinants for the typical distribution of acrosomal tyrosine-phosphorylated proteins. In addition, conception rates as a result of AI using cryopreserved spermatozoa tended to be associated with percentages of epididymal spermatozoa with typically distributed SPACA1 proteins. Results from sucrose gradient centrifugation fractionation experiments indicate SPACA1 proteins are sperm membrane raft-associated proteins. Based on these results, it is hypothesized that there is an association between bull subfertility when semen is used for AI and epididymal dysfunctions in the arrangement of membrane lipid rafts during sperm maturation.

Preservation of Epididymal Stallion Sperm in Liquid and Frozen States: Effects of Seminal Plasma on Sperm Function and Fertility

Three separate experiments were conducted to improve preservation of stallion epididymal sperm. In the first one, two different cooling extenders (Kenney and Gent) were compared. Sperm viability and motility patterns were assessed in 10 different epididymal sperm samples after 0 hours, 24 hours, 48 hours, 72 hours, and 96 hours of preservation at 4°C. No significant differences were observed in any of the evaluated parameters either between extenders or throughout the storage period. The second set of experiments was designed to determine whether supplementing thawing medium (INRA Freeze) with seminal plasma had any impact on the quality of frozen-thawed epididymal sperm. Ten epididymal frozen-thawed sperm samples coming from separate stallions were used and different functional parameters (sperm membrane integrity and lipid disorder, motility, intracellular Ca2+ levels, and intracellular concentrations of peroxides and superoxides) were evaluated after incubation with or without 50% seminal plasma. Supplementing thawing medium with seminal plasma had no impact on sperm function and survival. The third experiment was an in vivo study. Twenty-five mares were inseminated with epididymal frozen-thawed sperm and seminal plasma, and 21 were bred with epididymal frozen-thawed sperm only. Pregnancy rates obtained for mares artificially inseminated with epididymal frozen-thawed sperm and seminal plasma were significantly (P < .05) higher than those observed when seminal plasma was not infused (64% vs. 19%). Taken together, our data indicate that the quality of epididymal stallion sperm can be maintained at 4°C for up to 96 hours. In addition, not only does supplementing frozen-thawed epididymal sperm with seminal plasma have any damaging effect on their quality but it may also improve pregnancy rates after artificial insemination.

The ABCA1 blocking agent probucol decreases capacitation in ejaculated dog spermatozoa

BackgroundThe ATP binding cassette (ABC) transporters participate in the cholesterol and phospholipid transport within and through cell membranes of many cells including spermatozoa. Cholesterol efflux is important for capacitation of spermatozoa. ABCA1 expression has been assessed in canine spermatozoa previously but its role in capacitation still has to be determined. The aim of the study was to test whether inhibition of ABCA1 (1) decreases capacitation in ejaculated and epididymal canine sperm samples and (2) decreases cholesterol efflux in the same samples. Twenty-one ejaculates and sperm from 22 epididymal tails were collected from healthy dogs. Motility was measured by CASA and viability assessed after staining with SYBR-14/PI. Samples from ejaculated sperm and sperm from epididymal tails were aliquoted. One part was incubated with the ABCA1 inhibitor probucol, the other served as a negative control. In all samples, capacitation was evaluated by chlortetracyclin (CTC) assay and cholesterol was measured by cholesterol efflux assay and colorimetric enzymatic assay.ResultsIn ejaculated sperm, blockade of ABCA1 with 100 µM of probucol/mL of sample resulted in a significantly higher percentage of uncapacitated and acrosome reacted spermatozoa (P < 0.001 and P = 0.031), capacitation was significantly decreased (35% in probucol samples vs 54.2% in controls, P < 0.001). In probucol inhibited sperm samples from epididymal tails, the percentage of capacitated spermatozoa did not differ between groups but the percentage of acrosome reacted spermatozoa increased significantly (P = 0.014). The cholesterol measurement revealed significantly lower cholesterol concentration in the probucol group when compared to the controls (P = 0.035), however only in ejaculated sperm samples.ConclusionsCTC assay and cholesterol measurement revealed significant differences between groups; we conclude that inhibition of ABCA1 significantly decreased capacitation and cholesterol efflux in ejaculated canine spermatozoa. The inhibition was not complete but ABCA1 is supposed to contribute to capacitation in canine ejaculated spermatozoa. ABCA1 is probably not important for capacitation of epididymal spermatozoa but might exert other functions during spermatozoa ripening.

The Effects of Glyphosate Based Herbicides on Rat Sperm Mitochondria

TPS 633: Health effects of pesticides, Johan Friso Foyer, Floor 1, August 27, 2019, 3:00 PM - 4:30 PM Background/Aim: While glyphosate is one of the most widely used herbicides in the world, there are concerns about its health effects. Glyphosate-based propriety formulations, such as Roundup®, contain unknown additives which may modify potential health effects and glyphosate has been classified as probably carcinogenic to humans. There are questions about its role as an endocrine disruptor and its impact on sperm health. We sought to evaluate the impacts of these chemicals on sperm by assessing damage to mitochondrial DNA, which is thought to be essential for sustaining sperm motility and for successful fertilization. Methods: Epididymal sperm samples were obtained from Sprague-Dawley rats. Eighteen animals were prenatally exposed and then orally dosed with glyphosate. Equal numbers were similarly exposed to Roundup® or served as controls. After weaning, 8 animals per group were treated for 6 weeks and the remaining 10 animals for 13 weeks. Total DNA was extracted from frozen sperm. LA-qPCR was performed to determine mitochondrial and genomic lesions per 10kb. RT-PCR was performed to determine mitochondrial copy number. Preliminary Results: Mitochondrial copy number averaged 100.14 for controls, 95.94 for glyphosate samples, and 95.36 for Roundup® samples. Compared to controls, relatively fewer lesions were found in animals dosed with Roundup® and relatively more lesions were found in animals dosed with glyphosate. Inferential and parametric statistical comparisons of mtDNA damage per group and according to sperm quality indicators will be presented. The differences between the glyphosate and Roundup® groups are particularly informative for understanding how surfactants and other purported inert ingredients may have a role in toxicity. Conclusions: These data suggest that there may be differential effects on sperm mitochondria stemming from exposure to glyphosate and its derivatives. These findings are suggestive and can stimulate more hypothesis-driven mechanistic studies in vitro and in vivo, and epidemiologic studies in populations.

A novel flush technique to simulate natural dispersal of spermatozoa in the female reproductive tract and expedite motility assessment of fresh ejaculated Merino (&lt;i&gt;Ovis aries&lt;/i&gt;) sperm

Motility is an important criterion in the assessment and quantification of the quality of ejaculated and epididymal sperm samples. Ovine sperm spermatozoa are particularly susceptible to cryodamage, and shortening the interval from collection to cryostorage may potentially minimize the negative effects of cryopreservation, thereby improving the post-thaw viability of ram spermatozoa. The use of the swim-up technique (SUT) in quantifying spermatozoa motility is well documented, especially for the isolation of highly motile spermatozoa for assisted reproductive purposes. However, this technique is time consuming and involves a swim-up period of 10 minutes before the motility of a sample is recorded by using computer-assisted sperm analysis (CASA) software such as the Sperm Class Analyser® (SCA®). The novel flush technique (FT) allows for capturing of sperm motility tracks via the SCA® system shortly after semen collection, that is, within a minute. This study compared fresh ejaculated sperm motility traits by using the SUT and FT. Motility evaluations were performed using 45 semen samples obtained from 15 adult Merino rams (Ovis aries) at weekly intervals. Motility recordings were captured at 100 frames per second, using a calibrated 20 μm deep Leja slide. The percentage total motile spermatozoa of samples subjected to the FT was 89.2%, which was significantly higher than that recorded by the SUT (83.9%). The results also indicated that the wobble (WOB) parameter showed significantly higher values when using the FT, and parameters curvilinear velocity (VCL) and amplitude of the lateral head displacement (ALH) indicated significantly higher values when using the SUT. Establishing the ideal spermatozoa concentration and analysis of sperm subpopulation motility characteristics would assist in the optimization of the FT, and its use in CASA motility analysis of ovine spermatozoa. Standardization of CASA analysis of ovine semen samples, which would enable the selection of quality spermatozoa samples for use in field insemination (fresh samples) or in vitro fertilization programs, and laparoscopic AI cryopreserved samples warrants further investigation.Keywords: Cryopreservation, merino, sperm class analyser, sperm motility, sperm separation technique

Effect of Amifostine on Sperm DNA Fragmentation and Testes after Radioiodine Treatment.

IntroductionRadioactive iodine (RAI) is commonly used for the treatment of hyperthyroidism caused by Graves’ disease or thyroid nodules. However, information available on the impact of RAI therapy on male gonadal function is scarce. This study aimed to determine any possible damage to testicular tissue and sperm quality caused by RAI therapy, and the radioprotective effect of amifostine against such damage.Material and MethodsIn total, 36 rats were randomly allocated to three groups, including a control group, RAI group (111 MBq Iodine-131), and RAI + amifostine group (111 MBq Iodine-131 and a single dose of 200 mg/kg amifostine). Blood and epididymal sperm samples were taken for hormone analyses and the evaluation of spermatological parameters. The TUNEL assay and haematoxylin-eosin were used to stain testicular tissue samples to detect histological changes and apoptosis.ResultsThe groups differed insignificantly for the testicular mass index and spermatozoa concentration. However, spermatozoa motility and percentage of viable spermatozoa were higher in the RAI + amifostine group, compared to the RAI group. Sperm DNA fragmentation and the index of apoptotic germ cells significantly decreased in the amifostine group, in comparison to the radioiodine group. While the testosterone levels showed no significant change, the follicle stimulating hormone (FSH) levels significantly decreased in the RAI + amifostine group.ConclusionAll histopathological parameters and some spermatological parameters showed that RAI therapy caused statistically significant damage of testicular tissue and this damage was reduced by amifostine.

Stallion sperm selection prior to freezing using a modified colloid swim-up procedure without centrifugation

The aims of this study were to: 1) develop a new method for stallion sperm selection using a modified swim-up procedure through a colloid and 2) evaluate its impact in good quality ejaculates from bad freezers in comparison to methods involving centrifugation such as single layer centrifugation and sperm washing. Ejaculates were processed before freezing using three different procedures: sperm washing (SW), colloid single layer centrifugation (SLC) and a modified colloid swim-up (SU). After semen processing, sperm recovery rates were measured and sperm were frozen. Post-thaw sperm motility (assessed by computer-assisted sperm analysis), normal forms and plasma membrane integrity (evaluated under bright-field and fluorescence microscopy respectively), and DNA fragmentation (assessed by the Sperm-Halomax kit) were compared between treatments. Sperm recovery rates were similar between SU and SLC but lower than SW. Sperm motility after thawing was lower in SU in comparison to SLC and SW, maybe due to the incomplete removal of seminal plasma before freezing. Sperm DNA fragmentation was lower in SU and SLC selection methods, particularly in SLC selected samples during the first 6h of incubation. The remaining sperm parameters assessed were similar among treatments. In conclusion, SLC is more suitable than SW and SU to process stallion semen prior to freezing, in particular when sperm DNA damage is suspected. Further studies are needed in order to determine the potential benefits of SU in samples where centrifugation is not necessary, such as epididymal sperm, ejaculate fractioning or post-thaw semen samples.

Cytoplasmic droplet acting as a mitochondrial modulator during sperm maturation in dogs

Motility acquisition during sperm maturation and passage through the epididymis is closely related to mitochondrial function and appears to occur in parallel with cytoplasmic droplet (CD) migration. However, such mechanism remains unclear in dogs. Thus, the aim of this study was to characterize the influence of sperm CD in the mitochondrial functionality during epididymal sperm maturation in dogs. Twenty-one adult dogs were submitted to elective bilateral orchiectomy. Testicles were stored for 18–24h at 5°C and epididymal sperm samples were then collected from different segments of the epididymis (caput, corpus and cauda). Samples were evaluated for computer-assisted motility analysis (CASA), presence of CD (eosin/nigrosin stain), ultrastructural CD analysis and sperm mitochondrial activity (3,3' diaminobenzidine technique) and membrane potential (JC-1 probe). Samples collected from the corpus epididymis showed higher motility and mitochondrial activity in comparison to the caput sperm. Moreover, corpus sperm had lower percentage of proximal droplets compared to caput samples, while mitochondrial membrane potential remained unchanged. Cauda samples showed higher motility, mitochondrial activity and potential, however, lower presence of sperm droplets (proximal and distal). In conclusion, the CD is essential for epididymal sperm maturation in dogs, showing important functions along the transit in the epididymis. In the corpus segment, the migration of the CD along the sperm midpiece provides a high mitochondrial activity and the onset of sperm motility. On the other hand, sperm from cauda epididymis lack CD but suffered lipid membrane changes which allow a maximum mitochondrial membrane potential and motility.

Effects of epididymis cold storage on frozen-thawed epididymal sperm quality in tomcats (Felis catus)

The effect of cold storage of testes and epididymides at 4 °C for 12 h on the cryopreservation capacity of epididymal feline sperm was evaluated. Ten domestic cats were castrated, and testes and epididymides collected. Specimens were randomly assigned to two groups: in Group A, epididymal samples were immediately processed and frozen in 0.25-ml straws; in Group B, both testes and epididymides were maintained in saline at 4 °C for 12 h and sperm was then processed and frozen. Motility, morphology, acrosome status, sperm viability and DNA integrity were assessed in epididymal sperm samples before freezing (baseline), at thawing (0 h) and 6 h post-thawing (6 h). Although values were lower in Group B, no significant intergroup difference was observed for any of the parameters tested either at baseline or at 0 h. However, significantly higher values (P &amp;lt; 0.05) were observed in Group A at 6 h for total sperm motility (29.0 ± 2.4% vs 13.0 ± 4.3%), sperm viability (35.2 ± 5.4% vs 15.4 ± 1.4%) and normal morphology (47.6 ± 0.8% vs 40.0 ± 2.1%). It was observed that motility and acrosome status of epididymal sperm are the most sensitive parameters when both types of sperm samples (from fresh epididymis or from 12 h cold-stored epididymis) are frozen-thawed. When sperm quality was assessed 6 h after thawing, spermatozoa precooled in the epididymides showed significantly lower values for motility, viability and morphology than spermatozoa from fresh epididymal samples.

Transcriptomic Variation during Spermiogenesis in Mouse Germ Cells.

To explore variations in the transcription activity during spermiogenesis, round and elongated spermatids were collected from ICR/CD1 model mice using laser capture microdissection (LCM) and cauda epididymal sperm samples. The transcripts were sequenced using RNA-seq, and the reads were mapped to mm9. The majority of the reads (70%) in the round and elongated spermatids were mappable to known and predicted exons, but that in sperm was only 9%. The results of the distribution of reads suggested that alternative splicing was more complicated in sperm than in round and elongated spermatids. In the 19,127 genes, we detected the expression of 5,104 de novo genes and 91,112 alternative splicing events, and 12,105 were differentially expressed. Gene ontology (GO), InterPro domains, and KEGG revealed changes in gene transcription, mitochondrial protein translation, cellular components, and energy metabolism during spermiogenesis. The results provided considerable information about alternative splicing events, differentiallly expressed genes (DEGs), and novel transcriptions during spermiogenesis in mice.

Choice of staining technique affects the morphological assessment of epididymal feline sperm

Morphological examination of spermatozoa remains a valuable tool for determining the quality of feline sperm for cryopreservation and/or for use in artificial insemination. Numerous staining techniques have been employed for feline sperm assessment, and several variations have also been reported. Forty-eight domestic cats were castrated and epididymal sperm samples were obtained. Sperm morphology was assessed using two staining methods, an air-dried stain (Giemsa-Wright) and a wet stain (eosin-nigrosin), to determine their relative abilities for estimation of the proportion of normal and abnormal spermatozoa. The effects of centrifugation and the cooling process on sperm morphology were also analysed. The mean percentage of morphologically normal spermatozoa was significantly lower in eosin-nigrosin-stained samples than in Giemsa-Wright-stained preparations (46.5% vs 53.4%; P < 0.01), both examined by conventional light microscopy. Results concerning head, midpiece and tail sperm abnormalities suggest that these methods differ in their capacity to identify sperm anomalies in cat. Although no significant differences were observed for secondary abnormalities, cytoplasmic droplets were significantly lower in Giemsa-Wright-stained samples (P < 0.01). Centrifugation or refrigeration of sperm samples did not affect the epididymal sperm morphology in evaluated samples. This study highlights the necessity of minimising morphological assessment variations in order to eliminate subsequent errors and to improve the reproducibility of sperm morphology assessment in different laboratories.

Selection of red deer spermatozoa with different cryoresistance using density gradients.

The objective of sperm selection media is selecting the best spermatozoa and to remove seminal plasma and diluent for using them in assisted reproductive techniques. It is known that individuals show different cryoresistance in response to the same freezing procedure. Our hypothesis was that the efficacy of selection media could be dissimilar for samples with different sperm quality after thawing. Epididymal sperm samples from mature Iberian red deer were collected and frozen. Males were classified as with high post-thaw sperm quality when sperm motility (SM)≥70%, or as with low post-thaw sperm quality when SM≤69%. Samples were centrifuged using the following density gradients (DG): Percoll® , Puresperm® and Bovipure™ , and several functional sperm parameters were assessed after sperm selecting and washing. Males classified with high sperm quality had higher post-thawing values (p>.05) for all parameters evaluated, except for linearity index, than those categorized as low sperm quality. After selection, some sperm characteristics improved (viability, apoptosis and mitochondrial activity) for both groups, showing the males with high sperm quality higher values in all sperm parameters except for kinematic traits and DNA fragmentation index (%DFI), regardless of DG. Bovipure™ yield lower values of sperm motility, viability, apoptosis and mitochondrial activity in relation to Percoll® and Puresperm® considering both quality groups. There was an interaction between the type of DG and sperm quality group for sperm viability (p=.040) and apoptosis (p=.003). Thus, Percoll® selected less live and more apoptotic spermatozoa than Puresperm® and Bovipure™ for males with low sperm quality. In conclusion, the DG are more efficient selecting spermatozoa from samples with high sperm quality, acting differently depending on initial sperm quality.

Pulmonary exposure to cellulose nanocrystals caused deleterious effects to reproductive system in male mice

ABSTRACTOver the past several years there has been an increased number of applications of cellulosic materials in many sectors, including the food industry, cosmetics, and pharmaceuticals. However, to date, there are few studies investigating the potential adverse effects of cellulose nanocrystals (CNC). The objective of this study was to determine long-term outcomes on the male reproductive system of mice upon repeated pharyngeal aspiration exposure to CNC. To achieve this, cauda epididymal sperm samples were analyzed for sperm concentration, motility, morphological abnormalities, and DNA damage. Testicular and epididymal oxidative damage was evaluated, as well as histopathology examination of testes. In addition, changes in levels of testosterone in testes and serum and of luteinizing hormone (LH) in serum were determined. Three months after the last administration, CNC exposure significantly altered sperm concentration, motility, cell morphology, and sperm DNA integrity. These parameters correlated with elevated proinflammatory cytokines levels and myeloperoxidase (MPO) activity in testes, as well as oxidative stress in both testes and epididymis. Exposure to CNC also produced damage to testicular structure, as evidenced by presence of interstitial edema, frequent dystrophic seminiferous tubules with arrested spermatogenesis and degenerating spermatocytes, and imbalance in levels of testosterone and LH. Taken together, these results demonstrate that pulmonary exposure to CNC induces sustained adverse effects in spermatocytes/spermatozoa, suggesting male reproductive toxicity.

Influence of different anaesthetic protocols over the sperm quality on the fresh, chilled (4°C) and frozen-thawed epididymal sperm samples in domestic dogs.

This study assessed the influence of three different anaesthetic protocols on semen quality obtained from the epididymis. Sixty male dogs undergoing to routine sterilization were assigned to three anaesthetic protocols: thiopental group (TG, n=20), propofol group (PG, n=20) and ketamine-dexmedetomidine group (KDG, n=20). Immediately after orchidectomy, the cauda epididymides and vas deferent ducts were isolated and then a retrograde flushing was performed to collect spermatozoa. In experiment 1, after the initial evaluation of the semen (sperm concentration, sperm motility and the percentages of live spermatozoa, abnormal spermatozoa and acrosome membrane integrity), semen samples were diluted in Tris-glucose-egg yolk extender and chilled for 48hr, and the sperm motility was assessed at 6, 24 and 48hr. In experiment 2, semen samples were diluted in Tris-glucose-egg yolk extender and chilled for 24hr, and then samples were frozen in two extenders with different glycerol concentrations, to reach a final concentration of 50-100×10(6) spermatozoaml(-1) , 20% egg yolk, 0.5% Equex and 4% and 5% glycerol, respectively. Mean values of total sperm concentration, sperm viability and the percentages of intact acrosome and abnormal spermatozoa were not significantly different between experimental groups, and therefore, the anaesthetic protocols assessed did not affect sperm parameters mentioned above. However, our study confirmed a detrimental effect of the use of thiopental (TG) over the total sperm motility (p<0.05) and progressive sperm motility (p<0.05) of the fresh and chilled epididymal sperm samples. The anaesthetic protocols including the application of propofol or ketamine-dexmedetomidine can be used to recover sperm in domestic canids without significant changes in sperm quality compared when semen is collected routinely and these techniques could be applicable to endangered wild canids.