Frozen-thawed Epididymal Spermatozoa Research Articles

Effect of Microfluidic and Density gradient techniques to select Frozen-thawed Epididymal Sperm from Bighorn rams.

Gamete preservation of wild species is a priority in conservation programs. However, field techniques to recover sperm from deceased animals may impair its final quality.The cryopreservation process also has its challenges, and the minimal required quality parameters may differ depending on the intended use of the semen.Often, postmortem harvested and cryopreserved sperm may need to undergo a selection process to improve the final quality of the sample. Certain sperm selection techniques that employ centrifugal forces can increase the risk of damaging the cells. In contrast, microfluidics uses hydrostatic pressure and capillary forces, avoiding the need for costly equipment and minimizing cell damage.This study aims to compare sperm motion parameters, morphology, and acrosome integrity of cryopreserved-thawed epididymal bighorn ram sperm using two commercial selection techniques, VetCount Harvester® device (MFD) and BovipureTM (NidaCon International AB, Möndal, Sweden) density gradient centrifugation (DGC).Sperm parameters associated with quality were, progressive motile sperms (PMS), straight velocity (VSL), average path velocity (VAP), curvilinear velocity (VCL), linearity (LIN), straightness (STR), beat cross frequency (BCF), amplitude of lateral head deviation (ALH) and plasma membrane integrity (PMI). Computer Assisted Sperm Analyzer was used for the semen analysis. Eosin/nigrosine and Spermac stains were used to assess morphology and acrosome integrity, respectively. Two veterinarians counted the total number of morphologically normal sperm with complete acrosome.Mean sperm concentration was higher (P < 0.01) before filtration (129.56 ±53.9 ×106/ml) than DCG (68.42 ±29.18 ×106/ml) and MFD (52.94 ±28.76 ×106/ml). PMS was higher (P <0.01) after selection using the DGC (79.70 ± 11.11) and MFD (81.23 ±5.64) compared to the no sperm-selected controls (45.36 ±14.67). Sperm kinetics parameters (VCL, VSL, VAP, BCF, LIN, and STR) were higher in the DGC (P<0.01). However, ALH (P<0.01) was significantly higher in the MFD and Control. Morphological normal sperm, acrosome integrity, and plasma membrane integrity were higher (P<0.01) in samples processed with DGC and MFD.In conclusion, microfluidics and single-layer centrifugation improved motion parameters, viability, and proportion of morphologically normal sperm with intact acrosomes from frozen-thawed sperm harvested from Bighorn epididymis. The VetCount Harvester® microfluidic device was comparable to DGC and could be an alternative option for sperm selection. Further research is needed to explore the effect of selected sperm using this technique on artificial insemination, re-cryopreservation, and fertility outcomes.

Equilibration time improves the sperm variables of wild ruminant ejaculated and epididymal sperm cryopreserved by ultra-rapid freezing

This work examines the effect of equilibration time with extender on ultra-rapidly frozen-thawed wild ruminant epididymal (origin: Iberian ibex) and ejaculated (origin: mouflon) sperm variables. Sperm samples were prepared either without prior equilibration, or equilibrated for 30 min before freezing. Higher quality (p < 0.05) frozen-thawed spermatozoa were obtained when equilibration was allowed, for ejaculated sperm in terms of sperm motility, acrosome apical ridge integrity, sperm viability, and percentage of normal cells, and for epididymal sperm in terms of linearity and straightness of sperm movement. The sperm head area, head perimeter, head length and head width were smaller (p < 0.01) in the equilibrated than non-equilibrated frozen-thawed epididymal sperm; no such dimensional changes were recorded for ejaculated sperm. In conclusion, equilibration prior to ultra-rapid freezing improves the cryoresistance of sperm cells, although viable sperm cells can be obtained without equilibration. The epididymal sperm showed greater cryoresistance, supporting the idea that it is more resistant to freeze-thawing than ejaculated sperm.

Effect of post-thaw addition of seminal plasma to cryopreserved epididymal equine spermatozoa

Seminal plasma (SP) has been reported to increase the immediate motility of frozen-thawed epididymal sperm and appears to be important for plasma membrane remodeling, capacitation, acrosomal reaction and fertilization. The objective of this study was to evaluate the effect of incubating equine epididymal frozen-thawed sperm with different concentrations of seminal plasma. Sperm samples were obtained from testes after castration of 40 young Silla Argentino stallions (24 to 36 months of age). Testicles were cooled (5°C) and after 24 h, the cauda epididymis were dissected and flushed (retrograde) with Kenney extender. Epididymal sperm were cryopreserved in EDTA-glucose with 5% DMF, 11% lactose, 20% egg-yolk and Equex, using a rapid temperature-descent curve. The samples were thawed at 37°C for 1 min and then incubated 10 minutes with different concentrations of SP (10%, 50%, 100%) vs. a control (0% SP). To create a pool of SP, Silla Argentino stallions of proven fertility were used. Seminal plasma was separated and stored at -18 °C until use. Semen parameters evaluated were sperm motility (Computer Assisted Semen Analysis; ISAS v1, Proiser), morphology (Diff Quick stain), viability and membrane function (combined hypoosmotic test and Coomassie blue stain). Data were analyzed using Kruskal Wallis test with P<0.05considered significant. Post-thaw incubation with increasing concentrations of seminal plasma significantly decreased sperm total and progressive motility (0%: 33.06 ± 2.02; 26.71 ± 1.90; 10%: 24.06 ± 1.74; 18.85 ± 1.59; 50%: 19.44 ± 1.71; 14.92 ± 1.53; 100%: 14.97 ± 1.57; 11.66 ± 1.48 respectively. Values are mean +/- SEM). However, the values of Beat-Cross Frequency, Linearity and Straightness were significantly higher when the spermatozoa were incubated with SP (BCF: 0%: 10.24 ± 1.47a; 10%: 10.67 ± 3.13a,b; 50%: 10.63 ± 2.31b; 100%: 11.45 ± 2.73b; STR: 0%: 0.59 ± 0.07a; 10%: 0.62 ± 0.09a,b; 50%: 0.64 ± 0.09b; 100%: 0.64 ± 0.12b; LIN: 0%: 0.23 ± 0.05a; 10%: 0.25 ± 0.06b; 50%: 0.26 ± 0.07b; 100%: 0.26 ± 0.07b). There was no effect on membrane function, acrosome status and sperm morphology, regardless of the concentration of SP used (P>0.05). The results indicate that there is no evidence of a beneficial effect of post-thaw supplementation with SP at the concentrations used in this study. It has to be considered that the main role of SP is not associated with sperm physiology but as a modulator in the female reproductive tract. Hence we are continuing in vivo studies to assess whether SP addition can improve pregnancy rates after artificial insemination with frozen-thawed epididymal sperm.

Influence of the Addition of Zinc Oxide Nanoparticles to Cryopreservation Medium for Dog Epididymal Spermatozoa

The purpose of this study was to assess the effect of different concentrations of zinc oxide nanoparticles (ZnONPs) introduced to an extender for frozen-thawed epididymal dog spermatozoa. Epididymides from 22 castrated dogs were minced and cultured in a Tris buffer. The recovered spermatozoa were diluted in Tris-Citric acid-Fructose (TCF) extender with different concentrations of ZnONPs (100 and 200 µg/mL) and control (0.0 µg/mL). Diluted samples were equilibrated at 5 °C for 2 hours before being packed in 0.25 mL straws and stored in liquid nitrogen (-196 °C). After thawing at 37°C for 30 seconds, sperm motility, viability, membrane integrity, acrosome integrity, DNA integrity, and lipid peroxidation by malondialdehyde (MDA) concentration were all measured. The results were presented as mean ± SEM. Adding 100 and 200 µg/mL ZnONPs to the cryopreservation medium significantly (P < .05) improved motility and membrane integrity compared to the control. Viability and acrosome integrity were considerably (P < .05) better at 100 µg/mL ZnONPs than at 200 µg/mL ZnONPs and the control. MDA concentration was significantly (P < .05) decreased at 100 µg/mL ZnONPs compared to 200 µg/mL ZnONPs and the control. When 100 µg/mL ZnONPs were compared to 200 µg/mL ZnONPs and the control, the percentage of DNA damage was significantly (P < .05) reduced. Consequently, adding 100 µg/mL ZnONPs to TCF extender resulted in a significant increase in the proportion of motility, viability, membrane-intact, and acrosome-intact dog epididymal sperm, as well as the preservation of DNA integrity and the prevention of lipid peroxidation at the membrane level.

The effect of epididymal sperm cryopreservation on neonatal birthweight following PESA-ICSI.

To compare the neonatal birthweight of singletons derived from ICSI cycles with fresh or frozen-thawed epididymal sperm in patients with obstructive azoospermia. A total of 436 singletons derived from ICSI cycles with fresh (n = 220) or frozen-thawed (n = 216) epididymal sperm in obstructive azoospermia evaluated from 2012 to 2018 in the retrospective study. Multivariate generalized linear model was used to analyze the association between epididymal sperm cryopreservation and neonatal birthweight. The crude birthweight and z-score in neonates derived from frozen-thawed epididymal sperm were significantly lower than those from fresh epididymal sperm (3186.57g vs 3303.61g and - 0.18 vs 0.08, respectively), with a mean difference of 117.04 (95% CI 32.36-201.72) g and 0.25 (95% CI 0.06-0.45). Adjusted for confounders including parental age and BMI, maternal ovarian reserve, paternal FSH and T levels, peak E2 during OPU cycles, frozen-thawed embryo transfer, embryo development stage, gestational age, maternal parity and child gender, the multivariate model suggested that singletons conceived from ICSI with fresh epididymal sperm was on average 91.21g heavier than those conceived from ICSI with frozen-thawed epdidiymal sperm (95% CI 12.72 to 166.7, P = 0.016). Cryopreservation of epididymal sperm may negatively affect birthweight.

Influence of pentoxifylline and caffeine on stallion epididymal sperm motility after thawing

Two substances at two concentrations each were added to frozen-thawed stallion epididymal spermatozoa to improve motility indicators, which were assessed 30, 60, 120, 180 and 240 min after thawing. Pentoxifylline was added at concentrations of 3.6 mM (1 mg/ml) and 7.18 mM (2 mg/ml). Both concentrations had a positive effect on total and progressive motility of spermatozoa throughout the study. The other substance, caffeine, was added at concentrations of 2 mM (0.4 mg/ml) and 5.5 mM (1 mg/ml). Both concentrations had a positive effect on total and progressive motility of spermatozoa only 30 and 60 min after thawing. Subsequently, 180 min after thawing, improvement was found only in total sperm motility.

32 An approach to invitro oocyte maturation and fertilization in threatened Saharan Dorcas gazelle (Gazella dorcas osiris) using frozen-thawed epididymal sperm cells

The Dorcas gazelle (Gazella dorcas), in the family Bovidae, is one of the smallest species of the genus Gazella. Its population is decreasing and it is classified as vulnerable by the International Union of Conservation of Nature Red List of Threatened Species. The subspecies Saharan Dorcas gazelle (G. dorcas osiris), originating in the central-western Saharan and Sahel regions, is almost extinct in the wild because of over-hunting. Thereon, assisted reproductive techniques (ARTs) could help in its population management. Here, we performed invitro maturation (IVM) of oocytes recovered from an adult female of Saharan Dorcas gazelle (10 years old) to attempt IVF with frozen-thawed epididymal sperm cells from an adult male (9 years old) of the same subspecies, following protocols for ARTs in cattle. Epididymal sperm was obtained after postmortem examination by cannulation of vas deferens with commercial semen extender Gent A (Minitub). Motility features were assessed using a computer-assisted sperm analysis system (Proiser SL), and viability was evaluated using eosin/nigrosin staining. After collection, viability (88.3%), total (17.8%) and progressive (8.7%) motility were assessed. A total of 820.5×106 sperm cells was obtained and diluted (1:1) in a semen extender with permeable cryoprotectants (Gent B, Minitub). Then, the sample was refrigerated in straws (200×106 mL−1; 0.5mL) at 4°C for 1h and exposed to liquid nitrogen vapors (LN2) for 10min before being directly plunged into and kept on LN2. A total of 35 oocytes were recovered by slicing, 27 of which were selected for IVM (24h, 38.5°C, 5% CO2 in humidified air). Motile sperm were obtained by centrifugation of thawed semen (10min, 1000×g) by performing a density gradient (BoviPure, Nidacon). Post-thawing viability (72.9%) and total (14%) and progressive (4%) motility were evaluated just before IVF. After IVM, 17 oocytes were incubated for IVF for 20h with 1×106 thawed sperm cells mL−1 (although the first polar body extrusion was observed only in 5 of the 17 oocytes before IVF; 29.4%). Also, 7 oocytes selected for IVM were vitrified and kept for the genetic bank, and 3 were fixed. Thereafter, all oocytes that underwent IVF were incubated for invitro embryo culture (38.5°C, 5% CO2, 5% O2 in humidified air) to avoid discarding any potential zygote. Cleavage was assessed at 48h post-IVF. From those, only 2 oocytes successfully achieved IVF out of the 5 that showed first polar body extrusion (40%), although cleavage was asymmetric and embryos did not progress. The rest of oocytes (12) did not achieved IVM, IVF, or cleavage. Results suggest that optimal protocols and appropriate individualized conditions for vulnerable nondomestic ungulates are required for successful ARTs. Future efforts are needed to improve preservation strategies for the gametes/embryo conservation of endangered species. JG and MRC contributed equally to this work. JG is supported by AGAUR (2018 FI_B 00236) and MRC by FPU (Training for Academic Staff) (FPU15/06029).

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.

78 Radio electric asymmetric conveyer treatment during prolonged cold storage of ovaries enhances developmental competence of oocytes in the domestic cat model

Hypothermic storage (4°C) of ovaries for long-distance transport holds great potential to expand access to fertility preservation in animals and humans (Duncan et al. 2016 Reproduction 152, 201-210). However, storage for prolonged periods (over 24h) leads to structural/functional changes in the ovarian tissue with a critical reduction of follicular viability and oocyte quality (Piras et al. 2018 Reprod. Biol. Endocrinol. 10, 16-76; Isachenko et al. 2009 Fertil. Steril. 91, 1556-1559). The radio electric asymmetric conveyer (REAC) is a novel technology platform for neuro and bio-modulation that optimize the ion fluxes and the mechanisms driving cellular asymmetry and polarization in biological structures (Maioli et al. 2016 Sci. Rep. 6, 28682). The REAC has already proved to be effective in promoting cell differentiation and reprogramming and to counteract the biological mechanisms linked to aging and the degenerative process (Berlinguer et al. 2017 Reprod. Biol. Endocrinol. 15, 11; Maioli et al. 2014 Age 36, 9-20). In the present study, the domestic cat was used as an experimental model to evaluate the effect of REAC treatment during ovary storage at 4°C for 48h on invitro developmental ability of oocytes retrieved from antral follicles. Ovaries harvested from healthy domestic queens during ovariectomy were randomly assigned to the REAC-treated (R: n=13) and untreated (C: n=13) groups. In detail, ovaries were maintained in 4mL of phosphate-buffered saline at 4°C for 48h. The REAC device was set at 2.4 GHz, and its conveyer electrodes were immersed into the phosphate-buffered saline. After 48h, ovaries were sliced to release cumulus-oocyte complexes, which were selected according to their morphological characteristics (Johnston et al. 1991 Biol. Reprod. 45, 898-906) for IVM (R: n=130; C: n=133). Matured oocytes were fertilised (IVF) with frozen-thawed epididymal spermatozoa and presumptive zygote were invitro cultured (IVC) for 7 days. On Day 2 and Day 7 of IVC, respectively, the number of embryos cleaved and developing to the blastocyst stage was determined. The IVM, IVF, and IVC were performed according to the procedure of Piras et al. (2018 Reprod. Biol. End. 16, 76). Data were analysed by chi-square test with STATA\IC 11.0. Maturation rate of oocytes did not differ between groups (R: n=59/130, 45.4%; C: n=66/133, 49.6%). Cleavage rate was higher (P&amp;lt;0.05) in the R group (n=34/59, 57.6%) compared with the C group (n=25/66, 37.9%). The percentages of blastocyst formation relative to the number of cleaved embryos (R: n=12/34, 35.3%; C: n=3/25 12.0%) and to the total number of MII oocytes (R: n=12/59, 20.4%; C: n=3/66, 4.5%) increased (P&amp;lt;0.05) after REAC treatment compared with the untreated counterpart. In conclusion, REAC treatment during cold storage of cat ovaries for 48h positively affected the quality of oocytes as assessed by invitro embryo production outcome. The REAC technology could provide a useful approach for the optimization of ovarian tissue transport conditions for fertility preservation especially for endangered species and patients with fertility-threatening conditions.

42 Vitrification of invitro-produced feline embryos

Preservation of feline embryos is useful in propagating endangered species, preserving valuable genetics, and supporting biomedical research. Although a wide variety of cryoprotectants (CP) and protocols are successfully used for vitrification of invitro-produced (IVP) embryos, there are often species-specific differences in viability of embryos post-warming. The purpose of this study was to evaluate the viability of IVP feline embryos after vitrification using two common CPs, propanediol (PrOH) or ethylene glycol (EG). Embryos were produced with oocytes and frozen-thawed epididymal sperm collected from local spay-neuter clinics using a published IVP protocol developed for producing domestic feline embryos. Day 7 early blastocysts (stage 5), blastocysts (stage 6), and expanded blastocysts (stage 7) were evaluated for quality (grade 1 or 2) and randomly assigned to one of three treatments: vitrification with PrOH (n=32), vitrification with EG (n=31), or control (n=47), which was allowed to continue in culture until Day 8. The vitrification protocol was as follows. The base medium for all vitrification media was a HEPES-buffered feline optimized culture medium (FOCMH). Embryos were placed in 0.5mL of equilibration medium (7.5% dimethyl sulfoxide, 7.5% PrOH or EG, 0.5M sucrose, 10% Ficoll, and 20% fetal calf serum (FCS)) for 5min at room temperature. Individual embryos were then moved to 20-μL drops of vitrification medium (15% dimethyl sulfoxide, 15% PrOH or EG, 0.5M sucrose, 10% Ficoll, and 20% FCS) at room temperature for 30s before being loaded onto Cryolock devices and plunged into liquid nitrogen. Warming was done using a 3-step process for all vitrified embryos. First, embryos were moved from liquid nitrogen directly to 0.5mL of 1M sucrose, 10% Ficoll, and 20% FCS at 37°C for 1min. Next, embryos were moved to 0.5mL of 0.5M sucrose, 10% Ficoll, and 20% FCS at 20°C for 3min. Finally, embryos were transferred to 0.5mL of FOCMH for 5min at 37°C. All warmed embryos were cultured in medium, optimized for feline embryos, with 5% FCS and evaluated for re-expansion of the blastocoele and progression in development at 24 and 48h. Results are from five replicates. Embryos vitrified in EG exhibited higher percentages of viable embryos 24h after warming (84%) than embryos vitrified in PrOH (59%; P&amp;lt;0.05). The continued embryonic growth of viable embryos after culture for 48h showed equivalent developmental rates, at 87, 96, and 100% for control, EG-treated, and PrOH-treated embryos, respectively (P&amp;gt;0.05). Results indicate EG is a more successful CP treatment for vitrification of feline embryos when evaluating viability 24h post-warming. We report a higher viability of embryos post-thaw than previous studies using the same CPs (Pope et al. 2012 Reprod. Domest. Anim. 47, 125). This may be due to the shorter exposure time to the CPs we used during the vitrification process. We conclude that EG and PrOH are effective CPs for Day 7 feline IVP embryos using this protocol. Further research is needed to increase treatment numbers and evaluate pregnancy rates from embryos transferred post-warming.

Combined Single-Straw Packaging of Cryopreserved Stallion Epididymal Sperm and Separated Homologous Seminal Plasma

The aim of the present study was the investigation of stallion epididymal sperm frozen with seminal plasma within the same straw separated by an air bubble before freezing. We hypothesized that mixing both components after thawing improves the sperm quality compared with conventionally frozen-thawed epididymal sperm. Cryopreservation of epididymal sperm allows the preservation of spermatozoa in case of death or termination of the breeding carrier. However, these sperm lack contact with the seminal plasma, which plays an important role in the development of sperm functionality and the regulation of uterine inflammatory response after insemination. Motion characteristics, morphology, and viability were assessed in epididymal sperm mixed with seminal plasma after thawing (group SP) and compared with conventionally packed cryopreserved epididymal sperm that had no contact with seminal plasma (group FT). Total and progressive motility, curvilinear velocity (VCL), and average lateral head displacement were significantly higher in group SP (P < .05). During post-thaw incubation, VCL decreased in group FT but not in group SP (P < .05), and beat cross-frequency was significantly lower in group FT than in group SP after post-thaw incubation (P < .05). Straightness did not differ between the groups (P > .05). Although the percentage of tail abnormalities and isolated sperm heads increased significantly only in group SP after cryopreservation (P < .05), the proportion of morphologically abnormal and nonviable sperm was not affected by seminal plasma (P > .05). Mixing both components after thawing increased sperm motility and velocity but did not negatively affect morphology or viability. This packaging system is promising for routine artificial insemination of stallion epididymal sperm.

Comparison of DNA fragmentation of frozen-thawed epididymal sperm of dogs using Sperm Chromatin Structure Analysis and Sperm Chromatin Dispersion test

The aim of this study was to compare sperm DNA fragmentation of frozen-thawed epididymal sperm of dogs using the SCSA (Sperm Chromatin Structure Assay) and SCDt (Sperm Chromatin Dispersion test). For this purpose, epididymis from neutered dogs were minced and incubated in a Tris-based extender. The recovered sperm were frozen in a two-step cooling protocol with Tris-based, egg yolk extender and increasing glycerol concentrations, and stored in liquid nitrogen. After thawing, each replica was incubated at 38 °C for 24 h. Sperm DNA fragmentation index (sDFi) was assessed by SCSA and SCDt at 0, 3, 6 and 24 h of incubation and compared within treatments. The relationship and agreement between techniques were evaluated by Pearson’s coefficient and Intraclass Correlation Coefficient (ICC). The results were expressed as mean ± standard error of the mean (SEM). Both techniques indicated there was a significant increase of DNA fragmentation after 24 h of incubation. Moderate correlation (r = 0.65; P<0.01) but lack of agreement (ICC = 0.451; P>0.05) was found between SCSA and SCDt. The lack of agreement indicates that SCSA and SCDt measure different aspects of DNA fragmentation. Four halo morphologies were detected after 24 h of incubation using the SCDt: un-fragmented DNA with a small halo, fragmented DNA with large halo and two new halo presentations never described before for dog sperm: receding sperm with a disappearing halo and “bald” sperm without chromatin dispersion halo around the core. Sperm without a halo of chromatin dispersion are not described by the manufacturer and are similar to un-fragmented sperm, which could lead to erroneous results when using the SCDt. Further studies with different incubation periods and including the new morphologies described in this study should be performed. In conclusion, although SCSA and SCDt can evaluate the changes in the sperm DNA fragmentation dynamics of frozen-thawed epididymal dog sperm, these provided different findings on sperm DNA fragmentation.

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.

Sex ratio of calves generated from artificial insemination using frozen-thawed epididymal spermatozoa of spotted buffalo

Sex ratio of calves generated from artificial insemination using frozen-thawed epididymal spermatozoa of spotted buffalo

Pentoxifylline effects on capacitation and fertility of stallion epididymal sperm

The aims of this study were to determinate whether pentoxifylline (PTX) increases the motion parameters of fresh and frozen-thawed equine epididymal spermatozoa, to evaluate the tyrosine phosphorylation of frozen-thawed epididymal sperm in the presence of PTX and to determine whether the PTX-treatment of stallion epididymal sperm prior to freezing improves the fertility response of mares to a reduced number of spermatozoa per insemination dose. Fifty epididymis were flushed with a skim milk based extender with or without PTX. The pre-treatment with PTX enhanced the sperm motility after being harvested (P<0.05); however the freeze-thaw process did not alter the sperm kinematics between control and treated samples (P>0.05). Plasma membrane integrity did not differ between control and PTX group after recovery and after thawing (P>0.05), as observed in tyrosine phosphorylation, which the PTX treatment did not alter the percentage of tail-associated immunofluorescence of cryopreserved epididymal sperm (P>0.05). For the fertility trial, different insemination groups were tested: 800×106 epididymal sperm (C800); 100×106 epididymal sperm (C100); 100×106 epididymal sperm recovered in an extender containing PTX (PTX100). The conception rates for C800; C100 and PTX100 were 68.7% (11/16); 31.5% (5/16) and 50% (8/16), respectively. The conception rate did not differ among groups (P>0.05), however, a low number of animals was used in this study. A trend toward significance (P=0.07) was observed between C800 and C100 groups. In conclusion, PTX has no deleterious effect on sperm motility, viability and capacitation of cryopreserved stallion epididymal sperm. The conventional artificial insemination with 100×106 sperm recovered with PTX ensures acceptable conception rates and maximize the limited number of doses of cryopreserved stallion epididymal sperm.

Developmental competence of Dromedary camel oocytes fertilized in vitro by frozen-thawed ejaculated and epididymal spermatozoa.

The present study aimed to compare the in vitro fertilizing capacity of frozen-thawed ejaculated and epididymal spermatozoa in order to standardize the semen preparation protocol for camel in vitro fertilization (IVF). Semen samples were collected from 7 Dromedary camels by means of artificial vagina (AV). Ten cauda epididymes were obtained from slaughtered adult camels, isolated, incised and rinsed for obtaining the sperm rich fluid. Ejaculated and epididymal spermatozoa were processed for cryopreservation. Fresh and frozen-thawed ejaculated and epididymal spermatozoa were evaluated for motility, livability, membrane and acrosomal integrities. Frozen-thawed ejaculated and epididymal spermatozoa were used to fertilize camel mature oocytes in vitro. The results showed that, the progressive motility of freshly collected epididymal spermatozoa was significantly (P<0.05) higher than ejaculated spermatozoa (49.25 ± 1.75 vs. 38.50 ± 1.50%, respectively). The viability index of epididymal spermatozoa was significantly (P<0.05) higher than that of ejaculated spermatozoa (96.63 ± 2.45 vs. 84.00 ± 4.08, respectively). The post-thaw acrosome and membrane integrities of epididymal spermatozoa were significantly (P<0.05) higher than those of ejaculated spermatozoa. Morula and blastocyst rates of camel oocytes in vitro fertilized by frozen-thawed epididymal spermatozoa (59.4 ± 0.8, 19.12 ± 0.7 and 10.29 ± 0.7%, respectively) were significantly (P<0.05) higher than those fertilized by frozen-thawed ejaculated spermatozoa (48.27 ± 3.1, 11.63 ± 1.1 and 5.43 ± 0.8%, respectively). In conclusion, the Dromedary camel frozen epididymal spermatozoa have the capacity to endure cryopreservation, fertilize oocytes and produce embryos in vitro better than ejaculated sperm.

In Vitro Fertilization Outcome After Intracytoplasmic Sperm Injection with Fresh and with Frozen-Thawed Epididymal Spermatozoa

&lt;p&gt;&lt;strong&gt;Introduction&lt;/strong&gt;&lt;/p&gt;&lt;p&gt;Testicular epididymal sperm aspiration (TESA) is one of the method to retrieve sperm from the testes in men with azoospermia. The aim of the study is to compare the In vitro fertilization (IVF) outcome of intracytoplasmic sperm injection (ICSI)-ET cycles with fresh testicular epididymal spermatozoa obtained on the same day with oocyte retrieval and with frozen-thawed testicular epididymal spermatozoa.&lt;/p&gt;&lt;p&gt;&lt;strong&gt;Material &amp;amp; Methods&lt;/strong&gt;&lt;/p&gt;&lt;p&gt;A retrospective comparative analysis of patients who underwent fresh TESA and frozen-thawed TESA in ICSI-ET cycles from January 2012 to December 2014 in Halim Fertility Center was done. Fresh testicular epididymal sperm aspiration (fresh TESA) was performed on the same day with oocyte retrieval in 28 cycles and the frozen-thawed testicular epididymal sperm aspiration (frozen-thawed TESA) was used in 30 cycles. &lt;/p&gt;&lt;p&gt;&lt;strong&gt;Results&lt;/strong&gt;&lt;/p&gt;&lt;p&gt;The two groups were comparable in terms of the ages of male and female patients, etiology of infertility and duration of infertility. Fertilization rates in fresh TESA group were 53,5% and in frozen-thawed TESA group, fertilization rates were 50%. There was no statistically significant difference between the groups. Clinical pregnancy rates in fresh TESA group were 35,7% and in frozen-thawed TESA group, clinical pregnancy rates were 26,7% and statistically there was no significant difference between the groups.&lt;/p&gt;&lt;p&gt;&lt;strong&gt;Conclusion&lt;/strong&gt;&lt;/p&gt;There is no significant difference in the in vitro fertilization outcome of intracytoplasmic sperm injection (ICSI)-ET cycles between fresh TESA and frozen-thawed TESA .

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.

In vitro fertilization using frozen-thawed feline epididymal spermatozoa from corpus and cauda regions

Epididymal sperm preservation offers a potential for rescuing genetic material from endangered or valuable animals after injury or death. Spermatozoa from corpus, as well as from cauda, have the capability to be motile and to undergo capacitation and can thus potentially be preserved for assisted reproductive technologies. In the present study, feline frozen-thawed epididymal spermatozoa from corpus and cauda regions were investigated for their ability to fertilize homologous oocytes and further embryo development in vitro. Epididymal spermatozoa from corpus and cauda of seven cats were cryopreserved and used for IVF. Cumulus–oocyte complexes (n = 419) were obtained from female cats after routine spaying. Frozen-thawed corpus epididymal spermatozoa showed similar properties of acrosome integrity, membrane integrity, and chromatin integrity as frozen-thawed spermatozoa from cauda except corpus spermatozoa showed lower motility (P < 0.05). The fertilizing capacity of frozen-thawed corpus epididymal spermatozoa was confirmed by similar number of embryos developing to the two- and four-cell stages compared with sperm from cauda (32.03% vs. 33.33%). However, oocytes fertilized with corpus spermatozoa had lower potential to develop to the blastocyst stage (6.79%) and had lower cell numbers compared to oocytes fertilized with cauda spermatozoa (14.08%). In conclusion, spermatozoa from corpus epididymis had a similar capability to fertilize homologous oocytes in vitro as sperm from cauda but resulted in fewer embryos developing to the blastocyst stage compared to spermatozoa from the cauda.

Dose-dependent effects of homologous seminal plasma on motility and kinematic characteristics of post-thaw stallion epididymal spermatozoa.

Preservation of epididymal spermatozoa is important to save genetic material of endangered species and breeds, or in case of unexpected injury, which will end the breeding career of valuable sires. Seminal plasma (SP) influences sperm quality in a dose-dependent manner and its addition to preserved semen immediately before insemination may be beneficial for sperm fertility. Increased plasma membrane stability of epididymal spermatozoa reduces freezing injury of cells, and the addition of SP after freezing and thawing might have activating and protecting effects on spermatozoa within the female genital tract. In this study, epididymal spermatozoa were harvested by retrograde flush of the epididymal cauda immediately after routine castration and frozen. Seminal plasma was collected from other six stallions. Homologous SP (SP from the same species, but from a different animal) was added to frozen-thawed epididymal spermatozoa at concentrations of 0, 5, 20, 50 and 80% SP. Addition of SP increased sperm motility and influenced kinematic values in a dose-dependent manner (p < 0.05). Motility improved at concentrations of 20 and 50% SP, but did not further increase at 80% SP. There was no difference in sperm motility among SP from six different donor stallions regardless of the concentrations of SP (p > 0.05). Total and progressive motility of ten frozen-thawed epididymal spermatozoa samples collected from different stallions after dilution with extender and 5, 20, 50 or 80% SP differed significantly (p < 0.05). In conclusion, addition of homologous SP to frozen-thawed stallion epididymal spermatozoa immediately improved motility in a dose-dependent manner regardless of semen quality of SP donor stallions. This might positively influence fertility when SP is added before insemination. Moreover, there seems to be a threshold level of SP concentration for optimal improvement of sperm motility.