Stallion Epididymal Sperm Research Articles

Cryopreservation and freezability of epididymal and ejaculated stallion spermatozoa

The aim of the present study was to compare the seminal attributes and freezability of the epididymal sperm recovered from castrated stallions and ejaculated semen collected through artificial vagina from the same stallions previously. The seminal quality parameters viz., viability, sperm morphology, plasma membrane functional integrity, acrosome integrity and mitochondrial membrane potential were studied, and recorded immediately after fresh semen collection/retrieval, before freezing, and after cryopreservation. Similar protocols were followed for freezing the epididymal and ejaculated semen. From the current study, it was observed that the sperm concentration of the epididymal recovered was significantly higher than that of fresh ejaculation. Other seminal parameters in thefresh sample, like total and progressive sperm motility, sperm membrane functional integrity, acrosome integrity and kinematic parameters like VSL, STR, and linearity were similar without any significant differences between both. However, mitochondrial membrane potential, BCF and linearity of the spermatozoa differed significantly between the groups after cryopreservation. From the current study, it can be concluded that the protocols used for cryopreserving the fresh ejaculates can be adopted for freezing the stallion epididymal spermatozoa without causing significant damage and through this method; elite stallion germplasm can be safely cryopreserved and conserved.

Cryopreservation and freezability of epididymal and ejaculated stallion spermatozoa

The aim of the present study was to compare the seminal attributes and freezability of the epididymal sperm recovered from castrated stallions and ejaculated semen collected through artificial vagina from the same stallions previously. The seminal quality parameters viz., viability, sperm morphology, plasma membrane functional integrity, acrosome integrity and mitochondrial membrane potential were studied, and recorded immediately after fresh semen collection/retrieval, before freezing, and after cryopreservation. Similar protocols were followed for freezing the epididymal and ejaculated semen. From the current study, it was observed that the sperm concentration of the epididymal recovered was significantly higher than that of fresh ejaculation. Other seminal parameters in thefresh sample, like total and progressive sperm motility, sperm membrane functional integrity, acrosome integrity and kinematic parameters like VSL, STR, and linearity were similar without any significant differences between both. However, mitochondrial membrane potential, BCF and linearity of the spermatozoa differed significantly between the groups after cryopreservation. From the current study, it can be concluded that the protocols used for cryopreserving the fresh ejaculates can be adopted for freezing the stallion epididymal spermatozoa without causing significant damage and through this method; elite stallion germplasm can be safely cryopreserved and conserved.

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.

Effect of Isolation Protocols and Cryoprotectants on Freezing of Stallion Epididymal Spermatozoa

BACKGROUND: The recovery of spermatozoa from the cauda epididymis may be the only option to obtain genetic material from elite stallions that had undergone castration or sudden death due to colic or severe injury. OBJECTIVE: To evaluate two different protocols for retrieval of stallion epididymal spermatozoa and to evaluate different cryoprotectants on the freezability of the epididymal spermatozoa. MATERIALS AND METHODS: Six epididymides from three stallions were collected immediately after routine castration under general anesthesia. In the first experiment, each epididymis (of two testes) of the same stallion were processed using different methods for retrieval of the epididymal spermatozoa and were pooled and cryopreserved either using 5% glycerol or 5% dimethyl formamide (DMF) as cryoprotectant. The semen quality parameters viz., progressive motility, HOST, viability and acrosome integrity were evaluated at the fresh, pre-freeze and post-thaw stages. RESULTS:Retrograde method of flushing of epididymis yielded significantly (ρ &lt; 0.05) higher concentration of the stallion sperm than that of the floating method. The qualitative semen parameters i. e., viability, plasma membrane integrity and acrosome integrity were found to be significantly restored using 5% DMF as cryoprotectant in comparison to when 5% glycerol was used. CONCLUSION: Retrograde flushing method of epididymis yielded significantly higher sperm concentration to that of the floating method, and 5% DMF as cryoprotectant provided acceptable freezability of stallion epididymal spermatozoa.

Effect of sperm and glycerol concentration on epididymal sperm motility parameters after thawing

The aim of this study was to evaluate the effect of different glycerol concentrations and different sperm concentrations on stallion epididymal sperm motility indicators after thawing. For statistical analysis, 25 stallions were used. Collection of the epidydimal spermatozoa was performed as a retrograde flush of cauda epididymis and ductus deferens within 12 h post castration. After evaluation, the resuspended spermatozoa were centrifuged, the supernatant removed and the spermatozoa resuspended in Gent semen extender to get three different groups with different concentrations of sperm (250 × 106 in ml, 500 × 106 in ml, 1000 × 106 in ml) and different final glycerol concentrations (2%, 4%, and 6%). Therefore, 9 different samples were finally obtained from each stallion. The spermatozoa were packed and placed in a fridge (4 °C) for 2 h, then placed in liquid nitrogen vapour (−80 to −100 °C) and after 25 min plunged into the liquid nitrogen and stored at −196 °C for at least 5 days. The selected straws were individually thawed in a 38 °C water bath for 30 s prior to post-freezing analysis. Motility indicators were assessed at 0, 60, 120, and 180 min after thawing. Parametric test was used for analysis; the measured indicators were total motility, progressive motility, curvilinear velocity, straightness, and average-path velocity. In this study, the best results were reached in samples diluted into a concentration of 1,000 × 106 in ml, regardless of the concentrations of glycerol.

Efficiency of Ringer B. Braun solution on stallion epididymal sperm motility and viability compared to the commercial extender within 72 hours of storage

Collection of epididymal stallion sperm offers the opportunity to retain and use genetic material from the males after elective castration or evenpost mortem. The aim of the present study was to evaluate the effect of the Ringer B. Braun solution on stallion epididymal sperm viability and motility during liquid storage for 72 h at 4 °C and at 20 °C. Eight stallions (3–10 years old) were used in the study. Sperm from the cauda epididymis was harvested immediately after routine castration. The sperm from epididymis was washed out and diluted with Ringer B. Braun solution and with a commercial semen extender which was used as a control. Samples from each horse were divided into two parts: one part of samples was stored at 4 ± 1 °C as recommended for the commercial semen extender, the other one was stored at 20 ± 1 °C. Sperm viability, motility, and pH were checked one hour after collection and dilution, and after 24, 48 and 72 h of incubation. The results of sperm viability, subjective and progressive sperm motility showed slightly higher results in semen samples diluted with Ringer B. Braun solution in one hour compared to the commercial extender, by 2.40 ± 0.49% for viability (P&gt; 0.05), by 0.30 ± 9.92% for subjective motility (P&gt; 0.05) and by 5.70 ± 6.07% for progressive motility (P&gt; 0.05). We suggest that Ringer solution could be used for a short term storage (1–24 h) of equine epididymal semen at a 4 °C temperature.

Influence of the Season and Region Factor on Phosphoproteome of Stallion Epididymal Sperm

Simple SummaryPhosphorylation and dephosphorylation of proteins are considered to be the most important processes in sperm maturation during the epididymal transit. We demonstrated that 27 proteins underwent phosphorylation both in and out of the breeding season. Differences in the phosphorylation status were demonstrated in the case of endoplasmic reticulum chaperone BiP, albumin, protein disulfide-isomerase A3, nesprin-1, peroxiredoxin-5, and protein bicaudal D homolog.Epididymal maturation can be defined as a scope of changes occurring during epididymal transit that prepare spermatozoa to undergo capacitation. One of the most common post-translational modifications involved in the sperm maturation process and their ability to fertilise an oocyte is the phosphorylation of sperm proteins. The aim of this study was to compare tyrosine, serine, and threonine phosphorylation patterns of sperm proteins isolated from three subsequent segments of the stallion epididymis, during and out of the breeding season. Intensities of phosphorylation signals and phosphoproteins profiles varied in consecutive regions of the epididymis. However, significant differences in the phosphorylation status were demonstrated in case of endoplasmic reticulum chaperone BiP (75 and 32 kDa), protein disulfide-isomerase A3 (50 kDa), nesprin-1 (23 kDa), peroxiredoxin-5 (17 kDa), and protein bicaudal D homolog (15 kDa) for season x type of phosphorylated residues variables. Significant differences in the phosphorylation status were also demonstrated in case of endoplasmic reticulum chaperone BiP and albumin (61 kDa), protein disulfide-isomerase A3 (50 kDa), and protein bicaudal D homolog (15 kDa) for region x type of phosphorylated residues variables.

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.

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.

Ejaculated compared with epididymal stallion sperm vitrification

The aim of this study was to evaluate the effect of trehalose and lactose extenders on ejaculated and epididymal stallion sperm vitrification. Ejaculated semen samples were collected from seven fertile stallions, and cauda epididymis samples were collected from ten stallion carcasses after slaughter. Both the ejaculated and the epididymis samples were diluted and vitrified using INRA 96® and bovine serum albumin as well as trehalose or lactose. As a control, ejaculated and epididymal samples were collected and frozen using the conventional method. Vitrification was performed by immersing sperm suspensions directly in LN2. After thawing or devitrification, there was assessment of samples for sperm motility using computer-assisted analysis. Viability was assessed using SYBR-14 and propidium iodide (PI) and acrosome integrity by fluorescein using isothiocyanate combined with peanut agglutinin (FITC-PNA) and PI. Epididymal sperm vitrification with trehalose (EPT) or lactose (EPL) resulted in greater progressive sperm motility than sperm of the control sample (EPC). After post-thaw/devitrification of sperm in the EPT group, sperm motility was greater (P<0.001) compared to that using EPL (50.72 ± 5.09% compared with 34.21 ± 3.02%). The results from assessment of ejaculated sperm samples after undergoing the vitrification process indicated cells were less viable (P<0.001) than the control (EJC) sample. In conclusion, vitrification of epididymal stallion sperm using trehalose might be a beneficial alternative for the long-term storage of sperm samples with great economic value. Spermatozoa from vitrified ejaculates of stallions, however, had lesser motility and viability rates than samples subjected to conventional freezing.

Effects of Different Freezing Protocols on Motility, Viability, Mitochondrial Membrane Potential, Intracellular Calcium Level, and DNA Integrity of Cryopreserved Equine Epididymal Sperm

The aim of the present study was to evaluate the effect of different freezing procedures on sperm motion, viability, the acrosome status, mitochondrial membrane potential (MMP), intracellular calcium content, and DNA integrity on epididymal stallion sperm. Therefore, the sperm of 10 healthy stallions was harvested by retrograde flushing after testectomy, diluted with a semen extender containing defined milk proteins and a freezing extender containing egg yolk and glycerol and frozen according to 4 different protocols, using a programmable freezer and a floating rack performing a slow (processes 1 and 2) or a fast cooling rate (processes 3 and 4, respectively). Post-thaw total motility and slow sperm values were lower when using process 4 compared with processes 1 and 2 (P < .05) after 1 hour of incubation. Progressive motility was lower in process 4 compared with process 1 immediately after thawing and after 1 hour of incubation (P < .05). The amount of rapid sperm was lower when using process 4 compared with process 1 immediately after thawing (P < .05). After 1 hour of incubation, the amount of rapid sperm was lower when using process 4 compared with processes 1 and 2 (P < .05). Higher values for viable sperm were seen in processes 1 and 2 compared with process 4 (P < .05) after 1 hour of incubation. Immediately after thawing, more viable sperm with high MMP (hMMP) were observed when using process 3 compared with process 2 (P < .05). After 1 hour of incubation, a significantly higher amount of viable hMMP sperm were detected when using processes 1 and 2 compared with process 4 (P < .05). Process 2 yielded a lower percentage of sperm containing low calcium (lCa) than process 3 immediately after thawing (P < .05). After 1 hour of incubation, the lowest amount of lCa sperm was observed using process 4 (P < .05). The subpopulation of viable/hMMP/lCa sperm was higher when using process 3 compared with process 2 immediately after thawing (P < .05). After 1 hour of incubation, the lowest amount of this subpopulation was detected in process 4 (P < .05). The DNA integrity was similar in all groups. In conclusion, a slow cooling rate with a controlled rate freezer resulted in best sperm quality after thawing. Using a floating rack in nitrogen vapor as an alternative to a programmable freezer, equilibration in a cooled environment is advantageous.

Comparison of the Effects of Five Semen Extenders on the Quality of Frozen-Thawed Equine Epididymal Sperm

Cryopreservation of epididymal sperm allows the saving of genetic material in case of unexpected death or emergency castration. The aim of the present study was the comparison of five different combinations of extenders commercially available for equine frozen semen processing for cryopreservation of epididymal sperm. Epididymal sperm were harvested from gonads of 10 healthy stallions after routine castration by retrograde flush technique. Then, samples were split and diluted with (1) INRA96 + INRA Freeze, (2) BotuSemen + BotuCRIO, (3) EquiPlus + Gent Freeze, (4) EquiPlus + EquiPlus Freeze, and (5) Gent + Gent Freeze. Extenders 1 and 2 showed higher values for total and progressive motility after thawing compared with extender 4 (P < .05). Extender 3 was in between 1 and 2 (P > .05), and extender 5 resulted in the lowest values (P < .05). The subpopulation of viable frozen-thawed sperm with high mitochondrial membrane potential and low intracellular calcium content was higher using extender 1 compared with extenders 3, 4, and 5 (P < .05) and higher in extender 2 compared with extenders 4 and 5 immediately after thawing (P < .05). After 1 hour of incubation, this subpopulation yielded the highest values in extender 2 (P < .05). Immediately after thawing, extender 1 yielded higher values for percentage of DFI and mean DFI than extenders 3, 4, and 5 (P < .05). Following 1 hour of incubation after thawing, sperm processed with extender 1 resulted in the highest values for percentage of DFI and mean DFI (P < .05). Using extender 2, mean DFI values were lower than those in extender 1 and higher than the extenders 3, 4, and 5 (P < .05). The study revealed that according to the examined sperm quality parameters, freezing extenders (extender 1, extender 2) using low concentrations of glycerol either combined with or without methylformamide were beneficial for cryopreservation of stallion epididymal sperm. For processing of stallion epididymal sperm, an extender containing milk proteins (extenders 1–4) for initial dilution after sperm harvesting is preferable to an extender including egg yolk (extender 5).

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.

The Effect of Pre- and Post-injection Procedural Factors on Blastocyst Rate After ICSI

Cryopreservation of epididymal sperm allows the saving of genetic material in case of unexpected death or emergency castration. The aim of the present study was the comparison of five different combinations of extenders commercially available for equine frozen semen processing for cryopreservation of epididymal sperm. Epididymal sperm were harvested from gonads of 10 healthy stallions after routine castration by retrograde flush technique. Then, samples were split and diluted with (1) INRA96 + INRA Freeze, (2) BotuSemen + BotuCRIO, (3) EquiPlus + Gent Freeze, (4) EquiPlus + EquiPlus Freeze, and (5) Gent + Gent Freeze. Extenders 1 and 2 showed higher values for total and progressive motility after thawing compared with extender 4 (P < .05). Extender 3 was in between 1 and 2 (P > .05), and extender 5 resulted in the lowest values (P < .05). The subpopulation of viable frozen-thawed sperm with high mitochondrial membrane potential and low intracellular calcium content was higher using extender 1 compared with extenders 3, 4, and 5 (P < .05) and higher in extender 2 compared with extenders 4 and 5 immediately after thawing (P < .05). After 1 hour of incubation, this subpopulation yielded the highest values in extender 2 (P < .05). Immediately after thawing, extender 1 yielded higher values for percentage of DFI and mean DFI than extenders 3, 4, and 5 (P < .05). Following 1 hour of incubation after thawing, sperm processed with extender 1 resulted in the highest values for percentage of DFI and mean DFI (P < .05). Using extender 2, mean DFI values were lower than those in extender 1 and higher than the extenders 3, 4, and 5 (P < .05). The study revealed that according to the examined sperm quality parameters, freezing extenders (extender 1, extender 2) using low concentrations of glycerol either combined with or without methylformamide were beneficial for cryopreservation of stallion epididymal sperm. For processing of stallion epididymal sperm, an extender containing milk proteins (extenders 1–4) for initial dilution after sperm harvesting is preferable to an extender including egg yolk (extender 5).

Effects of seminal plasma and flash-freezing on DNA structure of stallion epididymal sperm exposed to different potentiators of DNA damage

The tolerance of sperm DNA structure to seminal plasma and freezing conditions has both clinical and basic biologic relevance. In this study, fresh (FS) or flash-frozen (FZ) stallion epididymal sperm were exposed (SP+) or unexposed (SP−) to seminal plasma. Sperm were then evaluated to monitor the degree of change in DNA structure following challenge with chemical (dithiothreitol-DTT), oxidative (iron sulfate; FeSO4) or enzymatic (DNase I) potentiators of DNA damage. For sperm not treated with potentiators (controls), there was no effect of SP treatment (SP− vs. SP+) or freezing treatment (FS vs. FZ; non-significant) on measures of any DNA assays (i.e., 8-hydroxy, 2′deoxyguanosine [8OHdG], TUNEL, or sperm chromatin structure [SCSA] assays). Group FZ was more susceptible than Group FS to potentiators of DNA damage. Percent 8OHdG-positive sperm was higher in Group FZ/SP− treated with FeSO4 than all other groups (P < 0.05). Percent TUNEL-positive sperm was similar among FZ/SP− groups treated with DTT, FeSO4, or DNase (non-significant) and was higher in these groups than all other treatments (P < 0.05). Percent COMP-αt was higher following treatment with DNase or DTT, as compared to their respective controls, regardless of prior exposure to SP (P < 0.05). Overall, sperm DNA structure was unaffected by seminal plasma or freezing treatment when samples were not exposed to potentiators of sperm DNA damage; however, marked differences were identified in DNA structure when sperm were challenged with chemical, oxidative or enzymatic treatments. These results highlight the importance of challenging DNA structure prior to analysis. The use of potentiators of DNA damage provided a model to evaluate sperm DNA structure following exposure of sperm to various experimental treatments.

The Effect of Four Different Commercial Semen Extenders on the Motility of Stallion Epididymal Sperm

To preserve epididymal sperm, only a limited number of sperm is available, and an optimum processing method that is applicable for the majority of stallions is therefore crucial for successful preservation. The aim of the present study was to evaluate the effect of four different extenders that are commercially available for chilled semen on the motion characteristics of stallion epididymal sperm. Sperm were harvested by retrograde flush from 20 epididymides after the routine castration of 10 stallions. Aliquots of sperm samples were diluted with each of the four extenders ([E1] skim milk-based, [E2] containing defined milk protein, [E3] containing egg yolk, and [E4] containing caseinate). Total motility (TMOT %) and progressive motility (PMOT %) assessed immediately after sperm harvesting and during prolonged storage were highest in extenders with selected milk proteins (E2: 54, 24–79 and 50, 19–78; E4: 57, 23–82 and 52, 14–80) compared to the skim milk-based extender (E1: 40, 1–66 and 37, 0–62) and the egg yolk-containing extender (E3: 21, 6–48 and 13, 2–40; median, min–max for TMOT and PMOT, stored for 48 hours at 4°C, respectively). Motility values were similar for extenders E2 and E4 during the entire storage period (P > .05), while extenders E1 and E3 yielded significantly lower values (P < .05). Using E1, motility increased during the storage period but did not reach values similar to those of E2 or E4. Curvilinear velocity (VCL), amplitude lateral head displacement (ALH), and beat-cross frequency (BCF) differed among all extenders after sperm harvesting (P < .05), but not after 24 (VCL, ALH) and 48 hours (VCL, ALH, BCF; P > .05). Based on the motility results, we recommend extenders containing defined milk protein to process epididymal sperm.

Cushioned and Single Layer Centrifugation Improve Epididymal Stallion Sperm Motility Postcentrifugation

Traumatic injuries and sudden death can prematurely end the breeding career of a stallion. In such cases, a final spermatozoa collection can be obtained by harvesting the cauda epididymis. Semen samples can then be used for fresh artificial insemination or cryopreserved. Centrifugation is a critical point of sperm cryopreservation processing and can be detrimental to spermatozoa. Colloid centrifugation approaches reduce this physical damage and can be used to select better quality sperm. The aim of this research was to determine the effect of cushioned and single layer centrifugation (SLC) on epididymal stallion sperm motility postcentrifugation. Eight stallions were submitted to bilateral orchiectomy and the resulting epididymal cauda (n = 16) were flushed with semen extender. After harvesting, the samples were submitted to three centrifugation protocols: conventional (20 minutes at 600×g), cushioned (20 minutes at 900×g), and SLC (20 minutes at 300×g). The pellets were resuspended and sperm parameters were evaluated. Sperm morphology was evaluated under a phase-contrast microscope, total motility (TM) and progressive motility (PM) were evaluated with computer-assisted semen analyses. The proportion of morphologically normal spermatozoa was 72.2% for SLC, 72% for cushioned, and 67% for conventional (P > .05). After conventional centrifugation, it was recorded a TM of 7.4% and PM of 2.7%. After cushioned centrifugation, it was recorded a TM of 13.9% and PM of 6.5%. After SLC, it was recorded a TM of 46% and PM of 32.1%. The motility of spermatozoa recovered by SLC (P < .05) and cushioned centrifugation (P > .05) were higher than those recovered by conventional centrifugation. Colloids, including cushioned and SLC, improved epididymal stallion sperm motility postcentrifugation.

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.

Effect of single layer centrifugation on motility of epididymal stallion sperm – preliminary results

Effect of single layer centrifugation on motility of epididymal stallion sperm – preliminary results

Effects of Two Different Cooling Devices for Testicles Transport on Stallion Epididymal Sperm Quality

This study evaluates the effects of two cooling devices and temperature for testicles storage on epididymal sperm quality after 24 hours; different levels of seminal plasma (0% and 10%) were evaluated on sperm after recovering. Testicles from six stallions were recovered immediately after castration (2) or at the slaughterhouse (4); of the same animal, one testicle was placed in Equitainer (+8°C), the other in a styrofoam box with ice (+3°C). After 24 hours, the temperature of parenchyma was measured, and testicles and epididymal were weighted. Sperm were flushed from the cauda epididymides with Kenney extender, total sperm number recorded and motility and viability evaluated immediately after flushing (T0) with or without 10% SP (G1 Eq 0%, G2 Eq 10%, G3 Ice 0%, G4 Ice 10%). Motility and viability were evaluated after 24 hours and 48 hours of storage at +4°C. Temperature of the parenchyma was lower in testicles stored in ice compared to Equitainer (3.2 ± 0.6°C and 8.6 ± 2.5°C, respectively; P < .05). Motility and viability at T0 were similar (P > .05) in G1 and G3, whereas addition of SP after recovery significantly improved motility only in samples stored in Equitainer (G2). Viability was higher (P < .05) in G2 than in G4. At T24 and T48, no differences (P > .05) in sperm quality were found between storage methods or samples with or without SP. In conclusion, equine testicles can be safely stored either at lower (+3°C) or higher (+8°C) temperature than +5°C. This can be useful, especially when testicles are shipped in a hot climate, where devices cannot guarantee optimal refrigeration conditions.