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The Lidia bovine breed is an important hallmark of the Spanish cattle industry. Bulls are selected based upon aggressiveness and epididymal sperm cryopreservation is the way to obtain and store their genetics. There are not specifically designed protocols yet to perform Lidia bull sperm cryopreservation. The present study aimed to determine if a tris-fructose-citrate-egg yolk (20% v/v; TFY) extender supplemented with 7% glycerol (TFY1) or 3.5% glycerol plus 3.5% dimethylformamide (DMF; TFY2) are suitable media for cryopreservation of epididymal Lidia bull sperm. Moreover, the effect of N-acetylcysteine (NAC), a potent antioxidant, was evaluated. The epididymis were stored at 4°C for 24, 48, 72 or 96 h, and both freezing media were tested as such or supplemented with 1 or 2.5 mM of NAC. Our data demonstrated that post-thaw viability was well maintained (TFY1: 50.8% ± 1.9 at 24 h and 52.4% ± 0.8 at 96 h and TFY2: 52.6% ± 1.6 at 24 h and 56.1% ± 1.8 at 96 h; mean % ± SEM; p>0.05) as also were total and progressive sperm motility, high mitochondrial membrane potential, ROS production, DNA status and acrosomal intactness of Lidia bull sperm up to 96 h of epididymal storage, all extender variations being similar (p>0.05). In conclusion, the use of TFY medium supplemented either with 7% glycerol alone or the combination of 3.5% glycerol and 3.5% DMF were equally safe choices for epididymal Lidia bull sperm cryopreservation, and NAC addition did not significantly improve sperm post-thaw quality.

The interest to develop assisted reproductive technologies and cryobanking for farm animal genetic resource conservation has recently increased. However, cryopreservation for ex-situ management of genetic diversity sometimes is not routinely feasible, owing to the lack of facilities (AI centres, laboratories) and expertise near the local breed farming area. In these cases, epididymal sperm obtained from slaughtered or castrated animals, associated with the possibility of managing rather long periods between animal death, sperm recovery and freezing, would increase the opportunities to create semen storages. This investigation addresses the pre-freeze/post-thaw quality of goat epididymal sperm as a function of testicle storage temperature (environment or +5°C) and time elapsed between animal’s death and sperm recovery (0, 24, 48, 72 h) to establish the optimal protocols for the recovery and cryopreservation of epididymal sperm in this species. Testicles of 50 mature bucks collected at the abattoir were divided in two groups: half of the testicles (n=50) were transported to the laboratory at environment temperature (E), whereas the remaining half (n=50) at a refrigeration temperature (R) of +5°C. In the two groups (E) and (R), one testicle from each pair was processed after slaughter forming the time 0 groups (0E and 0R). The contralateral testicle was processed after 24, 48 or 72 h of storage, at the corresponding temperature. Sperm motility and kinetic parameters, viability and morphology were assessed in pre-freeze and post-thaw samples. Until 48 h postmortem, both E and R temperatures are able to maintain good pre-freeze epididymal sperm quality. After 48 h postmortem, R temperature is fundamental to reduce epididymal sperm quality decay in pre-freeze samples. Moreover, testicle refrigeration also has a positive impact on post-thaw samples, allowing a lower decline through time considering total motility, kinetics parameters, sperm viability and sperm abnormalities. Therefore, when sperm cryopreservation is not immediately practicable, goat testicles should be transported and stored at 5°C up to a maximum of 48 h postmortem to ensure an acceptable sperm quality.

Context:Ionidium suffruticosum(L.) Ging (Violaceae) is an important medicinal plant widely used as a herbal traditional medicine in Ayurveda for the treatment of infertility. Currently, little pharmacological information is available on its male fertility properties following prolonged use. Objective:To investigateI. suffruticosumleaf extracts for male fertility parameters. Materials and methods:The ethanol lyophilized fraction was administered orally on carbendazim-induced sub-fertility rats (250 mg/kg body weight for 28 days). The effects of fractions on rat’s fertility parameters i.e., body and testes weight, sperm motility, sperm vitality, epididymal sperm counts, its morphology, enzyme and antioxidant stress and histopathology were studied and compared with clomiphene citrate. Results:The sub-fertile male rats treated withI. suffruticosumleaf extract increased the body weight of 7 g, testis weight of 97 mg, increased cauda epididymal sperm counts of 34.2 × 106sperm/mL, motility of sperm 46% and vitality 28% also increased and normal sperm morphology also improved up to 32%. The carbendazim-treated group showed loss in body weight of 33 g, testis weight of 851 mg, decreased epididymal sperm counts of 15 × 106sperm/mL, with sluggish motility and a highly significant fall in the live sperms of about 57%. Discussion and conclusion:The leaf fraction ofI. suffructicosumincreased the testicular weight, spermatogenesis, sperm counts, lessened sperm agglutination, and increased testicular oxidative biomarkers, SOD, and CAT. This study therefore supports the usage ofI. suffructicosumin traditional medicine for infertility. [ABSTRACT FROM PUBLISHER]

This study was conducted to optimize the cryopreservation of epididymal bison sperm harvested in the field. In the first experiment, epididymal bison sperm were treated with or without seminal plasma (n = 6) and cooled to 5 °C over 2 hours. In a separate experiment, glycerol was added at different times and sperm was held at 5 °C for different periods of time before cryopreservation (n = 11). In addition, epididymal sperm frozen with and without seminal plasma (n = 6) and after 4, 24, and 48 hours (n = 5) of equilibration at 5 °C, were evaluated for their in vitro fertilizing ability. Post-thaw motility of bison epididymal sperm was similar when cryopreserved with or without seminal plasma or when glycerol was added at either 0, 4, 24, or 48 hours before freezing (P > 0.05). However, sperm incubated at 5 °C for 24 hours before freezing exhibited higher percentages of motile sperm (44% vs. 35% for 4 hours or 48 hours, P < 0.05). Fertilization rates of bison oocytes were not different for any treatments. Chilling the whole epididymis for 24 or 48 hours resulted in complete loss of sperm viability. In conclusion, bison epididymal sperm can be chilled outside of the epididymis for at least 48 hours before cryopreservation without compromising post-thaw sperm motility providing flexibility for technicians performing field collections. [ABSTRACT FROM AUTHOR]

This study compared three cryopreservation protocols on sperm functions, IVF outcomes, and embryo development. Epididymal spermatozoa cryopreserved using slow‑cooling (18% w/v raffinose, RS‑C) were compared with spermatozoa vitrified using 0.25 M sucrose (SV) or 18% w/v raffinose (RV). The motility, vitality, and DNA damage (TUNEL assay) of fresh control (FC) spermatozoa were compared with post-thawed or warmed RS‑C, RV, and SV samples. Mouse oocytes (n = 267) were randomly assigned into three groups for insemination: RV (n = 102), RS‑C (n = 86), and FC (n = 79). The number and the proportion of two‑cell embryos and blastocysts from each treatment were assessed. Sperm motility (P < 0.01) and vitality (P < 0.05) were significantly reduced after vitrification compared with slow‑cooled spermatozoa. However, DNA fragmentation was significantly reduced in spermatozoa vitrified using sucrose (15 ± 1.8% [SV] vs 26 ± 2.8% [RV] and 27 ± 1.2% [RS‑C]; P < 0.01). Although the number of two‑cell embryos produced by RS‑C, RV, and FC spermatozoa was not significantly different, the number of blastocysts produced from two‑cell embryos using RV spermatozoa was significantly higher than FC spermatozoa (P = 0.0053). This simple, small volume vitrification protocol and standard insemination method allows successful embryo production from small numbers of epididymal spermatozoa and may be applied clinically to circumvent the need for ICSI, which has the disadvantage of bypassing sperm selection. [ABSTRACT FROM AUTHOR]

Spotted buffalo, an exotic species that exists in Tana Toraja, South Sulawesi, Indonesia, is getting extinct due to high number of slaughtered during a funeral ceremony, called Rambu Solo’, as well as special treatments that do not allow the male spotted buffaloes perform natural mating activity. According to that, the research was trying to start conservation program by collected the cauda epididymal sperm soon after slaughtered. Two egg yolk-based extenders with different buffers, tris hydroxyl amino methane (TEY20) and trisodium citrate dehydrate (CEY20), were used as comparison to evaluate the post-thawed epididymal sperm quality and fertilizing capacity in artificial insemination program. The results showed that the post-thawed progressive motility of epididymal sperm was 40% and 39.17%, while viability was 65.99% and 63.26% and membrane integrity was 65.43% and 63.03% in TEY20 and CEY20 extenders, respectively. The success rate of pregnancy was 46.67% using post-thawed epididymal sperm in TEY20 and 40% using the one in CEY20 extenders. In conclusion, tris-based and citrate-based extenders have similar ability to maintain the epididymal sperm quality and its fertilizing capacity.

Objective: This study aims to evaluate the protective effects of American ginseng administered by gastric intubation on sperm vital quality in adult male rats treated with cyclophosphamide (CP).Materials and Methods: In this experimental study, 28 Adult male Wistar rats were assigned to four groups, seven rats in each. The animals allocated to control, CP treated, Ginseng treated and CP-Ginseng treated groups. Rats were treated with CP (6.1 mg/kg/day, i.p) for 6 weeks. American ginseng was used at a dose of 500 mg/kg/day during treatment. Sperm analysis (motion, count, morphology and viability) were evaluated at the end of the experiments. Sperm motion was assessed by Computer-Assisted Sperm Analysis (CASA). The data were analyzed using GB stat software. Probability values of p

AbstractFor evaluation of citric acid and citrate effects on bovine epididymal protoplasmic droplets, fifty bovine testes were collected in the October 2007 till June 2008 from Urmia slaughterhouse and transported to the laboratory in a cool container filled with 5 °C ice pack. Caudal epididymis was incised and sperm cells were put into Petri dishes containing hams f10 media with 10% fetal calf serum (FCS), which were kept in 37 °C, CO2 incubator. Then sperm cells were counted and 50-milion per mL concentration was prepared. After this stage, three dilutions of citric acid (0.1, 0.2, 0.3 N) and one dilution of citrate (1N), based on normal osmolarity and normal pH were added to a micro tube containing 25 milion per mL sperm. Then one-step eosin-nigrosin staining in 30-60-120-240-360 minutes was performed and slides were evaluated with 1000-x phase contrast microscope and 200 sperm cells per slide were counted. The results revealed significant difference between blank and citric acid 0.3 N. The proportion of protoplasmic droplet in group consisting of 0.3 N acid citric in 120-240-360 minutes, was significantly lower than that of blank (P < 0.05). There was no significant difference between citrate – blank and citric acid 0.1N-blank groups, but after 240 minutes significant difference was observed between blank & citric acid 0.2 N (P < 0.05). In conclusions citric acid based on dilution and time duration can reduce the proportion of bovine epididymal sperm cytoplasmic droplets.

Hypobaric hypoxia is of interest due to an increase of human populations working at high altitude. Testicular damage is related to the physiological response (neoangiogenesis) to increased intrascrotal blood flow as temperature rises. Hypoxia is a stress factor with overproduction of reactive oxygen species (ROS). The effect of hypoxia in mice reproductive parameters is analyzed. Animals were exposed to simulated hypoxia of 4,200 meters above sea level (m.a.s.l.) in a chamber for 33.2 days, both to continuous (HH) or intermittent hypoxia (HI) with an intermittency period of 4 days hypoxia /4 days normoxia (500 m.a.s.l.). The anti-inflammatory drug Ibuprofen was administered to a group of mice to control vasodilation and increased blood flow. Melatonin was administered to another group of mice as a potent ROS scavenger. Animals in both HH and HI exposure were compared to normoxic non-treated controls. There was a hematological response in hypoxia, with an increase in hematocrit and reticulocytosis. There was also increased teratozoospermia. This damage was more pronounced in HH than HI, suggesting that alternating normoxic periods permits compensation for the effects of hypoxia. In both hypoxia systems, the level of lipoperoxidation and the instability of DNA increased. In HH, there was a reduction of teratozoospermia in melatonin-treated mice. Ibuprofen presented a protective effect on the same parameters as melatonin with both HI and HH. The quality of sperm DNA, fragmentation, unpacking and DNA stability diminished. In conclusion, reproductive damage elicited by HH or HI was partially ameliorated by simultaneous treatment with antiflogistic and/or antioxidant agents.

Quality of epididymal and ejaculated sperms of spotted buffalo in three different extenders (i.e. Andromed, Andromed supplemented with 0.2% dextrose, and Andromed supplemented with 0.4% dextrose) was studied. The results showed that there was no significantly different (P > 0.05) quality of ejaculated and epididymal sperms. The motility percentage of post thawing epididymal and ejaculated sperms was 41-46% and 41-45%, respectively. And percentage of cytoplasmic membrane integrity of the post thawing epididymal and ejaculated sperms was 66-67% and 47-54% respectively. There was also no significant different (P > 0.05) in the percentage of cytoplasmic membrane integrity between the extenders of both ejaculated and epididymal sperms. This result suggested that epididymal sperms were reliable for artificial insemination as good as ejaculated sperms.

Quality of epididymal and ejaculated sperms of spotted buffalo in three different extenders (i.e. Andromed, Andromed supplemented with 0.2% dextrose, and Andromed supplemented with 0.4% dextrose) was studied. The results showed that there was no significantly different (P > 0.05) quality of ejaculated and epididymal sperms. The motility percentage of post thawing epididymal and ejaculated sperms was 41-46% and 41-45%, respectively. And percentage of cytoplasmic membrane integrity of the post thawing epididymal and ejaculated sperms was 66-67% and 47-54% respectively. There was also no significant different (P > 0.05) in the percentage of cytoplasmic membrane integrity between the extenders of both ejaculated and epididymal sperms. This result suggested that epididymal sperms were reliable for artificial insemination as good as ejaculated sperms.

The aim of this research was to observe the quality of spotted buffalo epididymal sperm after storage at 4 °C as liquid semen for 12 and 24 hours in andromed containing sucrose 0.2% and 0.4%. Sperm was collected from epididymal tissues by combination of slicing and pressing methods. The parameters observed were the percentage of progressive motility and live sperm. The results showed that the percentage of progressive motility of the liquid semen after storage for 12 hours in andromed (A), andromed + sucrose 0.2% (P1) and andromed + sucrose 0.4% (P2) were 48.33%; 53.33% and 55% (P>0.05), respectively. In addition, the percentage of motility after 24 hours of storage in those extenders were 45%; 46.67% and 46.67% (P>0.05), respectively. The percentage of live sperm in A, P1 and P2 after 12 hours of storage were 70.33%; 73% and 73.33% while after 24 hours of storage, the percentage of live sperm in those extenders were 66.33%; 68.67% and 69.67%, respectively. In conclusion, the addition of 0.2% and 0.4% sucrose in andromed extender could maintain the quality of the spotted buffalo epididymal sperm after storage for 12 and 24 hours at 4 °C.

h e objective of this study was to obtain the viability and plasma membrane integrity of the spotted buff alo epididymal sperm after addition of dextrose into Andromed® extender. Spermatozoa that have been collected from cauda epididymis were diluted with Andromed® extender as control (K) and Andromed® + 0.2% dextrose (P1) and Andromed® + 0.4% dextrose (P2) as treatments. h e results showed that the quality of epididymal spermatozoa decreased during cryopreservation process. h e percentage of motility after thawing in P1 (46%) and P2 (46.67%) were signifi cantly higher (P

The aim of this study was to investigate the effect of three concentration of citric acid on motility patterns of bovine epididymal sperms. For this purpose, 50 pairs of bovine testicles were collected immediately after slaughter form urmia abattoir and transferred to the laboratory alongside 5oc ice pack. Epididymal tail sperms were collected with a few incisions in vascular areas and transferred to hams f10 milieu with 10% fetal calf serum and counted after 15 minutes of incubation at 37oc in Co2 incubator. Concentrations of 50 million sperms per ml were proposal and in the normal sperm pH rang of 6.7-7.4, 0.1, 0.2 and 0.3 normal concentration of citric acid were added to sperm continuity micro tubes (normal concentration of acid equals 7 mg/ml of bovine serum) and at 15, 30, 45, 60, 90, 120, 180, 240 and 360 minutes the motility patterns of epididymal sperms were evaluated using the computer assisted sperms analyzing (CASA) method. Data were analyzed with one-way ANOVA using the SPSS 15 software. The results indicated significant differences in various indices of sperm motility patterns (Curvilinear Velocity, Straight-line Velocity, Average Path Velocity, Mean Angel Degree, Amplitude of Lateral Head Displacement, Beat-Cross Frequency, Linearity, Wobble) particularly at 0.3 normal concentration of citric acid compared with the control.

Abstract: In laboratory mice, sperm quality is usually assessed in spermatozoa collected from the cauda epididymidis of freshly sacrificed males. Percutaneous epididymal sperm aspiration (PESA) is a non-terminal alternative that would allow repeated sperm collection for sperm quality assessment in living males. To test whether PESA is a suitable method to assess sperm quality, we compared sperm traits between samples collected by PESA vs the commonly applied terminal cauda epididymidis dissection. The collected sperm samples were analyzed using computer-assisted sperm analysis and various parameters, including sperm motility, swimming velocity and morphology, were determined. We were able to retrieve motile sperm from all mice using PESA and the terminal cauda epididymidis dissection. Based on computer-assisted sperm analysis, however, sperm motility and swimming velocity were significantly lower after PESA compared to samples obtained by cauda epididymidis dissection. In addition, we found significantly more morphological abnormalities in PESA samples, probably induced as a side effect of the sampling technique. Although sperm samples collected by PESA are successfully used for in vitro fertilization, we cannot recommend PESA as a suitable method to assess sperm quality in mice, since the procedure seems to impair various sperm traits., Lay Summary: In mice, sperm quality is usually assessed in sperm collected from the epididymis (organ where ripe sperm is stored) of euthanized males. However, there is one non-terminal and minimal invasive alternative to collect sperm, called percutaneous epididymal sperm aspiration (PESA), which allows repeated sample collections from the same individual. Given that individual sperm quality is variable and can change according to various factors, PESA could allow to track sperm quality over time and would be highly appreciated in different research fields. Here, we tested the suitability of PESA to determine sperm quality by comparing sperm samples collected by PESA vs the commonly applied terminal epididymis dissection. We used computer-assisted sperm analysis to determine various sperm quality traits. Surprisingly, we found that sperm collected by PESA showed significantly reduced motility, swimming velocity and more morphological abnormalities compared to sperm samples collected by epididymis dissection. Thus, we cannot recommend PESA as a suitable method to determine sperm quality traits as the procedure itself seems to affect collected sperm cells.

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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. [ABSTRACT FROM AUTHOR]

Epididymal alpaca sperm represent an alternative model for the study of alpaca semen. The objective of this study was to characterize the normal values of some functional variables in epididymal alpaca sperm using imaging flow cytometry. Alpaca testicles ( n = 150) were processed and sperm were recovered from the cauda epididymides. Only 76 samples with acceptable motility and sperm count were considered for assessment by imaging flow cytometry. Acrosome integrity and integrity/viability were assessed by FITC-PSA/PI and FITC-PNA/PI. Mitochondrial membrane potential (MMP) was assessed by MitoTracker CMXRos and MitoTracker Deep Red FM. Lipid peroxidation was evaluated using BODIPY 581/591 C11. Results show that the mean values for acrosome-intact sperm were 95.03 ± 6.39% and 93.34 ± 7.96%, using FITC-PSA and FITC-PNA, respectively. The mean values for acrosome-intact viable sperm were 60.58 ± 12.12% with FITC-PSA/PI and 58.81 ± 12.94% with FITC-PNA/PI. Greater MMP was detected in 65.03 ± 15.92% and 59.52 ± 19.19%, using MitoTracker CMXRos and MitoTracker Deep Red FM, respectively. Lipid peroxidation was 0.84 ± 0.95%. Evaluation of acrosome-intact and acrosome-intact viable sperm with FITC-PSA/PI compared with. FITC-PNA/PI or MMP with MitoTracker CMXRos compared with MitoTracker Deep Red FM were correlated ( P < 0.05). The MMP using MitoTracker CMXRos was the only variable correlated ( P < 0.05) with sperm motility ( r = 0.3979). This report provides a basis for future research related to alpaca semen using the epididymal sperm model. [ABSTRACT FROM AUTHOR]

Epididymal sperm preservation can be used to avoid the total loss of genetic material in threatened species. Spermatozoa from the corpus, as from the cauda, are motile and can undergo capacitation. Thus, they can potentially be preserved for assisted reproductive technologies. However, cryopreservation of spermatozoa has a direct detrimental effect on sperm quality. The aim of this study was to compare the chromatin stability and the survival rate of spermatozoa from the corpus and cauda epididymis after cryopreservation. Epididymal spermatozoa were collected and cryopreserved from the corpus and cauda of 12 domestic cats. Sperm motility, progressive motility, membrane integrity, acrosome integrity, and DNA integrity were evaluated before and after freezing thawing. The average total number of spermatozoa collected from the corpus was lower (10.2 × 10 6 ± 7.4) than that from the cauda epididymis (24.9 × 10 6 ± 14.4; P = 0.005). The percentage of spermatozoa with intact DNA did not differ significantly whether it was collected from the corpus or cauda regions and did not decrease after freezing thawing in either region. However, motility of spermatozoa from both regions was affected by the freezing thawing process with a significant decline in motility after thaw compared with fresh spermatozoa. A significant difference in the percentage of motile sperm between the corpus and cauda was observed after the freezing thawing process (P < 0.001). Although sperm motility was lower in postthaw spermatozoa from the corpus epididymidis than from the cauda, the rate of the reduction did not differ between regions. This study indicates that the cryopreservation process does not have a negative effect on chromatin stability of feline epididymal spermatozoa. Spermatozoa from the corpus region have a similar freezability as spermatozoa from the cauda region. Therefore, preservation of spermatozoa from the corpus and the cauda epididymidis might be of value in preserving genetic material from endangered or valuable felids. [ABSTRACT FROM AUTHOR]

The aim of this study was to evaluate relationships among body weight, sexual secondary traits and epididymal semen quality in young bucks. Body weight (BW), scrotal circumference (SC), horns diameter (HD), and horns length (HL) were recorded monthly on 54 young Alpine bucks, from the second to the eighth month of life. After slaughter, testicles weight (TW) and epididymal weight (EW) were recorded and epididymal sperm quality was evaluated. Epididymal sperm recovery was performed by using the retrograde flushing technique and sperm concentration, motility, viability and morphology were evaluated. Positive relationships between BW with TW ( P < 0.001) and EW ( P < 0.05), and SC with TW ( P < 0.05) were observed. Some effects of secondary sexual traits on epididymal semen quality parameters were detected: BW and TW on sperm production (concentration, total number of sperm and number of semen doses) ( P < 0.05), and SC on the presence of immature sperm ( P < 0.05). In particular, it was shown that in goat species horns size is related to semen quality in terms of sperm production (concentration, total number of sperm and number of semen doses) and presence of normal sperm ( P < 0.05). In conclusion, scrotal circumference and horns size could be used as parameters to select animals for breeding. [ABSTRACT FROM AUTHOR]