Search Results (21)

The conservation of small and genetically vulnerable brown bear populations, such as the Cantabrian subpopulation in Spain, depends on developing species-specific assisted reproductive technologies and genetic resource banks. However, the lack of standardized sperm collection and cryopreservation protocols hinders their application. This study provides the first comparative analysis of sperm quality and proteomic profiles from three different origins: epididymal, pre-ejaculated, and ejaculated. Sperm quality parameters —motility and kinetic, viability, apoptosis, and oxidative stress— and protein expression were assessed. Although yields were similar, marked differences were observed in sperm quality and protein profiles. Sixty-three proteins involved in metabolism, stress response, and oxidative balance were differentially expressed depending on sperm origin. Epididymal sperm showed the highest viability and motility, lowest apoptosis, and a proteomic profile indicative of active spermatogenesis and enhanced oxidative stress defense. In contrast, ejaculated sperm had increased oxidative stress and reduced expression of metabolic proteins, while pre-ejaculated sperm exhibited lower motility, likely due to urine contamination and mitochondrial protein alterations, despite comparable viability and apoptosis. These findings offer novel insights into brown bear sperm biology and highlight the importance of sperm origin in developing optimized assisted reproduction strategies, ultimately supporting ex situ conservation efforts for this species. Full article

This study evaluated the post-thaw motility and in vitro fertility of ejaculated and epididymal semen from Pantaneiro bulls and characterized cell-free DNA (cfDNA) in fresh seminal plasma. Semen from five bulls was collected via electroejaculation or post-mortem epididymal extraction. Fresh semen parameters and cfDNA concentrations were assessed before cryopreservation. Post-thaw sperm kinetics were evaluated using CASA at 0 and 6 h of incubation, and in vitro embryo development was analyzed following IVF. Data were assessed using ANOVA and logistic regression. Ejaculate samples exhibited more morphological defects than epididymal samples (15.8% vs. 1.8%, p ≤ 0.05). Post-thaw, epididymal semen showed higher total (87.2% vs. 32.4%) and progressive (67.1% vs. 14.4%) motility at 0 h (p ≤ 0.05), and higher motility at 6 h (38.9% vs. 11.0%, p ≤ 0.05). In vitro fertility did not differ significantly between ejaculated (n = 525 oocytes) and epididymal (n = 500 oocytes) semen groups in terms of cleavage (49.6% vs. 44.2%) and blastocyst formation on D7 (26.1% vs. 22.2%, p > 0.05). cfDNA concentration in fresh semen ranged from 11.4 to 50.9 ng/µL. These findings indicate that epididymal sperm from Pantaneiro bulls retain high post-thaw motility and fertility. Additionally, cfDNA characterization in seminal plasma contributes to indigenous cattle preservation and advances in male fertility research. Full article

Sexual rest (SR) in bulls leads to the accumulation of senescent spermatozoa in the extragonadal reserves, potentially affecting semen quality and reproductive efficiency. Therefore, this study aimed to investigate the impact of SR on the seminal plasma proteome and oxidative status of Nellore bulls. Six adult bulls were subjected to 195 days of SR and sequential semen collections using the electroejaculation method. The ejaculates were analyzed to assess sperm quality. Seminal plasma from the first and last ejaculates was evaluated for oxidative status and proteomic profile using LC-MS. The results revealed significant improvements in sperm motility, vigor, and antioxidant enzyme activity (superoxide dismutase and catalase) in the last ejaculate compared to the first. Conversely, higher levels of oxidative markers, such as malondialdehyde and carbonyl proteins, were observed in the first ejaculate. Proteomic analysis identified 156 proteins, with 28 differentially abundant between ejaculates. The first ejaculate showed a higher abundance of proteins linked to acrosomal exocytosis and energy metabolism, while proteins associated with sperm motility and immune modulation were elevated in the last ejaculate. These findings suggest that SR induces oxidative stress and proteomic alterations in seminal plasma, negatively affecting sperm quality, emphasizing the need for strategic reproductive management in bulls. Full article

Assisted reproduction techniques play a significant role in veterinary medicine, and although they are widely used in domestic animals, they are also becoming increasingly relevant in clinical practice for wild felids, especially in the conservation efforts for endangered species. In this study, the result of two semen collection techniques, urethral catheterization after pharmacological induction (Ur.Ca.P.I.) and electroejaculation, are described, aiming to provide new practical information about sperm collection using the Ur.Ca.P.I. technique and electroejaculation in tigers and leopards, describing the authors’ experience and presenting new data and observations. The following descriptive study included two subjects of Panthera tigris species and two of Panthera pardus. These subjects, after general anesthesia, underwent sperm collection initially with Ur.Ca.P.I. and, subsequently, with electroejaculation. Sampling was made possible in both species thanks to the use of electroejaculation. Sperm volumes in leopards ranged from 0.3 to 0.5 mL and in tigers from 0.5 to 2.177 mL. Sperm concentration in leopards ranged from 136 × 10⁶ to 280 × 10⁶ sperm/mL, and in tigers, from 21.5 × 10⁶ to 354 × 10⁶ sperm/mL. Urethral catheterization gave positive results in leopards, with sperm volumes ranging from 25 up to 150 µL and a concentration ranging from 110 × 10⁶ up to 1082 × 10⁶ sperm/mL. In tigers, unlike in leopards, the use of the Ur.Ca.P.I. technique encountered difficulties that did not allow satisfactory results to be obtained. Therefore, it would be useful to test the feasibility of urethral catheterization on a larger group of individuals in order to have more meaningful feedback. Finally, because electroejaculation always allowed semen collection in tigers, with a higher sperm quality than samples collected by Ur.Ca.P.I., we currently consider it the technique of choice for the collection of semen material in this species. Full article

The objective of the present study was to determine the effects that the reproductive season has on the motility, kinematics, morphology, and sperm morphometry of Brahman bulls evaluated with a commercial CASA system. The experiment was carried out at the Costa Rica Institute of Technology from March to August 2021. A total of eight Brahman bulls were used. A total of 28 ejaculates were collected in the pre-mating period (PMP), during it (DMP), and after it (AMP) using an electroejaculator. The sperm concentration was measured with the Accuread photometer. The motility was measured using a Spermtrack^® counting chamber. The analyses were performed with the CASA-Mot ISAS^®v1 system. The morphology was analyzed using a microscope with a negative phase contrast objective. Morphometry was evaluated with the CASA-Morph. The sperm concentration did not present differences between the PMP and AMP; however, it was significantly higher than DMP (p > 0.05). Regarding the progressiveness variables, linearity on forward progression (LIN), straightness (STR), and wobble (WOB) were higher (p < 0.05) DMP. A kinematic principal component analysis grouped all the variables into three factors and an effect on the reproductive period was found (p < 0.05) in the parameters of the head and middle part of the sperm, such as width and perimeter, which were greater in the PMP. The length of the sperm head in the PMP and DMP did not show differences; however, both were larger (p < 0.05) than AMP. The insertion distance of the middle piece of the sperm was significantly greater than DMP. Finally, the PMP contained cells with a larger insertion angle (p < 0.05) than AMP. These findings are important to understand the implications of reproductive status on sperm quality and to consider them in andrological evaluations. Full article

This study aimed to evaluate the proteomic profile of seminal plasma from young Nellore bulls. We used 20 bulls aged between 19.8 and 22.7 months, divided into two groups according to the results of the Breeding Soundness Evaluation (BSE): approved (FIT n = 10) and not approved (UNFIT n = 10). The scrotal perimeter was measured and a semen collection was performed through electroejaculation. The percentage of sperm motility, mass motility, and sperm vigor were calculated using conventional microscopy, and the percentage of sperm abnormalities was calculated using phase-contrast microscopy of all ejaculates. Seminal plasma was separated from spermatozoa using centrifugation and processed for proteomic analysis by LC-MS/MS. Seminal plasma proteins were identified using MASCOT Daemon software v.2.4.0 and label-free quantification analysis was carried out by SCAFFOLD Q+ software v.4.0 using the Exponentially Modified Protein Abundance Index (emPAI) method. Functional classification of proteins was performed based on their genetic ontology terms using KOG. Functional cluster analysis was performed on DAVID. There were no differences in scrotal perimeter and physical semen characteristics between FIT and UNFIT groups of bulls. The percentage of sperm abnormalities was higher (p < 0.05) in the UNFIT group of bulls. A total of 297 proteins were identified for the two groups. There were a total of 11 differentially abundant proteins (p < 0.05), two of them more abundant in FIT bulls (Spermadhesin-1 and Ig gamma-1 chain C region) and nine in UNFIT bulls (Vasoactive intestinal peptide, Metalloproteinase inhibitor 2, Ig lambda-1 chain C regions, Protein FAM3C, Hemoglobin beta, Seminal ribonuclease, Spermadhesin 2, Seminal plasma protein BSP-30kDa, and Spermadhesin Z13). Spermadhesin-1 was the protein with the highest relative abundance (36.7%) in the seminal plasma among all bulls, corresponding to 47.7% for the FIT bulls and 25,7% for the UNFIT bulls. Posttranslational modification, protein turnover, and chaperones were the functional categories with the highest number of classified proteins. Protein functional annotation clusters were related to Phospholipid efflux, ATP binding, and chaperonin-containing T-complex. The differentially abundant proteins in the group of FIT bulls were related to sperm capacitation and protection against reactive species of oxygen. In contrast, differentially expressed proteins in the group of UNFIT bulls were related to motility inhibition, intramembrane cholesterol removal and oxidative stress. In conclusion, the proteomic profile of the seminal plasma of FIT bulls presents proteins with participation in several biological processes favorable to fertilization, while the proteins of the seminal plasma of UNFIT bulls indicate a series of alterations that can compromise the fertilizing capacity of the spermatozoa. In addition, the relative abundance of spermadhesin-1 found in the seminal plasma of young Nellore bulls could be studied as a reproductive parameter for selection. Full article

Stallion sperm analysis is indicated for infertility diagnosis, pre-sale expertise, production of fresh or frozen doses, and frozen straw quality control. Various collection methods are described, and numerous assays can be performed on semen. Determining an approach for each of these cases is challenging. This review aims to discuss how to obtain relevant clinical results, answering stallion owners’ concerns. Semen can be collected with an artificial vagina on a phantom or a mare, by electro-ejaculation under anesthesia, or after pharmacological induction. The collection method influences the semen volume and concentration, while the total sperm number depends on the testicular production and collection frequency. In the seminal plasma, acidity, pro-oxidant activity, and some enzymes have repercussions for the semen quality and its conservation. Moreover, non-sperm cells of seminal plasma may impact semen conservation. Motility analysis remains a core parameter, as it is associated with fresh or frozen dose fertility. Computer-assisted motility analyzers have improved repeatability, but the reproducibility between laboratories depends on the settings that are used. Morphology analysis showing spermatozoa defects is useful to understand production and maturation abnormalities. Staining of the spermatozoa is used to evaluate viability, but recent advances in flow cytometry and in fluorochromes enable an evaluation of multiple intracellular parameters. Spermatozoa protein expression already has clinical applications, for example, as a fertility and freezing ability predictor. At present, stallion semen analysis ranges from macroscopic evaluation to assessing spermatozoa proteins. However, clinically, all these data may not be relevant, and the lack of standardization may complicate their interpretation. Full article

The Bengal tiger (Panthera tigris tigris) is critically endangered, so assisted reproductive technologies, including artificial insemination, are important conservation tools. For wild and domestic felids, electroejaculation (EE) is the most common semen collection method, with protocols optimized to obtain sufficient amounts of viable sperm for artificial insemination. However, less attention has been paid to ensuring animal wellbeing during the process. This study examined the effects of three EE protocols (Low, 2–5 volts; Medium, 3–6 volts; High, 4–7 volts) on semen quality, testicular size, serum testosterone, creatine kinase (CK), and malondialdehyde (MDA) concentrations, and serum cortisol as a proxy for stress. Blood samples were collected before, during, and after each EE series. Seminal plasma pH, and sperm motility, viability, and morphology were evaluated after each procedure. Seminal plasma and sperm pellet MDA concentrations were also determined. Primary sperm abnormalities and seminal plasma MDA were higher in the Low compared to Medium and High voltage groups (p < 0.05). Serum CK in the High voltage group increased during the EE series (p < 0.05), suggesting the potential for muscle damage. However, no significant changes were observed for serum cortisol, testosterone, or MDA concentrations. Results suggest the Medium voltage protocol produced good quality samples at lower voltages than the High protocol with no negative effect on muscle function, which might be better for animal welfare. Full article

Sperm quality decreases over time, so bull semen may need to be preserved after field collection. However, the effect of handling such semen samples from commercial farms and placing them in very short–term storage has not been elucidated. Therefore, ejaculate from 25 bulls from 1 dairy and 14 beef cattle farms were collected under farm conditions and evaluated for semen quality during the first two hours after collection. Two commercial extenders (AndroMed^® and BIOXcell^®) and two different storage temperatures (5 °C and room temperature) were used to evaluate the influence on semen quality and sperm kinetics in ejaculates grouped into three evaluation times, based on time since collection (Time 1: <75 min, n = 7; Time 2: 75–105 min, n = 11; and Time 3: 105–120 min, n = 7). Classical semen parameters, sperm motion kinetics by CASA and colony-forming units were assessed. The differences between both extenders in curvilinear and straight–line velocities (VCL and VSL) for the different time groups (Time 2 and Time 3) were statistically significant for p < 0.05. AndroMed^® showed lower VSL, straightness and linearity in sperm compared to BIOXcell^® (p < 0.05). In conclusion, AndroMed^® induced more curvilinear movement, while BIOXcell^® stimulated straighter motility. Full article

The semen of domestic mammals is conventionally collected with an artificial vagina (AV) for artificial insemination (AI) or for short- or long-term storage. However, the procedure has certain drawbacks: animal training is not feasible in extensive animal care systems nor among wild species, as the trained animals sometimes fail to mount. Hence, there is a need for alternative semen collection methods. Electroejaculation (EEJ) and epididymal sperm recovery (ESR) are the two effective alternatives to AV. However, in recent years, animal welfare campaigners have called for the ban, in certain EU countries, of EEJ due to its inhumane nature. In this review, alternative methods of sperm collection (by EEJ and ESR, their qualities, and their freezing techniques) are highlighted, as well as the effects of EEJ on pre-freeze and post-thaw ram sperm quality parameters and the animal welfare progress made in EEJ between the 20th and 21st centuries. Additionally, the techniques for enhancing post-thaw sperm quality prior to freezing and for the freezing of EEJ and ESR spermatozoa are explored. ESR and EEJ are reliable alternatives to AV on certain occasions. EEJ is ideal for semen collection in wild or untrained animals, breeding soundness examinations, collection outside of the breeding season, and culling. At the same time, ESR is ideal in cases of castration, accidental death of elite sire, or postmortem for gene conservation purposes or assisted reproductive technologies (ARTs) studies. Full article

In the presented study, the effects of ROCK inhibitor Y-27632, antifreeze protein III, and boron at two different doses were investigated on the spermatological parameters of Ankara buck semen after freeze–thawing. Ejaculates were collected from bucks using an electroejaculator during the breeding season. The ejaculates that showed appropriate characteristics were pooled and used in the dilution and freezing of semen. The extender groups were formed by adding two different doses of three different additives (ROCK inhibitor Y-27632, 5 and 20 µM; antifreeze protein III, 1 and 4 µg/mL; boron, 0.25 and 1 mM) to the control extender. The semen was diluted with the different extenders at 35–37 °C and loaded into straws. Sperm samples frozen in liquid nitrogen vapors, following equilibration, were stored in liquid nitrogen. It was observed that extender supplementation improved post-thaw motility of Ankara buck semen after freeze–thawing. Differences were significant (p < 0.01) for 5 and 10 µM doses of ROCK inhibitor (71.82% and 74.04 % motility), as well as for 0.25 and 1 mM doses of boron (76.36% and 72.08% motility), compared to the control group (66.15% motility). With respect to the evaluation of acrosomal integrity and mitochondrial activity after freeze–thawing, although supplementation provided protection at all doses, the efficacy was not statistically significant (p > 0.05). It was observed that DNA damage was improved by antifreeze protein III at 1 µg/mL (1.23% ± 0.23%) and by boron at all doses (0.25 mM: 1.83% and 1 mM: 1.18%) compared to the control group (3.37%) (p < 0.01), following the thawing process. In the present study, it was determined that some additives added to the extender provided significant improvements in buck spermatozoa motility and DNA damage after thawing. Full article

Spinal cord injury (SCI) is a relevant medical and social problem. According to the World Health Organization, the commonly estimated worldwide annual incidence of SCI is 40 to 80 cases per million population. After the SCI experience, most men present with sexual dysfunction (erectile dysfunction (ED) and ejaculatory dysfunction), fertility problems (such as impaired spermatogenesis, abnormalities in sperm viability, motility, and morphology), and systemic disorders such as genitourinary infection and endocrine imbalances. The best options available for managing the ejaculatory disorders in patients suffering from SCI are penile vibratory stimulation (PVS) and electroejaculation (EEJ). Furthermore, the treatment of ED in SCI patients consists of medical therapies including phosphodiesterase 5 inhibitors (PDE5i), intracavernosal injections (ICI), vacuum erection devices (VEDs), and surgical as penile prosthesis (PP). This review provides a snapshot of the current evidence for the mechanisms of sexual dysfunction and infertility in SCI patients, discusses the best management strategies for these conditions, and offers our perspective on the direction of future research. Full article

The aim of our research was to compare three Slovak sheep breeds in the quality parameters of cryopreserved sperm. The ejaculates of Slovak Dairy (SD), Native Wallachian (NW), and Improved Wallachian (IW) sheep rams (n = 12) were collected by electro-ejaculation. Heterospermic samples were created from suitable ejaculates, separately for each breed (at least 90% of total and 80% of progressive motility). Samples were equilibrated in a Triladyl^® diluent and frozen by automated freezing. Sperm samples were subjected to the motility, morphology, (CASA), viability and apoptosis (DRAQ7/Yo-Pro-1), fertilizing capability (penetration/fertilization test (P/F) in vitro) and acrosomal status (transmission electron microscopy) assays before freezing and after thawing. It was found that there were no significant differences (p < 0.05) between the evaluated breeds in motility, viability, apoptosis, morphological properties, and fertilizing ability of cryopreserved sperm. Significant differences occurred in acrosomal status. Our results demonstrate that the use of the selected cryopreservation protocol is suitable for at least three different sheep breeds, which can greatly benefit the biodiversity protection and simplifies the creation of an animal genetic resources gene bank. Full article

Assisted reproductive technologies can aid conservation efforts via support of ex situ population management and preservation of genetic material. Data from 38 sperm collection attempts from 17 polar bears (1–5 procedures/bear) were evaluated. Sample collections were attempted via electroejaculation (EEJ; n = 6), urethral catheterization (UC; n = 25), or sperm rescue (SR; n = 7) during the breeding season (Jan. 1-May 21; n = 27) and nonbreeding season (May 22-Dec. 31; n = 11). Sperm retrieval was successful in 1 EEJ (16.7%), 18 UC (72.0%) and 4 SR (57.1%) collections. Initial sperm motility and viability were 50.0% and 77.0% for EEJ, 64.3 ± 7.4% and 80.9 ± 3.8% for UC, and 56.7 ± 8.8% and 80.5 ± 0.5% for SR. UC and SR were more likely to be successful during the breeding season (84.2–100%) than the nonbreeding season (25.0–33.3%). Testicular tumors were observed in four males (57%) during SR. In total, 13 samples were cryopreserved (n = 1 EEJ, 9 UC, and 3 SR) with egg-yolk-based equine extender (EQ) or OptiXcell (OP). For both extenders, post-thaw motility and viability were reduced by 20–60% and 30–65%, respectively. Further efforts to optimize procedures are warranted, but this summary provides data useful for enhancing the success of polar bear sperm collection and cryopreservation. Full article

The preservation of rhinoceros semen is vital for captive breeding programs. While successful collection and cryopreservation of rhinoceros semen has been reported, the volume and quality of semen produced is often low due to the high viscosity associated with ejaculates collected via electroejaculation. Reducing semen viscosity would enable access to previously unusable spermatozoa from viscous fractions and could improve quality post-thaw. The enzyme papain successfully reduced the viscosity of camelid semen but has yet to be tested in wildlife species. This study assessed the influence of papain on the in vitro quality of rhinoceros spermatozoa during cryopreservation using advanced semen assessment. In experiment 1, the motility of spermatozoa from the viscous fraction of an ejaculate, either untreated or treated with papain and its inhibitor E-64 prior to cryopreservation, was assessed post-thaw. In experiment 2, spermatozoa from papain-treated viscous fractions were compared to spermatozoa frozen from untreated sperm-rich fractions pre-freeze, as well as after 0, 1.5 and 3 h of incubation post-thaw (37 °C). Papain significantly increased the quantity of spermatozoa collected from ejaculates, as well as the motility prior to freezing. Papain also improved the post-thaw motility, velocity, linearity and straightness of samples compared to sperm-rich samples, with no detriment to sperm viability, lipid membrane disorder, production of ROS or DNA integrity (p < 0.05). Results show the benefit of supplementing rhinoceros spermatozoa with papain prior to cryopreservation on sperm cryosurvival and demonstrates the potential of using papain to improve the success of cryopreservation protocols, not only for the rhinoceros, but also for other wildlife species. Full article