Search Results (33)

Omega-3 polyunsaturated fatty acids (n-3 PUFAs) are recognized for their beneficial effects on male fertility. This study evaluated the protective effects of dietary n-3 PUFAs from extruded linseed, alone or combined with the alga Padina pavonica, against in vitro lipopolysaccharide (LPS)-induced sperm dysfunction in rabbits. Twelve bucks were fed for 60 days a control diet (CNT), a diet containing 5% extruded linseed (L), or 5% extruded linseed plus 0.2% P. pavonica extract (LPP). Ejaculates were exposed in vitro to increasing LPS concentrations (0, 400, and 600 µg/mL), and sperm motility was evaluated at 0, 1, 2, and 4 h using computer-assisted sperm analysis. LPS markedly impaired sperm motility in the CNT group, increasing the percentage of static spermatozoa (p < 0.001) and reducing sperm progressive motility (p < 0.001), with complete immobility observed at 600 µg/mL after 4 h. Conversely, sperm from L and LPP groups maintained significantly higher progressive motility, lower static sperm, and improved kinematic parameters throughout the LPS challenge (p < 0.05). Dietary n-3 PUFA supplementation also attenuated LPS-induced TLR4 activation and reduced lipid peroxidation, as indicated by lower seminal TBARS levels. No histological alterations were detected in the male reproductive tract. These findings indicate that n-3 PUFA supplementation, particularly linseed combined with algae, mitigates LPS-induced sperm dysfunction in vitro. Full article

The utility of bioresponsive multifunctional hydrogel compositions for biomedical applications is rapidly increasing due to the diverse array of biological stimuli that can profoundly alter physicochemical gel properties that benefit therapeutic interventions. The purpose of this research is to explore a bioresponsive hydrogel as a drug-free bioengineering concept to fortify the natural physical contraceptive barriers at the cervicovaginal junction. The results of this research demonstrate that a hydrogel comprising 4% (w/w) Carbopol^® 974P and 4% (w/w) polyvinylpyrrolidone (CP4%/PVP4%) undergoes bioresponsive structural changes in the presence of simulated seminal fluid, pH 7.7, (SFS) that increases the work required to spread the gel under physiologically relevant vaginal conditions. Combination of this bioresponsive hydrogel with liquified human semen at a volumetric ratio of 1:5 dramatically reduces in vitro sperm migration by 97%. Simultaneously, total sperm motility decreases from 72.0 ± 9.9% to 7.9 ± 13.7%, which is significantly below the WHO criteria defined for male fertility. Safety assessments performed in vitro and in vivo underline a robust vaginal safety profile comparable to approved vaginal products. Moreover, the results from an exploratory animal study performed with female New Zealand White rabbits suggest that the drug-free physical barrier established intravaginally after exposure of the bioresponsive CP4%/PVP4% hydrogel to alkaline semen seems at least equivalent in the prevention of pregnancy in vivo to the VCF^® Gel (Apothecus Pharmaceuticals, Ronkonkoma, NY, USA), a marketed spermicidal on-demand product containing nonoxynol-9. Full article

Artificial insemination (AI) in rabbits depends largely on chilled semen storage, but the physiological responses to chilling and associated biochemical changes in seminal plasma (SP) remain poorly understood, particularly across breeds. This study aimed to compare the semen preservation capacity of Algerian local population (LAP) and New Zealand White (NZW) rabbits and to explore the relationship between SP oxidative stress biomarkers and sperm traits during 72 h of chilled storage at 5 °C. Semen pools (nine/breed) were evaluated at 0, 4, 24, 48, and 72 h for motility, viability, and acrosome status. Oxidative stress markers were also assessed in the SP, including malondialdehyde (MDA), reactive oxygen metabolites (ROMs), superoxide dismutase (SOD), glutathione peroxidase (GPX), and catalase (CAT). LAP sperm showed higher motility (p < 0.001) and viability (p < 0.05), particularly between 4 h and 48 h, and exhibited a lower rate of acrosome reaction (p < 0.001) from 48 h to 72 h. Lower SOD and higher CAT activity in LAP (p < 0.001), correlated with MDA and acrosome status, respectively, may reflect a more balanced antioxidant response. Lipid peroxidation did not appear to be the main factor driving sperm deterioration (p > 0.05). These results demonstrate that LAP rabbits exhibit better resilience to chilled storage compared to NZW and highlight the potential value of CAT and SOD activities as indicators of sperm resilience during chilled storage. Further studies are required to validate and extend these findings, with the aim of improving semen preservation strategies. Full article

The aim of this study was to analyze how recombinant rabbit NGF (Nerve Growth Factor) encapsulated in chitosan (rrβNGFch) affects sperm viability, motility, capacitation, acrosome reaction (AR), kinetic traits, and apoptosis after 30 min and 2 h of storage. Specific intracellular signaling pathways associated with either cell survival, such as protein kinase B (AKT) and extracellular signal-regulated kinases 1/2 (ERK1/2), or programmed cell death, such as c-Jun N-terminal kinase (JNK), were also analyzed. The results confirmed the effect of rrβNGFch on capacitation and AR, whereas a longer storage time (2 h) decreased all qualitative sperm traits. AKT and JNK did not show treatment-dependent activation and lacked a correlation with functional traits, as shown by ERK1/2. These findings suggest that rrβNGFch may promote the functional activation of sperm cells, particularly during early incubation. The increase in capacitation and AR was not linked to significant changes in pathways related to cell survival or death, indicating a specific action of the treatment. In contrast, prolonged storage negatively affected all sperm parameters. ERK1/2 activation correlated with capacitation, AR, and apoptosis, supporting its role as an NGF downstream mediator. Further studies should analyze other molecular mechanisms of sperm and the potential applications of NGF in assisted reproduction. Full article

The cryopreservation of rabbit semen is a valuable strategy for genetic resource preservation and efficient artificial insemination, but outcomes remain inconsistent, partly due to variations in sperm concentration per dose. This study aimed to evaluate the in vivo effects of different sperm concentrations (15, 25, 35, 55, and 75 million per straw) on fertility, prolificacy, and offspring growth in nulliparous and multiparous does. A total of 384 rabbit females were inseminated using frozen–thawed semen, and their reproductive performance was compared with fresh semen. Fertility and kindling rates varied with sperm concentration and parity: nulliparous does showed the highest fertility at 15 million sperm/straw (84.4%), while multiparous does reached peak values at 25–55 million/straw (78.1–81.3%). Litter size and live-born kits were consistently higher in multiparous than in nulliparous does. Offspring body weight at 19 and 60 days was influenced by both sperm concentration and maternal parity, with better growth generally observed in multiparous groups. Weaning success remained high across all groups. Our results indicate that sperm concentrations ranging from 15 to 35 × 10⁶/straw are the most suitable for cryopreservation, as they maintain high fertility, prolificacy, and offspring growth, comparable to fresh semen. These results confirm that optimizing sperm concentration during cryopreservation improves reproductive efficiency and that tailoring insemination strategies to the physiological status of the female enhances outcomes. The results provide useful recommendations for improving cryopreservation techniques in rabbit breeding programs. Full article

High-fat diets (HFDs) have been found to compromise male fertility, with cholesterol dysregulation being a key factor. Sterol regulatory element-binding protein 2 (SREBP2) is a crucial transcription factor that regulates cholesterol biosynthesis and uptake, playing an essential role in maintaining cholesterol homeostasis in the testes. This study investigated the dynamics of SREBP2 and cholesterol levels during rabbit spermatogenesis under HFD conditions. Our findings reveal that SREBP2 expression fluctuates throughout the seminiferous epithelium cycle. However, HFDs induce stage-specific disruptions in cholesterol balance, leading to sperm with increased membrane cholesterol, a reduced sperm count in semen analysis, impaired motility, abnormal morphology, and decreased functionality. In the control group, SREBP2 expression patterns underscored its critical role in normal spermatogenesis. Interestingly, supplementation with extra virgin olive oil (EVOO) reversed the negative effects of HFD, normalizing SREBP2 expression and cholesterol content, which improved sperm quality. These findings emphasize the importance of stage-specific analysis in understanding how dietary fat impacts male fertility and suggest that EVOO may serve as a potential nutritional intervention to protect reproductive health. Full article

One hazardous material that occurs naturally in the environment and induces oxidative stress is cadmium (Cd). Epidemiological data revealed that exposure to cadmium in the workplace and environment might be linked to many illnesses and serious testicular injuries. Aims: It is taught that antioxidants can protect different organs against environmental toxic compounds. Therefore, the current investigation aims to show the role of antioxidants (gallic acid and selenium) in the protection against cadmium toxicity, including the architecture of the testes, semen properties, steroid hormones, protein expression of cytochrome P450 [CYP 19 and 11A1] contributing to the production of steroid hormones, and antioxidant enzyme activities, in male rabbits. Methods: Male rabbits were given cadmium orally three times/week [1 mg/kg BW] for twelve weeks. In addition, gallic acid (20 mg/kg) or selenium (1 mg/kg BW) was administered two hours before cadmium treatment. This investigation included a spectrophotometer, histopathology, and Western immunoblotting techniques. Results: Cadmium treatment significantly reduced sperm counts, testosterone, and estrogen levels after four, eight, and twelve weeks of treatment. In addition, after a 12-week treatment of rabbits with cadmium, the activity of 17β-hydroxysteroid dehydrogenase and antioxidant enzymes, including catalase, superoxide dismutase, glutathione reductase, glutathione peroxidase, and glutathione S-transferase, as well as the glutathione levels, were inhibited in the testes tissue. On the other hand, following cadmium treatment, rabbit’s testes showed a discernible increase in free radical levels. Interestingly, the activity of antioxidant enzymes and level of free radicals were recovered in rabbits treated with gallic acid or selenium before cadmium treatment. In addition, after 12 weeks of cadmium treatment, the steroidogenic protein expressions of CYP 11A1 and CYP 19 were upregulated and downregulated in the testes, respectively. Interestingly, after pretreatment of rabbits with either gallic acid or selenium for two hours before cadmium administration, the downregulated CYP11A1 was restored to normal levels. In the histopathological investigation, immature spermatozoids and sloughed spermatogonium cells were observed in cadmium-treated rabbits’ testes. On the other hand, pretreatments of rabbits with gallic acid or selenium mitigated and alleviated the adverse effects of cadmium on testes architecture and increased the production of healthy sperm. Conclusions: The lower levels of steroid hormones could be due to the downregulation of CYP11A1, inhibition of 17β-hydroxysteroid dehydrogenase, antioxidant enzyme activities, and the induction of free radical levels. Furthermore, the pretreatment of rabbits with gallic acid or selenium mitigated the adverse effects of cadmium on the tissue architecture of testes and steroid hormone levels. Full article

Lipopolysaccharide (LPS)-induced inflammation impairs sperm function; however, its impact on ejaculated rabbit sperm remains unexplored. This dose-response study aims to determine the LPS concentration that negatively affects sperm motility in vitro, while also providing the first identification of TLR4 localization on rabbit spermatozoa. Additionally, it evaluates malondialdehyde (MDA) levels in seminal plasma as an indicator of oxidative stress. Sperm motility was analyzed using computer-assisted sperm analysis (CASA) after incubation with increasing LPS concentrations (0, 50, 100, 200, 400, 600, and 800 µg/mL) at multiple time points (0, 1, 2, and 4 h). LPS doses ≥ 400 µg/mL significantly reduced progressive and non-progressive motility, as well as curvilinear velocity (all p < 0.001), while increasing the proportion of static spermatozoa (p < 0.05). Receiver operating characteristic (ROC) analysis identified 300 µg/mL as the threshold dose for motility decline. Immunofluorescence revealed TLR4 localization in the midpiece of sperm tails, with weak labeling in control samples and a marked increase after 4 h of incubation with 400 μg/mL LPS. MDA levels were assessed using the thiobarbituric acid reactive substances (TBARS) assay with a colorimetric kit, showing no significant effect of LPS treatment. No correlation was found between MDA and other semen parameters. ccThese findings identify TLR4 on rabbit sperm for the first time and establish a threshold LPS dose for future in vitro studies. Full article

Background/Objectives: The use of antibiotics in livestock contributes to antimicrobial resistance, highlighting the need for alternative solutions. Among these, chelating agents, like ethylenediaminetetraacetic acid (EDTA) and Chitosan, have shown potential in reducing bacterial contamination in seminal doses used in artificial insemination (AI), while preserving sperm quality. The objective of this study was to evaluate the potential use of EDTA and Chitosan as alternatives to antibiotics for the liquid storage of rabbit seminal AI doses. Methods: EDTA (20 mM) and Chitosan (0.05%) were tested both individually and in combination, by adding them to the semen extender, and their effects were compared with extenders containing antibiotics or none. The extenders were evaluated for microbial resistance and their ability to maintain sperm quality in vitro during refrigeration at 16 ± 1 °C for 72 h. To assess antimicrobial efficacy, Enterococcus faecalis was used. Seminal doses stored for 24 h were used for insemination under commercial conditions, and fertility rate and total kits born were evaluated. Results: Adding 0.05% Chitosan to the extender resulted in sperm parameters and bacterial load comparable to those achieved with antibiotics during refrigerated storage, yielding similar fertility rate and total kits born outcomes 24 h post-storage. In contrast, the use EDTA alone or in combination with Chitosan was less effective at controlling Enterococcus faecalis than the antibiotic extenders, which also resulted in a reduction of sperm total motility over storage period (0–72 h) and negatively impacted fertility rate and total kits born. Conclusions: Chitosan’s protective effect on sperm function, combined with its antimicrobial activity, makes it a promising alternative antimicrobial agent for the liquid storage of rabbit seminal AI doses. Full article

The influence of sericin supplementation and chilling temperatures (4 and 15 °C) on the short-term storage quality and fertility of rabbit semen was assessed over 72 h of storage. In experiment 1, pooled semen (five replications) was diluted to a concentration of 50 M mL⁻¹, and assigned to control, 0.1%, and 0.5% sericin groups, stored at 4 or 15 °C. Sperm motility, sperm kinematics, viability, and membrane and acrosome integrity were assessed at 0, 24, 48, and 72 h of storage. Experiments 2 and 3 were conducted to observe the effect of sericin on bacterial growth and in vivo fertility. The results of experiment 1 showed that sericin treatment, storage temperature, and storage duration influenced progressive and total sperm motility. The storage duration affected all sperm kinematics variables, whereas VCL, VSL, VAP, and BCF sperm kinematics altered in response to sericin treatment and storage temperature. Similarly, significant effects of sericin treatment, storage temperature, and storage duration were observed for acrosome integrity, sperm membrane integrity, and sperm viability. The results of experiment 2 indicated the antimicrobial effects of sericin when sperm were stored at 15 °C for 72 h. While promising pregnancy outcomes were observed with sericin-treated sperm for 72 h, these results were not significantly different. This study conclusively demonstrates that sericin treatment enhances the quality of rabbit semen when stored at lower temperatures for longer durations. The antimicrobial effects of sericin could be a contributing factor to the improved in vitro and in vivo fertility of rabbit sperm. Full article

Cryopreservation plays a critical role in animal breeding and the conservation of endangered species, but it often compromises sperm characteristics such as morphology, motility, and viability due to oxidative stress. This study explores the antioxidative effect of Mito-Tempo (MT) and Berberine (BER) to enhance post-thaw sperm quality in rabbits. Pooled rabbit sperm samples were supplemented with different concentrations (0.0, 0.5, 5, 10, 50 µmol/L) of MT and BER. Sperm motility was evaluated using computer-assisted semen analysis, while viability, apoptosis, reactive oxygen species (ROS) levels, acrosome integrity, and mitochondrial function were assessed through flow cytometry. The results revealed that MT at 5 and 10 µmol/L and BER at 10 µmol/L significantly improved total and progressive motility, mitochondrial activity, and sperm viability compared to the control group. Furthermore, 10 µmol/L BER enhanced acrosome integrity, while both 5 µmol/L MT and 10 µmol/L BER effectively reduced ROS levels and apoptosis. This study is the first to demonstrate the protective effects of MT and BER on rabbit sperm during cryopreservation. By mitigating oxidative stress and reducing apoptosis, these antioxidants markedly improved post-thaw sperm quality, positioning MT and BER as promising agents for improving sperm cryosurvival. Full article

Incorporating of agro-industrial co-products into animal nutrition could represent an opportunity to lessen the environmental impact of the food production chain. One such co-product is a hempseed cake originating from cold pressing hemp seeds to extract oil for human consumption. The aim of the present study was to evaluate the action of hempseed cake in the diet on male rabbit reproductive and some non-reproductive indexes. Male rabbits were fed either a standard diet (control group; C; n = 10) or a diet enriched with hempseed cake (experimental group E5 with 5% of a hempseed cake; n = 10, and experimental group E10 with 10% of a hempseed cake; n = 10) in 100 kg of the milled complete feed mixture. Rabbit weight gain, sperm concentration, motility, progressive motility, and sperm quality were evaluated using CASA and flow cytometry. Feeding with a hempseed cake, given at both tested concentrations, had no effect on weight gain per week and the total average weight gain compared to the control group (p > 0.05). Hempseed cake addition had no effect on sperm concentration in ejaculate, sperm motility, and progressive motility (p > 0.05). Selected haematological and biochemical indexes were examined. The E5 group showed positive tendencies in hepatic profile parameters, while in the E10 group the tendencies were opposite, though within the reference values. Based on our results, no negative effects of hempseed cake feeding on rabbit reproduction and health status were found, and we can recommend the use of hempseed cake at doses up to 10% in the nutrition and feeding of rabbits. Therefore, agro-industrial co-products can decrease the feeding cost. Full article

Silver nanoparticles possess valuable physical, chemical, and biological properties, rendering them widely applied as bioactive agents in the industry. Nonetheless, their influence on the natural environment and on living organisms remains unclear. Therefore, this study aims to investigate the impact of polymer composites containing silver nanoparticles on sperm cells. The nanosilver polymer composites were chemically synthesized, employing sodium alginate as the stabilizer. The reducing agents employed were solutions comprising sodium borohydride and xylose. The concentration of silver nanoparticles obtained after synthesis was 100 parts per million. The examined biological species were rabbit sperm cells. The impact of nanosilver on the sperm was assessed through the elucidation of the toxicity profile, comet test, and analysis of morphological characteristics of the animal cells. The results of the study demonstrate a twofold impact of polymer composites infused with silver nanoparticles on domestic rabbit sperm when obtained through chemical synthesis using two reducing agents (xylose and sodium borohydride) at a 10 ppm concentration. The comet test showed no harmful effect on the DNA integrity of rabbit sperm by the tested compounds. Twenty-four-hour exposure of rabbit spermatozoa to silver nanoparticles, obtained by reducing xylose and borohydride, induced significant secondary changes in the morphological structure of male reproductive cells. These findings indicate the potential reproductive toxicity of silver nanoparticles. Full article

Studying proteins associated with sex chromosomes can provide insights into sex-specific proteins. Membrane proteins accessible through the cell surface may serve as excellent targets for diagnostic, therapeutic, or even technological purposes, such as sperm sexing technologies. In this context, proteins encoded by sex chromosomes have the potential to become targets for X- or Y-chromosome-bearing spermatozoa. Due to the limited availability of proteomic studies on rabbit spermatozoa and poorly annotated databases for rabbits compared to humans, a bioinformatic analysis of the available rabbit X chromosome proteome (RX), as well as the human X (HX) and Y (HY) chromosomes proteome, was conducted to identify potential targets that could be accessible from the cell surface and predict which of the potential targets identified in humans might also exist in rabbits. We identified 100, 211, and 3 proteins associated with the plasma membrane or cell surface for RX, HX, and HY, respectively, of which 61, 132, and 3 proteins exhibit potential as targets as they were predicted to be accessible from the cell surface. Cross-referencing the potential HX targets with the rabbit proteome revealed an additional 60 proteins with the potential to be RX targets, resulting in a total of 121 potential RX targets. In addition, at least 53 possible common HX and RX targets have been previously identified in human spermatozoa, emphasizing their potential as targets of X-chromosome-bearing spermatozoa. Further proteomic studies on rabbit sperm will be essential to identify and validate the usefulness of these proteins for application in rabbit sperm sorting techniques as targets of X-chromosome-bearing spermatozoa. Full article

This study aimed to investigate the impact of sperm concentrations on the in vitro quality of cryopreserved rabbit semen. The semen pools (n = 8, from 80 donors) were split into five aliquots with final sperm concentrations of 15, 25, 35, 55, and 75 × 10⁶ per straw. The sperm motility parameters (CASA system) and membrane integrity (flow cytometric analysis) were both evaluated at various stages of the cryopreservation process: fresh semen dilution, cooling, equilibration, and immediately after and 30 min post-thawing. The results indicated the significant influence of the sperm concentration on the total motility (TM) and progressive motility (PM), with a consistent decline in all sperm variables over the time points. Notably, the semen with a final concentration of 15 × 10⁶ exhibited a higher TM and PM after cooling and equilibration. The post-thawing quality (TM, PM) was higher (p < 0.05) in the mid-range sperm concentrations of 25 × 10⁶ (49.9% and 19.7%) and 35 × 10⁶ (46.2% and 19.7%) compared to the other concentrations. This study demonstrated that the sperm concentration per straw played a significant role in specific phases of the cryopreservation process. These findings contribute valuable insights for refining and standardizing the cryopreservation protocol for rabbit semen, emphasizing the importance of the sperm concentration. Full article