Search Results (13)

κ-Carrageenan is a sulfated polysaccharide from red seaweed with substantial antioxidant activities. This study aimed to investigate the effect of κ-Carrageenan treatment on frozen–thawed (FT) porcine semen quality. Therefore, the spermatozoa were diluted and cryopreserved in a freezing extender supplemented with 0 (control), 0.2, 0.4, 0.6, and 0.8 mg/mL κ-Carrageenan. Sperm kinematics were assessed immediately after thawing (AT) and post-incubation for 120 min. The viability, acrosome integrity, lipid peroxidation, mitochondrial membrane potential (MMP), and intracellular caspase activity were measured AT. The results indicated that 0.2 mg/mL κ-Carrageenan increased total and progressive motility AT and post-incubation for 120 min (p < 0.05). Moreover, the viable sperm percentage and MMP after 0.2 mg/mL treatment were higher than those after control and other κ-Carrageenan concentration treatments. The proportion of acrosome-intact spermatozoa was significantly higher after 0.2 and 0.4 mg/mL κ-Carrageenan treatment than that after control and other κ-Carrageenan concentration treatments. The intracellular caspase activity was not significantly different among the experimental groups. However, the MDA concentration after 0.2 mg/mL κ-Carrageenan treatment was lower (p < 0.05) than that after the control treatment. Taken together, adding κ-Carrageenan to the porcine semen freezing extender improved the FT sperm quality mainly by influencing membrane stability and protecting against oxidative stress. Full article

Artificial insemination (AI) is used frequently in the breeding of sport horses, apart from Thoroughbreds. Most AIs are carried out with cooled semen rather than frozen semen because of the difficulties in identifying a protocol that is suitable for freezing most ejaculates and the necessity to inseminate close to ovulation because of the short life of the thawed spermatozoa. More widespread use of frozen semen would improve biosecurity, allow greater choice of stallions, and offer more flexibility when managing deliveries of semen to the stud. It would even decrease the amount of antibiotics used in semen extenders, since the volume of frozen semen is smaller than when cooled semen is inseminated. However, there is considerable variability in the cryosurvival of spermatozoa from different stallions, leading to the classification of stallions as good or bad freezers. Improvements could be made at the level of stallion nutrition, the semen collection regimen, the extender, the removal of seminal plasma, and the cooling protocol, among others. Stallion sperm membranes are highly susceptible to lipid peroxidation, but research on antioxidants has failed to identify an additive that would benefit all stallions. In the future, biomarkers for sperm freezability could be used as an aid in identifying suitable ejaculates for cryopreservation. Full article

An urgent need to boost the sustainability and efficiency of animal production exists, owing to the growing global population. Enhancing the global fertility of animals, especially cattle, is essential to ameliorate this issue. Artificial insemination and sperm cryopreservation have a considerable and favorable influence on the quantity and quality of the cattle produced. Sperm cryopreservation is crucial for livestock production because it promotes and accelerates genetic diversity and the worldwide dispersion of animals with enhanced genetics. Owing to the importance of cryobiology in reproductive technologies, researchers are developing new approaches, and they are testing cryoprotectant drugs to enhance sperm cryosurvival. However, the viability of sperm after freezing is low and widely varies across breeding yaks. These faults are crucial because they impede advances in reproductive biotechnology and the study of mammalian gametes at a fundamental level. Using chemicals, researchers have developed and enhanced various extenders with varying degrees of efficiency to reduce cryodamage and oxidative stress. In this article, we review the cryopreservation of yak semen, the development of extenders, the difficulties faced during cryopreservation, and the evaluation of semen quality using various methodologies. This review might be helpful for researchers exploring semen cryopreservation in the future, as demand for enhanced cryopreservation exists to boost the post-thaw viability and fertility of sperm. Full article

The hybrids of female channel catfish (Ictalurus punctatus) and male blue catfish (I. furcatus) account for >50% of US catfish production due to superior growth, feed conversion, and disease resistance compared to both parental species. However, these hybrids can rarely be naturally spawned. Sperm collection is a lethal procedure, and sperm samples are now cryopreserved for fertilization needs. Previous studies showed that variation in sperm quality causes variable embryo hatch rates, which is the limiting factor in hybrid catfish breeding. Biomarkers as indicators for sperm quality and reproductive success are currently lacking. To address this, we investigated expression changes caused by cryopreservation using transcriptome profiles of fresh and cryopreserved sperm. Sperm quality measurements revealed that cryopreservation significantly increased oxidative stress levels and DNA fragmentation, and reduced sperm kinematic parameters. The present RNA-seq study identified 849 upregulated genes after cryopreservation, including members of all five complexes in the mitochondrial electron transport chain, suggesting a boost in oxidative phosphorylation activities, which often lead to excessive production of reactive oxygen species (ROS) associated with cell death. Interestingly, functional enrichment analyses revealed compensatory changes in gene expression after cryopreservation to offset detrimental effects of ultra-cold storage: MnSOD was induced to control ROS production; chaperones and ubiquitin ligases were upregulated to correct misfolded proteins or direct them to degradation; negative regulators of apoptosis, amide biosynthesis, and cilium-related functions were also enriched. Our study provides insight into underlying molecular mechanisms of sperm cryoinjury and lays a foundation to further explore molecular biomarkers on cryo-survival and gamete quality. 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

κ-Carrageenan is a plant polysaccharide derived from red seaweeds reported to possess potential medicinal and antioxidants activities. The present study aimed to identify the cryoprotective effects of κ-carrageenan on the quality of frozen-thawed canine semen. Twenty-eight ejaculates were collected and diluted in a Tris egg-yolk-free extender supplemented with various concentrations of κ-carrageenan (0.0%, 0.1%, 0.2%, 0.3%, and 0.5%). The addition of κ-carrageenan to the extender at a 0.2% concentration induced a significant increase in the total motility (TM) and the rapid progressive motility (RPM) of canine sperm. Among the experimental groups, the highest percentage of sperms with intact acrosomes was found in the 0.5% κ-carrageenan group (p < 0.05). Apoptosis levels were significantly lower in the 0.1% and 0.2% κ-carrageenan treatment. Moreover, sperm in the κ-carrageenan supplemented group showed a significantly higher expression of antiapoptotic (Bcl-2) and lower expression of NADPH oxidase (NOX5), spermine synthase (SMS), and spermine oxidase (SMOX) genes than those in the control group. In conclusion, the addition of κ-carrageenan to the freezing extender improved the overall efficiency of frozen-thawed dog spermatozoa. Full article

Oocyte cryopreservation plays important roles in basic research and the application of models for genetic preservation and in clinical situations. This technology provides long-term storage of gametes for genetic banking and subsequent use with other assisted reproductive technologies. Until recently, oocytes have remained the most difficult cell type to freeze, as the oocytes per se are large with limited surface area to cytoplasm ratio. They are also highly sensitive to damage during cryopreservation, and therefore the success rate of oocyte cryopreservation is generally poor when compared to noncryopreserved oocytes. Although advancement in oocyte cryopreservation has progressed rapidly for decades, the improvement of cryosurvival and clinical outcomes is still required. This review focuses on the principles, techniques, outcomes and prospects of oocyte cryopreservation in domestic animals and humans. Full article

Advanced paternal age is associated with increased sperm reactive oxygen species (ROS) and decreased fertilization and pregnancy rates. Sperm washing during infertility treatment provides an opportunity to reduce high sperm ROS concentrations associated with advanced paternal age through the addition of idebenone. Sperm from men aged >40 years and older CBAF1 mice (12–18 months), were treated with 5 µM and 50 µM of idebenone and intracellular and superoxide ROS concentrations assessed. Following in vitro fertilization (IVF), embryo development, blastocyst differentiation, DNA damage and cryosurvival, pregnancy and implantation rates and fetal and placental weights were assessed. Five µM of idebenone given to aged human and mouse sperm reduced superoxide concentrations ~20% (p < 0.05), while both 5 and 50 µM reduced sperm intracellular ROS concentrations in mice ~30% (p < 0.05). Following IVF, 5 µM of idebenone to aged sperm increased fertilization rates (65% vs. 60%, p < 0.05), blastocyst total, trophectoderm and inner cell mass cell numbers (73 vs. 66, 53 vs. 47 and 27 vs. 24, respectively, p < 0.01). Treatment with idebenone also increased blastocyst cryosurvival rates (96% vs. 78%, p < 0.01) and implantation rates following embryo transfer (35% vs. 18%, p < 0.01). Placental weights were smaller (107 mg vs. 138 mg, p < 0.05), resulting in a larger fetal to placental weight ratio (8.3 vs. 6.3, p = 0.07) after sperm idebenone treatment. Increased sperm ROS concentrations associated with advanced paternal age are reduced with the addition of idebenone in vitro, and are associated with improved fertilization rates, embryo quality and implantation rates after IVF. Full article

Sperm cryopreservation represents a powerful tool for livestock breeding. Several efforts have been made to improve the efficiency of sperm cryopreservation in different ruminant species. However, a significant amount of sperm still suffers considerable cryodamage, which may affect sperm quality and fertility. Recently, the use of different “omics” technologies in sperm cryobiology, especially proteomics studies, has led to a better understanding of the molecular modifications induced by sperm cryopreservation, facilitating the identification of different freezability biomarkers and certain proteins that can be added before cryopreservation to enhance sperm cryosurvival. This review provides an updated overview of the molecular mechanisms involved in sperm cryodamage, which are in part responsible for the structural, functional and fertility changes observed in frozen–thawed ruminant sperm. Moreover, the molecular basis of those factors that can affect the sperm freezing resilience of different ruminant species is also discussed as well as the molecular aspects of those novel strategies that have been developed to reduce sperm cryodamage, including new cryoprotectants, antioxidants, proteins, nanoparticles and vitrification. Full article

The molecular mechanism regulating embryo development under reduced oxygen tension remains elusive. This study aimed to identify the molecular mechanism impacting embryo development under low oxygen conditions. Buffalo embryos were cultured under 5% or 20% oxygen and were evaluated according to their morphological parameters related to embryo development. The protein profiles of these embryos were compared using iTRAQ-based quantitative proteomics. Physiological O₂ (5%) significantly promoted blastocyst yield, hatching rate, embryo quality and cell count as compared to atmospheric O₂ (20%). The embryos in the 5% O₂ group had an improved hatching rate of cryopreserved blastocysts post-warming (p < 0.05). Comparative proteome profiles of hatched blastocysts cultured under 5% vs. 20% O₂ levels identified 43 differentially expressed proteins (DEPs). Functional analysis indicated that DEPs were mainly associated with glycolysis, fatty acid degradation, inositol phosphate metabolism and terpenoid backbone synthesis. Our results suggest that embryos under physiological oxygen had greater developmental potential due to the pronounced Warburg Effect (aerobic glycolysis). Moreover, our proteomic data suggested that higher lipid degradation, an elevated cholesterol level and a higher unsaturated to saturated fatty acid ratio might be involved in the better cryo-survival ability reported in embryos cultured under low oxygen. These data provide new information on the early embryo protein repertoire and general molecular mechanisms of embryo development under varying oxygen levels. Full article

The aim of our study was to test the effectiveness of a simple semen cryopreservation procedure, developed for cultivated salmonid, on the wild salmonid of the Mediterranean area and to evaluate the effect of different thawing rates and sperm-to-egg ratios. The semen of five individual males was diluted into a final extender concentration of 0.15 M glucose and 7.5% methanol and loaded into 0.25 mL plastic straws, and a final sperm concentration of 3.0 × 10⁹ sperm/mL was obtained. After equilibration, the straws were frozen by exposure to liquid nitrogen vapor at 3 cm above the liquid nitrogen level for 5 min. The semen was thawed at 40 °C/5 s or 10 °C/30 s. The sperm cryosurvival was evaluated by examining in vitro the sperm motility parameters using the CASA system, followed by fertilization trials in vivo, using three different sperm-to-egg ratios 6 × 10⁵, 4.5 × 10⁵ and 3 × 10⁵:1. The applied cryopreservation procedure resulted in remarkably high (85.6%) post-thaw sperm total motility, when the semen was thawed at 40 °C/5 s, whilst the highest fertilization rate (53.1%) was recorded for a sperm-to-egg ratio of 4.5 × 10⁵:1. According to these outcomes, the cryopreservation procedure that was tested turned out to be effective for the wild population of Mediterranean brown trout and practical for the creation of the first European semen cryobank foreseen as part of our “LIFE” Nat.Sal.Mo. project. Full article

Oxidative stress during freeze–thaw procedures results in reduced semen fertility. A decrease in free radical levels can improve the post-thaw sperm quality. We examined the effects of myoinositol supplementation in freezing medium on the structure and function of cryopreserved dog sperm. Pooled ejaculates were diluted with buffer without or with myoinositol (1 or 2 mg/mL). Analysis of fresh semen revealed that the optimal concentration of myoinositol was 1 mg/mL, and this concentration was used in further experiments. Post-thaw semen quality in the myoinositol-supplemented group was superior (p < 0.05) compared with that in the control group in terms of motility (57.9 ± 0.4% vs. 47.8 ± 0.2%), sperm viability (57.5 ± 0.5% vs. 44.6 ± 0.6%), intact plasma membrane (56.6 ± 0.4% vs. 46.2 ± 0.6%), and acrosome membrane (59.3 ± 0.5% vs. 51.8 ± 0.5%). In addition, sperm in the myoinositol-supplemented group showed a significantly lower expression of pro-apoptotic (BAX) and mitochondrial reactive oxygen species (ROS) modulator (ROMO1) genes but higher expression of anti-apoptotic (BCL2), and protamine-related (PRM2 and PRM3) genes compared with that in the control group. Therefore, myoinositol supplementation before freezing can protect against oxidative stress and improve post-thaw dog sperm quality. Full article

The aim of our study was to test the effects of different non-permeating cryoprotectants (NP-CPAs), namely low-density lipoproteins (LDLs), sucrose, and egg yolk, and thawing rates on the post-thaw semen quality and fertilizing ability of the native Mediterranean brown trout. Pooled semen samples were diluted 1:3 (v:v) with 2.5%, 5%, 10%, or 15% LDL; 0.05, 0.1, or 0.3 M sucrose; or 10% egg yolk. At the moment of analysis, semen was thawed at 30 °C/10 s or 10 °C/30 s. The post-thaw semen quality was evaluated, considering motility, the duration of motility, viability, and DNA integrity. Significantly higher values of motility and viability were obtained using egg yolk/10 °C for 30 s, across all treatments. However, LDL and sucrose concentrations affected sperm cryosurvival, showing the highest post-thaw sperm quality at 5% LDL and 0.1 M sucrose. Based on the in vitro data, egg yolk, 5% LDL, and 0.1 M sucrose thawed at 10 °C or 30 °C were tested for the in vivo trial. The highest fertilization and hatching rates were recorded using egg yolk/10 °C (p < 0.05). According to these in vitro and in vivo results, egg yolk emerged as the most suitable NP-CPA and 10 °C/30 s as the best thawing rate for the cryopreservation of this trout sperm, under our experimental conditions. Full article