Search Results (52)

The cryopreservation of Chengde polled goat semen plays a critical role in conserving genetic resources, enhancing the utilization efficiency of superior breeding bucks, and advancing artificial insemination techniques. However, spermatozoa are vulnerable to oxidative stress during the freezing process, which can significantly compromise sperm motility. In this study, pooled ejaculates from multiple bucks were divided into six groups, including a control group cryopreserved with conventional extender and five treatment groups supplemented with sericin at concentrations of 0.2%, 0.4%, 0.6%, 0.8%, and 1.0% (w/v). The results demonstrated that supplementation of the semen cryoprotectant with 0.6% sericin significantly improved post-thaw sperm viability to 65.25% in Chengde hornless goats, while concurrently reducing both the sperm abnormality rate (p < 0.05) and intracellular ROS levels (p < 0.05). Integrated TMT proteomics and LC/MS metabolomics further compared the 0.6% sericin group with the frozen control group and identified 162 differentially expressed proteins and 109 differential metabolites between the sericin supplementation and frozen control groups. Functional analysis revealed the significant enrichment of differential metabolites, such as glutamine, in the alanine, aspartate, and glutamate metabolism pathway, concomitant with the marked upregulation of antioxidant proteins including LRP8, GSTM3, and SIRT2. Thus, 0.6% sericin enhances cryotolerance primarily by improving sperm viability, reducing oxidative damage, and sustaining energy metabolism. These findings indicate that sericin enhances cryotolerance by reducing oxidative damage and supporting metabolic function, providing preliminary molecular insights for improving goat semen cryopreservation. Full article

This study evaluated the repeatability of selected sperm parameters in Salernitano stallions housed on the same farm. Semen was collected weekly for four weeks, and sperm kinetics, mitochondrial activity, and oxidative/nitrosative status were assessed before and after freezing the sperm with HF-20 and INRA Freeze. Pre-freezing, significant individual variability was observed, with low repeatability for semen volume (r = 0.32), total motility (r = 0.38), curvilinear velocity (r = 0.32), and lipoperoxidation (r = 0.36). Post-thaw, sperm frozen with INRA Freeze showed significant inter-stallion differences and low-to-moderate repeatability across kinematic parameters, mitochondrial membrane potential, nitric oxide, and lipoperoxidation, whereas those frozen with HF-20 showed repeatability only for progressive motility and intracellular H₂O₂. An assessment of freezability revealed significant inter-stallion variability and low-to-moderate repeatability for most kinematic traits in sperm frozen with INRA Freeze. Age influenced specific parameters in both fresh and frozen–thawed semen. Kinematic traits were strongly intercorrelated and associated with mitochondrial activity, as well as with lipoperoxidation, the latter being significantly related to H₂O₂ and nitric oxide levels. Although the overall post-thaw differences between extenders were not statistically significant, INRA Freeze enabled clearer discrimination among stallions. The generally low-to-moderate repeatability observed in this study suggests that extender choice can influence cryopreservation outcomes, and supports the need for tailored protocols. Full article

In response to the growing interest in less conventional alcoholic beverages, this study aimed to identify novel yeast strains suitable for sake production, with a focus on their potential application in bioflavouring. Commercially available strains of bottom-fermenting brewing yeasts (Saccharomyces pastorianus), a cryotolerant wine yeast (Saccharomyces bayanus), and a wild wine yeast (Torulaspora delbrueckii) were evaluated. The quality characteristics of sake obtained using non-conventional yeasts were compared with sake produced using Saccharomyces cerevisiae K7, one of the most commonly used strains in sake brewing. Sake made with non-conventional yeasts exhibited differences in fermentation kinetics, chemical composition, and sensory properties. Wine yeasts produced sake with the most favorable ester profile, markedly distinct from those obtained with other yeast strains used in the study. Compared to the conventional strain, the concentrations of the key contributors to the fruity/floral aroma, namely 3-methylbutyl acetate and ethyl hexanoate, in sake produced with S. bayanus were higher by 249.5% and 199.3%, respectively. The wine yeast S. bayanus may be considered the most promising strain for sake production due to its ability to generate elevated levels of volatile aroma compounds associated with Ginjo-ka characteristics, as well as its effectiveness in supporting a consistent and efficient alcoholic fermentation process. Full article

Currently, there is a need to improve the cryopreservation process for male gametes, especially for patients with low cryotolerance during sperm cryopreservation. Methods: The content and size of donor extracellular vesicles (EVs) in seminal plasma (SP) were assessed using nanoparticle tracking analysis (NTA), CD marker analysis, and transmission electron microscopy (TEM). Patient ejaculates were exposed to cryopreservation with or without prior co-culture with SP EVs and were not exposed to cryopreservation. The interaction of SP EVs with spermatozoa was assessed by TEM. Apoptotic, necrotic and late apoptotic cells, and mitochondrial functional activity were detected by flow cytometry. Results: NTA showed the highest concentration of SP EVs with a size of 80 nm, corresponding to small EVs. The binding of SP EVs to spermatozoa occurred along the entire plasma membrane, with an increased concentration of SP EVs at the neck and upper third of the sperm head. A significant increase in sperm motility was observed in the EVs co-culture group after cryopreservation/thawing. Flow cytometry showed a significant difference in the JC-1 Red/JC-1 Green ratio, indicating a higher mitochondrial membrane potential in the EVs exposure group. Conclusions: SP EVs have a protective function during human sperm cryopreservation. Full article

The in vitro production of embryos has significant potential to enhance animal productivity. However, further refining of this technology is required for its widespread adoption and cost-effectiveness. The objectives were to evaluate the effects of L-carnitine (LC) on the maturation, lipid content, and Hippo signaling of oocytes, and the cryotolerance of the resulting embryos. Abattoir-derived oocytes were in vitro matured using fetal bovine serum (FBS), bovine serum albumin (BSA), or FBS + 1.5 or 3.0 mM LC. The maturation rates did not differ among FBS (83%) and FBS with LC (1.5 or 3.0 mM; 82 and 80%, respectively). In contrast, the BSA group exhibited a significantly lower maturation rate of 71% compared to the other groups. The lipid content of matured oocytes (assessed using Nile red staining) was significantly reduced in the BSA group and in the FBS + LC groups, compared to the FBS group. The blastocyst-stage embryos were cryopreserved, and their cryotolerance was evaluated by assessing their ability to re-expand and hatch after thawing. The embryos from the FBS + LC groups showed a numerically higher re-expansion rate at 24 h (78.8%), compared to the BSA (74.0%) and FBS groups (57.7%). The expression of Hippo signaling pathway genes was not significantly affected by LC, indicating that LC enhanced cryotolerance and reduced lipid content without impacting oocyte maturation or the Hippo signaling pathway. Full article

(1) Background: Cryopreservation of epididymal spermatozoa in dogs is challenging due to their lower cryotolerance compared to ejaculated spermatozoa. Given the limited sperm volume obtained per individual, efficient recovery and preservation techniques are essential. (2) Methods: This study assessed sperm collection and cryopreservation methods from the cauda epididymis of ten dogs undergoing routine elective castration. After dissection and mincing, the cauda epididymidis tissue was incubated in 0.9% saline at 38 °C for either 10- or 30-min. Samples were analyzed for concentration and motility using AndroVision^® software (CASA; AndroVision™; Minitüb GmbH) (Tiefenbach, Germany). Additional evaluations included histological examination, hypoosmotic swelling test, live/dead staining, and morphological assessments. Three extenders, custom-made Tris-Fructose-Citrate (Tris), custom-made Uppsala, and commercial Optixcell^®, were used for cryopreservation and compared for post-thaw sperm quality. (3) Results: No significant differences were found between the 10- and 30-min incubation groups regarding sperm motility, viability, or histological integrity. The total sperm counts were 292 × 10⁶ ± 175 × 10⁶ for the 10 min group and 233 × 10⁶ ± 162 × 10⁶ for the 30 min group (p = 0.56). Histological sections revealed no significant difference in residual intraluminal spermatozoa between groups, indicating that 10 min of incubation is sufficient for effective sperm migration. Post-thaw sperm motility was significantly higher with Uppsala (17.2 ± 12.2%) and Optixcell^® (11.7 ± 6.5%) compared to Tris (4.7 ± 4.8%). Morphological abnormalities were lowest in samples preserved with Optixcell^® (37.5 ± 10.1%, p = 0.005). (4) Conclusion: A 10 min incubation period is adequate for efficient recovery of epididymal sperm in dogs. Among the tested extenders, Uppsala and Optixcell^® demonstrated superior cryoprotective effects, resulting in better post-thaw motility and reduced morphological abnormalities compared to Tris. Full article

Equine semen preservation is fundamental to modern equine reproduction, supporting breeding programs, genetic conservation, and industry sustainability. However, significant challenges persist, including temperature sensitivity, oxidative stress, bacterial contamination, individual variability, and lack of standardized preservation protocols. These factors contribute to reduced sperm viability and fertility following cryopreservation. This review examines critical obstacles in equine semen preservation, focusing on cryopreservation sensitivity, molecular damage mechanisms, economic constraints, and seasonal quality variations. We analyze the molecular and structural alterations (e.g., oxidative stress, membrane damage, and DNA fragmentation) and their impact on cryopreservation success. The review evaluates evidence-based enhancement strategies, including nutritional supplementation and genetic approaches, for improving semen quality. Nutritional interventions that utilize antioxidants, polyunsaturated fatty acids (PUFAs), and nutraceuticals have demonstrated promising results in enhancing sperm motility, preserving membrane integrity, and improving overall semen quality. Additionally, we discuss key candidate genes associated with equine semen-quality traits, including sperm motility, viability, and cryotolerance. The integration of nutritional supplementation and genetic selection strategies presents viable pathways for optimizing equine semen preservation techniques. These combined approaches offer potential solutions for overcoming current limitations, ultimately supporting sustainable breeding programs and advancing genetic conservation efforts in the equine industry. Full article

Lasia spinosa Thwaites (LST) has emerged as a potential supplement for enhancing male reproductive performance. This study evaluated the effects of long-term oral supplementation with LST on hematological parameters, semen characteristics, ultrasonographic measurements of the prostate gland and testes, and the cryopreservation potential of canine sperm. Six healthy male dogs received oral LST supplementation at a dosage of 10 mg/kg body weight once daily for 7 days (short-term). After a three-month washout period to ensure full physiological recovery, the same dogs underwent a long-term supplementation protocol (60 days). In the short-term trial, no clinically significant changes were observed in hematological or serum biochemical parameters, including complete blood count, alanine aminotransferase, creatinine, blood urea nitrogen, total protein, and albumin; all parameters were within normal reference ranges. Serum testosterone levels and semen characteristics were also unaffected (p > 0.05). During the long-term treatment, blood profiles and testosterone levels remained stable. Although prostatic and testicular volumes increased slightly, the changes were not statistically significant (p > 0.05). A significant increase in semen volume was observed (p < 0.05), while other semen parameters showed no significant differences. Notably, post-thaw sperm motility significantly improved at both 15 min and 4 h after thawing, and sperm viability was significantly enhanced at 4 h post-thaw (p < 0.05), suggesting a potential protective effect of LST during cryopreservation. These findings indicate that LST supplementation is physiologically safe and may improve canine sperm quality during freezing and thawing, supporting its potential application in reproductive health management. Full article

This study provides a comparative analysis of the post-thaw sperm lipidomic profiles of Alpine and Spanish–Creole goat breeds to explore breed-specific differences in fatty acid composition and their implications for sperm function and reproductive efficiency. Lipids were extracted from cryopreserved semen samples of Alpine (n = 7) and Spanish–Creole (n = 4) mature bucks and subsequently analyzed by gas chromatography–mass spectrometry (GC-MS), with 21 fatty acids identified within the two breeds. Eight of these fatty acids, namely 13:0, 16:0, 18:0, 24:0, 14:1, 18:1 (cis-9), 24:1, and 18:2 showed significant differences (p < 0.05). The levels of 16:0, 18:0, 24:0, 18:1 (cis-9), 18:1, and 18:2 were higher in the Alpine breed, whereas the levels of 13:0, 14:1, and 24:1 were higher in the Spanish–Creole breed (p < 0.05). Of those, 16:0, 18:1 (cis-9), and 18:2 were both statistically and biologically significant (p < 0.05, FC > 2). Concentrations of the total fatty acids, total saturated fatty acids (Total-SFA), and total polyunsaturated fatty acids (Total-PUFA) were significantly higher in the Alpine breed, whereas the concentrations of the total cis-monounsaturated fatty acid (Total cis-MUFA) were significantly higher in the Spanish–Creole breed (p < 0.05). Network and pathway analyses revealed that 16:0, 18:1 (cis-9), and 18:2 contributed to the most central nodes of the lipidomic network, which may support membrane stability and cryotolerance. The lipidomic differences observed between breeds may be attributed to both genetic and environmental factors and may provide valuable tools for enhancing breeding strategies, artificial insemination programs, and sperm cryopreservation techniques. Full article

Oxidative stress (OS) induced by an imbalance in reactive oxygen species (ROS) levels in vitro impairs embryonic development. Here, we assessed the effects of alpha-lipoic acid (ALA) in in vitro production media on OS reduction, embryonic development, and cryotolerance of bovine embryos. We evaluated the effects of adding different concentrations of ALA (2.5, 5, 10, and 25 μM) to in vitro maturation (IVM) or in vitro culture (IVC) medium on embryonic development. We also determined the effects of adding ALA (25 μM) to the IVM and IVC medium in the same routine on the development and quality of embryos, ROS levels, and cryotolerance. Embryos were produced in vitro using conventional protocols for each treatment. The inclusion of ALA in the IVM and IVC media did not affect the development or quality of embryos; however, it reduced ROS levels in grade II embryos and increased hatching after 12 h on day 7 in grade I embryos and on day 8 in grade II embryos after warming. These findings prompt questions regarding the potential of ALA in improving embryo metabolism, considering the initial embryo recovery in the first few hours of embryo warming. Full article

Specialty Saccharomyces cerevisiae strains have emerged as key contributors to innovations across various industries, offering unique functionalities that extend beyond conventional applications. This review explores the diverse roles of specialty S. cerevisiae in nutrition, winemaking, and bioethanol production. In the field of nutrition, yeast biomass serves as a sustainable and nutrient-dense source of proteins, vitamins, and bioactive compounds, presenting potential as a functional food ingredient. S. cerevisiae can bioaccumulate trace elements like selenium, zinc, and chromium, offering health benefits, but challenges in toxicity and biomass recovery must be addressed for safe use in supplements. In winemaking, S. cerevisiae enhances flavor profiles, improves fermentation efficiency, and reduces undesirable compounds, contributing to premium wine quality. The potential of S. cerevisiae in novel applications is vast, including the development of low-alcohol wines, cryotolerant strains for improved fermentation at lower temperatures, and reduced chemical additives, highlighting its versatility in enhancing wine quality and sustainability. Furthermore, specialty S. cerevisiae plays a pivotal role in bioethanol production, with strain selection and further improvement leading to enhanced yield and efficiency, particularly from lignocellulosic biomass. By examining the latest innovations in each of these areas, this review highlights the versatility and potential of specialty S. cerevisiae in advancing sustainable development and enhancing product quality across sectors. Full article

Background/Objectives: Semen cryopreservation is routinely performed in fertility clinics for a variety of reasons, including fertility preservation and storage of donor sperm, yet the freeze–thaw process leads to cellular damage via ice crystal formation, osmotic shock, and supraphysiological levels of oxidative stress. Sperm resistance to damage during the freeze–thaw process varies widely, yet the intrinsic factors associated with sperm cryotolerance are largely unknown. The study aimed to investigate whether poor chromatin condensation renders sperm vulnerable to DNA fragmentation and cell death induced by the freeze–thaw process. Methods: Participants (n = 51) from the general community who met the inclusion criteria collected a semen sample after 3–8 days of abstinence. Neat semen samples underwent traditional semen analysis, aniline blue (AB)-eosin staining for chromatin condensation, the terminal deoxynucleotidyl transferase dUTP nick end labelling (TUNEL) assay for DNA fragmentation, and the Annexin V assay for apoptosis/necrosis, prior to being cryopreserved using the liquid nitrogen vapour method and stored at −196 °C. Stored samples were later thawed at room temperature and processed using density gradient centrifugation. Motile sperm concentration, DNA fragmentation and apoptosis/necrosis were analysed in post-thaw samples. Results: As indicated by a significant interaction effect in linear mixed models, an increased proportion of AB-positive sperm in the pre-freeze sample exacerbated the adverse effect of freezing on sperm DNA fragmentation (p = 0.004), late apoptosis (p = 0.007), and necrosis (p = 0.007). AB-staining was positively correlated with all three parameters in the post-thaw sample (all r_s ≥ 0.424, all p < 0.01) and remained significant after adjusting for neat sperm concentration (all partial r_s ≥ 0.493, all p < 0.01). Similarly, AB-staining was significantly correlated with the percentage point change in sperm DNA fragmentation (r_s = 0.366, p = 0.014) and necrosis (r_s = 0.403, p = 0.009), both of which remained significant after adjusting for neat sperm concentration (both partial r_s ≥ 0.404, both p < 0.01), and borderline significantly correlated with percentage point change in late apoptosis (r_s = 0.307, p = 0.051). Conclusions: Sperm with poorly condensed chromatin may be more susceptible to cellular damage during the freeze–thaw process, independent of pre-freeze sperm concentration. These findings may help to explain the intrinsic variation in sperm resistance to cryodamage within and between individuals that is poorly understood. Full article

Preimplantation embryo culture, pivotal in assisted reproductive technology (ART), has lagged in innovation compared to embryo selection advancements. This review examines the persisting gap between in vivo and in vitro embryo development, emphasizing the need for improved culture conditions. While in humans this gap is hardly estimated, animal models, particularly bovines, reveal clear disparities in developmental competence, cryotolerance, pregnancy and live birth rates between in vitro-produced (IVP) and in vivo-derived (IVD) embryos. Molecular analyses unveil distinct differences in morphology, metabolism, and genomic stability, underscoring the need for refining culture conditions for better ART outcomes. To this end, a deeper comprehension of oviduct physiology and embryo transport is crucial for grasping embryo–maternal interactions’ mechanisms. Research on autocrine and paracrine factors, and extracellular vesicles in embryo–maternal tract interactions, elucidates vital communication networks for successful implantation and pregnancy. In vitro, confinement, and embryo density are key factors to boost embryo development. Advanced dynamic culture systems mimicking fluid mechanical stimulation in the oviduct, through vibration, tilting, and microfluidic methods, and the use of innovative softer substrates, hold promise for optimizing in vitro embryo development. Full article

Plant cryobanks play a significant role in modern science and breeding. They contribute to the recovery of lost species, the emergence of new plant varieties, and help preserve and explore the diversity of the plant world. The IPPRAS Cryobank collection is constantly supplemented with new samples, while, at the same time, the stored samples are being monitored. In order to test seed germination, seeds of Allium and Veratrum species were thawed. Rare Allium species seeds, such as A. nutans, A. schoenoprasum, and A. victorialis were stored in liquid nitrogen for 17, 19, and 30 years, respectively. Long-term cryopreservation decreased germination rates for A. nutans from 96.55 to 50.00%, for A. schoenoprasum from 72.00 to 62.75%, and for A. victorialis from 90.00 to 83.05%. Seeds of a rare medicinal species, Veratrum lobelianum, were stored in liquid nitrogen for 18 years; the seed germination rate during this storage period has been significantly decreased from 75.00 to 14.81%. V. nigrum seeds were also collected and frozen in liquid nitrogen for 3 days. Short-term cryopreservation did not result in a statistically significant change in germination rates (from 79.71 to 82.69%). The seeds of an endangered ornamental species, Cypripedium calceolus, were collected and kept frozen for 3 days. After cryopreservation, the seeds were planted on three different media, as follows: ½ MS, MS with 10% coconut milk, and BM1. On ½ MS medium, 24.98% seeds formed protocorms, while on MS medium with 10% coconut milk, this number was 10.02%, and on BM1 medium, it was 15.02%, respectively; however, after 2.5 months, all of the protocorms died. Thus, it appears that the existing protocol for seed cryopreservation of C. calceolus needs further improvement. The size, weight, and free water content (WC) of six previously cryopreserved Stipa species and three Allium species were measured. For all the Allium and Stipa species studied, we found no correlation between seed size, WC, and cryotolerance. We also found no correlation between the life form, which reflects the water requirement of the species, and cryotolerance. Full article

This study was conducted to investigate the genotypic and phenotypic characteristics of lactic acid bacteria (LAB) associated with forage plants in the native grassland of western Inner Mongolia and to evaluate their effects on alfalfa silage fermentation. Forage plants and their spontaneous fermentation silages were analysed using culture-based techniques for LAB isolation; the phenotypic properties and 16S rDNA and pheS or rpoA gene sequences of the isolates were evaluated; alfalfa was ensiled with four additive combinations: Lactiplantibacillus plantarum subsp. plantarum (GI19), Lact. plantarum subsp. plantarum and Pediococcus pentosaceus (GI19+GI51), GI19 and 20 g/kg fresh matter of sucrose (GI19+S), and GI19+GI51+S, for 60 d. A total of 73 strains belonging to 16 species were isolated. All isolates grew at 5–45 °C and in 3.0% NaCl, and most of them grew in 6.5% NaCl. Enterococcus faecalis and Lact. plantarum were 26.03% and 17.81% of the total isolates, respectively. All additives improved the silage quality, while GI19+S was more effective for alfalfa ensiling with a higher lactic acid content and lower pH, undesirable microorganism counts, and acetic acid and NH₃-N contents than remnant additives. In conclusion, the LAB species were diverse, and most of them possessed good cryotolerance and osmotolerance; GI19+S was the optimal inoculant for alfalfa fermentation improvement. Full article