Search Results (107)

Semen cryopreservation is associated with sperm vulnerability to oxidative stress and ice crystal-induced damage, adversely affecting in vitro fertilization (IVF) success. This study aimed to investigate the effects of freezing diluent supplemented with antioxidant limonin (Lim), myo-inositol (MYO), and the ice crystal formation inhibitor L-proline (LP) through sperm motility, morphological integrity, and antioxidant capacity. The Lim (150 mM), MYO (90 mM), and LP (100 mM) significantly ameliorated the quality of post-thaw sperm in Debao boar, and combined treatment of these agents significantly enhanced sperm motility, structural integrity, and antioxidant capacity compared with individual agents (p < 0.05). Notably, the combined use of these agents reduced glycerol concentration in the freezing diluent from 3% to 2%. Meanwhile, the integrity of the sperm plasma membrane, acrosome membrane, and mitochondrial membrane potential was significantly improved (p < 0.05), and the result of IVF revealed the total cell count of the blastocysts was also greater in the 2% glycerol group (p < 0.05). In conclusion, the newly developed freezing diluent for semen, by adding Lim (150 mM), MYO (90 mM), and LP (100 mM), can enhance the quality of frozen–thawed Debao boar sperm and reduce the concentration of glycerol from 3% to 2% as high concentrations of glycerol can impair the quality of thawed sperm and affect in vitro fertilization outcomes. In conclusion, the improved dilution solution formulated demonstrated efficacy in enhancing the quality of porcine spermatozoa following cryopreservation and subsequent thawing. Full article

Subfertile boars often go undetected until they cause significant reproductive losses. Current semen quality assessments are limited in their ability to predict fertility, highlighting the need for complementary biomarkers. This study explored whether semen freezability could serve as an indirect indicator of boar fertility. Eighteen boars were classified based on historical fertility records and semen freezability, assessed by post-thaw quality. Fresh and post-thaw semen samples were analyzed using the CASA system and fluorescence microscopy. High-fertility boars showed significantly better motility and functional sperm parameters in fresh semen compared to low-fertility boars. However, these differences were mostly lost after cryopreservation. Conversely, boars with good freezability had consistently better post-thaw semen quality, though this did not correlate directly with higher fertility outcomes. Notably, a combined analysis revealed that boars with both high fertility and poor freezability had the lowest post-thaw semen quality. This suggests that cryopreservation may expose hidden sperm defects not detectable in fresh semen. Total motility was the only parameter associated with both fertility and freezability. In conclusion, while freezability alone may not directly predict fertility, it may help identify low-performing males. The combined assessment of fresh semen motility and freezability could support more effective boar selection strategies. Full article

This study optimized the cryopreservation protocol for cashmere goat semen by testing centrifugation speeds (750, 1000, 1250, 1500 rpm) for seminal plasma removal and L-proline concentrations (10, 30, 50 mmol/L) in a freezing extender. Semen from six 3-year-old breeding bucks of Inner Mongolia cashmere goats was evaluated post-thaw in terms of motility, membrane integrity, antioxidant capacity, and artificial insemination (AI) outcomes (n = 130 does). The results demonstrated that the group that underwent centrifugation at 1250 rpm saw significantly improved sperm motility (p < 0.05), curvilinear velocity (VCL, p < 0.05), and straight-line velocity (VSL, p < 0.05) compared to the other groups. The addition of 30 mmol/L L-proline further enhanced post-thaw sperm motility (p < 0.05), plasma membrane integrity (p < 0.05), and acrosome integrity (p < 0.05), while significantly reducing reactive oxygen species (ROS, p < 0.05) and malondialdehyde (MDA, p < 0.05) levels. This group also exhibited the highest antioxidant capacity, as indicated by elevated levels of total antioxidant capacity (T-AOC), superoxide dismutase (SOD), catalase (CAT), and glutathione peroxidase (GSH-Px) (p < 0.05). AI trials revealed that semen treated with 1250 rpm centrifugation and 30 mmol/L L-proline achieved the highest kidding rate (56.82%), significantly outperforming the control group (37.21%, p < 0.05). Meanwhile, no significant differences were observed in prolificacy or offspring sex ratio (p > 0.05). In conclusion, this study demonstrates that combining 1250 rpm centrifugation for seminal plasma removal with the addition of 30 mmol/L L-proline to the freezing extender significantly improves the quality of cryopreserved cashmere goat semen and enhances AI outcomes. Full article

The cryopreservation of boar sperm effectively extends its storage period but often leads to increased reactive oxygen species (ROS) production, compromising sperm quality. Plant extracts, rich in bioactive compounds such as polyphenols and flavonoids, have been shown to reduce ROS. Djulis (Chenopodium formosanum), also known as the “ruby of cereals”, is nutritionally rich and holds potential as a cryoprotective additive. This study aimed to determine the optimal concentration of extracts from different parts of djulis, including unhulled seeds and stems, for effective boar semen cryopreservation. Fresh semen from Taiwan indigenous boars was diluted with a modified GLT-cryoprotectant extender containing glycerol, low-density lipoprotein (LDL), and trehalose. The experimental groups included DSS25, DSS50, DS25, and DS50—representing djulis unshelled seed at 25 mg/mL and 50 mg/mL, and djulis stem at 25 mg/mL and 50 mg/mL in distilled water, respectively—alongside a control group without additives. Post-thaw assessments included sperm motility, kinetic parameters, viability, acrosome integrity, and the antioxidant properties of djulis extracts, such as DPPH radical scavenging activity and total phenolic acid content. Results showed that total motility (TM) was significantly higher in the DSS25 (48.8 ± 3.9), DSS50 (49.0 ± 6.7), and DS50 (49.0 ± 2.4) groups compared to the control group (31.3 ± 4.8). Similarly, progressive motility (PM) was significantly improved in DSS25 (27.5 ± 2.7) and DSS50 (26.8 ± 4.1) versus the control (12.8 ± 3.2). However, for straightness (STR), the control group (87.8 ± 1.3) exhibited significantly higher values than the DS50 group (83.5 ± 1.3) (p < 0.05). Viability and acrosome integrity showed no significant differences across groups. In conclusion, djulis extracts positively influence sperm motility and forward movement, with 1% djulis extract confirmed to enhance the quality of cryopreserved semen. Future research will focus on determining the optimal dosage of djulis extract for improved cryopreservation outcomes. Full article

Chicken semen cryopreservation is crucial for utilizing high-quality cockerel genetics, but semen is highly sensitive to cryoinjury, leading to poor preservation outcomes. This study aimed to establish a theoretical foundation for selecting cockerels for semen cryopreservation through serum testing and to improve semen quality via DNA methylation editing. Semen and serum samples were collected from 102 Xiaoshan cockerels, with semen cryopreserved and thawed following standardized protocols. Post-thaw semen quality and serum testosterone (T) levels were assessed. Eight cockerels were selected based on motile sperm quality, and whole-genome bisulfite sequencing (WGBS) was used to analyze sperm DNA methylation. The results showed a significant positive correlation between serum T levels and sperm motility. There were notable differences in sperm motility and serum T levels between high-quality and low-quality semen groups but no differences in estradiol (E₂), superoxide dismutase (SOD), or glutathione peroxidase (GSH-Px) levels. A total of 217 differentially methylated regions (DMRs) and 116 differentially methylated genes (DMGs) were identified. Key genes such as PRKACB (protein kinase, cAMP-dependent, catalytic, beta) and ACSL1 (long-chain-fatty-acid--CoA ligase 1) were associated with sperm motility. These findings provide important insights for improving semen cryopreservation and contribute to breeding practices and the development of cryoprotectants. Full article

Sperm cryopreservation and assisted reproduction are powerful tools for the conservation of endangered species. The domestic cat has been a useful model for studying wild felid reproductive biology due to the limited availability of endangered individuals for experimental research. Here, we investigate the effect of cryodiluents (TEST vs. Biladyl) and the timing of glycerol addition (before vs. after refrigeration, in one vs. three steps, respectively) on post-thaw sperm quality (motility, acrosome integrity) and their subsequent in vitro fertilization (IVF) ability with homologous oocytes. The results showed no statistically significant differences in sperm traits when samples were cryopreserved in TEST or Biladyl, or when glycerol was added in one or three steps. Motile sperm and intact acrosomes were significantly correlated before and after cryopreservation, indicating consistent relationships in fresh and thawed samples. The use of Biladyl significantly reduced IVF rates after cryopreservation compared to fresh sperm. Cryopreservation in TEST led to IVF rates that were not significantly different from those of fresh sperm. Using swim-up after thawing, or adding 1 mM pentoxifylline, did not enhance IVF results. Overall, a TEST cryodiluent with 4% glycerol added in one step is a reliable option for preserving epididymal cat spermatozoa. Full article

This study aimed to investigate the effects of adding different levels of punicalagin or oleuropein to TRIS diluent on the quality of frozen–thawed semen from Najdi rams. Semen was diluted using TRIS-based diluter with 15% egg yolk (control group); supplemented with 0.1, 0.5, or 1 mg/100 mL punicalagin (in Experiment 1); or supplemented with 1, 2.5, or 5 mg/100 mL oleuropein (in Experiment 2). The collected semen was evaluated and cryopreserved, with the motility and concentration of sperm assessed using a CASA system. The results showed that the total motile spermatozoa (TMS), percentage of progressive motile spermatozoa (PMS), curvilinear velocity (VCL), rectilinear velocity, average path velocity (VAP), linearity coefficient, straightness index, minor defects, and sperm vitality were higher in the 0.1 mg/100 mL punicalagin group (p < 0.05) than in other groups. HOST% post-thawing was significantly higher in all punicalagin groups compared to the control group (p < 0.001). The percentages of PMS, TMS, VCL, minor defects, and sperm vitality were higher in the 1 mg/100 mL oleuropein group (p < 0.05) than in other groups. Oleuropein supplementation at 5 mg/100 mL decreased VAP in cooled sperms, while all levels increased VAP post-thawing. HOST-positive sperms% post-thawing was higher in all oleuropein-treated groups than the control group (p < 0.001). Moreover, oleuropein nonsignificantly increased the acrosome integrity in cooled sperms, while higher studied concentrations of oleuropein (2.5 and 5 mg/100 mL) decreased the acrosome integrity in frozen sperms. In conclusion, adding punicalagin (0.1 mg/100 mL) or oleuropein (1 mg/100 mL) to TRIS diluent improved the quality of frozen–thawed semen from rams. Full article

Cryopreservation is a widely used technique for long-term semen storage; however, it can induce oxidative stress, compromising sperm quality. This study examines the effect of Kaempferia parviflora (KP) supplementation in semen extenders on the post-thaw sperm quality of Thai native bulls. Fresh semen was collected and evaluated for motility, viability, and concentration prior to cryopreservation. Semen samples were allocated to four treatment groups: 0 mg/mL (control), 0.5 mg/mL, 1 mg/mL, and 1.5 mg/mL KP. Subsequently, the samples were frozen, then thawed, and analyzed for sperm motility, viability, membrane integrity, and oxidative stress. The results showed that supplementation with 1.0 mg/mL KP significantly improved sperm motility (46.29 ± 2.66%) and viability (43.42 ± 2.15%) compared to the control (40.77 ± 2.76% and 38.63 ± 2.66%, respectively). Membrane integrity was also enhanced (47.64 ± 1.18% vs. 41.85 ± 1.98% in the control), while oxidative stress levels were reduced (MDA concentration: 2.33 ± 0.29 µM/mL vs. 2.73 ± 0.33 µM/mL in the control). However, the highest concentration (1.5 mg/mL KP) negatively affected sperm quality, with reduced motility (36.97 ± 3.32%), viability (30.88 ± 3.02%), and membrane integrity (35.64 ± 1.61%). These findings suggest that 1 mg/mL KP is the optimal concentration for improving post-thaw semen quality in cryopreservation protocols for Thai native bulls. Full article

The supplementation of freezing medium with crocin results in an amelioration of post-thawing sperm quality, as determined by motility and viability. This study aimed to examine the molecular mechanisms underlying the ameliorative effect of crocin. Bovine spermatozoa were cryopreserved in a freezing medium supplemented with 0, 0.5, or 1 mM of crocin. Sperm lysates were evaluated for their redox status and the expression of proteins implicated in the heat stress response (HSR) and apoptosis. Crocin protected spermatozoa from the accumulation of superoxide anion and ameliorated their post-thawing antioxidant capacity in terms of ROS scavenging activity and glutathione content. Moreover, crocin decreased the levels of inducible nitric oxide synthase (iNOS), while it increased superoxide dimsutase-1 (SOD-1) levels. These effects were associated with an inhibition of apoptosis, as evidenced by a decreased Bax/Bcl-2 protein ratio and decreased levels of caspase-cleaved substrates. Finally, crocin affected the heat shock response of spermatozoa, since it upregulated the levels of heat shock proteins (Hsp) 60, 70, and 90. In conclusion, the addition of crocin to the freezing medium ensured controlled amounts of ROS, enhanced the antioxidant capacity of spermatozoa, and upregulated the anti-apoptotic proteins and Hsps, thus contributing to the maintenance of cellular homeostasis. Full article

Egg yolk, commonly employed as a cryoprotectant in semen cryopreservation, contains large particle matter that can diminish semen quality post thaw and complicate its quality assessment. For this reason, we designed a centrifugal treatment of chicken egg yolk to evaluate its effect on the cryopreservation of porcine semen. The control group (CG) was prepared with a dilution of chicken egg yolk by conventional mixing treatment, and the experimental group (EG) used a dilution of centrifugally treated chicken egg yolk for the ultra-low-temperature cryopreservation of porcine semen. The freezing process was carried out by conventional freezing methods. The spermatozoa were subsequently assessed for various parameters, including motility, acrosome integrity rate, plasma membrane integrity rate, antioxidant indexes, apoptosis rate, and the expression of apoptosis-related genes. The results showed that, post freeze–thawing, the motility, viability, VSL, and VCL of the spermatozoa in the EG were significantly higher than those observed in the CG (p < 0.05). Additionally, the acrosome integrity and plasma membrane integrity of the spermatozoa in the EG were significantly enhanced compared to the CG (p < 0.05). Furthermore, the EG exhibited significantly lower MDA content and sperm apoptosis rate (p < 0.05), while demonstrating significantly higher T-AOC and CAT levels (p < 0.05) relative to the CG. In comparison to the CG, the EG exhibited a significant reduction in the gene expression of TNF-a and Bax in the spermatozoa (p < 0.05), whereas the expression levels of CAT and Bcl-2 were significantly elevated (p < 0.05). In conclusion, the dilution solution formulated through the centrifugal processing of chicken egg yolk demonstrated efficacy in enhancing the quality of porcine spermatozoa following cryopreservation and subsequent thawing. Full article

This study investigated the effects of various cryoprotectant combinations on post-thaw sperm quality, biomolecular changes, DNA methylation, and pregnancy rates using Boer goat semen. Synchrotron-based Fourier-transform infrared spectroscopy (SR-FTIR) was used to assess biomolecular changes. A Tris-based extender supplemented with 5% glycerol was used in combination with different concentrations of cryoprotectants, including 1% and 3% soybean lecithin and 10% and 18% egg yolk, with Andromed^® serving as the control. SR-FTIR analysis revealed that the combination of 5% glycerol and 18% egg yolk (T4) resulted in significantly higher levels of lipids, ester lipids, and secondary protein structures (α-helix) compared with those under the other treatments (p < 0.05). Analysis of the principal component analysis (PCA) score plot and correlation loadings revealed a positive association between the cryoprotectant combination of T4 and increased levels of lipids and ester lipids, as well as enhanced sperm motility, progressive motility, and viability. Furthermore, this combination achieved a pregnancy and parturition rate of 66.67%, which was notably higher than the rate achieved with Andromed^® (37.50%). Moreover, T4 did not show a significant difference in DNA methylation levels compared to Andromed^® and fresh sperm (p > 0.05). Overall, the results indicated that specific cryoprotectant combinations play a key role in enhancing the biomolecular and functional integrity of freeze-thawed Boer goat semen. Full article

Cryopreservation of native Thai bull semen often results in significant post-thaw quality reduction, underscoring the need for effective cryoprotective strategies. This study investigated the effect of Moringa oleifera leaf extract (MOLE) as an antioxidant supplementation by incorporating four MOLE concentrations (0–1.5% [w/v]) into a standard semen extender, followed by cryopreservation using liquid nitrogen vapor freezing. Data were analyzed using a randomized complete block design with Tukey’s post hoc test (p < 0.05). Post-thaw analysis of semen revealed that 1 mg/mL MOLE significantly enhanced total sperm motility, progressive sperm motility, sperm viability, and sperm plasma membrane integrity compared to the control and other MOLE concentrations (p < 0.05). This concentration also improved the amplitude of lateral head displacement and curvilinear velocity and reduced malondialdehyde levels in semen samples (p < 0.05), indicating reduced lipid peroxidation. Higher MOLE concentrations negatively impacted semen quality. In conclusion, supplementation with 1 mg/mL MOLE markedly improved post-thaw semen quality and reduced lipid peroxidation, suggesting its potential as an antioxidant for enhancing reproductive outcomes in native Thai bulls. Full article

The main objective of this study was to investigate boar-to-boar variations in the quality characteristics of sperm from the sperm-rich fractions (SRFs) and whole ejaculates (WEs) following freezing–thawing. Several sperm attributes, such as motility patterns analyzed by the computer-assisted sperm analysis (CASA) system, mitochondrial function, membrane integrity, and DNA fragmentation were used to compare the cryo-survival of sperm from SRFs and WEs from boars with good and poor semen freezability (GSF and PSF, respectively). In this study, boars with post-thaw total motility (TMOT) more than 30% (>30%) were classified as having GSF, while those with post-thaw TMOT less than 30% (<30%) were classified as having PSF. Principal component analysis 1 (PCA1), which is the main component of the sample variation, explained approximately 75% of the variance between the GSF and PSF groups, reaffirming the reliability of post-thaw TMOT as a reliable criterion used to classify the animals. Most of the post-thaw sperm parameters of the SRFs and WEs were positively correlated. Furthermore, scatter plot analyses show stronger relationships between the analyzed post-thaw parameters of the frozen–thawed (FT) sperm of SRFs than those of WEs. Individual boar variations or the sperm source had marked effects on the quality characteristics of FT sperm. The higher TMOT, velocity straight line (VSL), and velocity average path (VAP) of FT sperm were more enhanced in the SRFs compared with the WEs of the PSF group. Furthermore, the mitochondrial function, membrane integrity, and DNA fragmentation of FT sperm were markedly higher in the SRFs than in the WEs, particularly for the poor freezability boars. We suggest that the freezability potential of sperm of the GSF group does not differ significantly between the SRFs and WEs, reaffirming that boar variability is an important factor that affects the cryo-survival of sperm. Full article

Due to oxidative damage and mitochondrial dysfunction, boar semen cryopreservation remains a significant challenge. This study investigates the effects of pyrroloquinoline quinone (PQQ), a mitochondrial-targeted antioxidant, on the post-thaw boar sperm quality during cryopreservation. Boar semen was diluted in a freezing extender containing different concentrations of PQQ (0, 10, 100, 1000, 10,000 nM). After freezing–thawing, the sperm motility, viability, acrosome integrity, mitochondrial activity, adenosine triphosphate (ATP) levels, DNA integrity, malondialdehyde (MDA) levels, reactive oxygen species (ROS) levels, superoxide dismutase (SOD) activity, mitochondrial transcription proteins levels, and fertilization capacity were assessed. The results show that 1000 nM PQQ supplementation to the freezing extender significantly enhanced post-thaw sperm motility, viability, and acrosome integrity compared to the control (p < 0.05). Additionally, 1000 nM PQQ increased mitochondrial membrane potential (MMP) and ATP levels, while decreasing MDA and mitochondrial ROS levels, and reducing DNA damage (p < 0.05). Furthermore, the levels of mitochondrial-encoded proteins were significantly elevated in the 1000 nM PQQ group compared to the control (p < 0.05). Interestingly, sperm in the 1000 nM PQQ group showed a higher binding rate to oviductal epithelial cells and the zona pellucida (ZP), indicating higher fertilization potential. These findings suggest that the use of mitochondria-target antioxidant, PQQ, can improve post-thaw boar sperm quality and fertilization via its capacity to reduce oxidative stress and protect mitochondrial function. Full article

Reproductive technologies, including sperm cryopreservation, offer conservationists enhanced capacity to genetically manage populations and improve the outcomes of conservation breeding programs (CBPs). Despite this potential, the post-thaw quality of amphibian sperm is highly variable following cryopreservation, and research focused on protocol refinement is needed. The aim of this study was twofold: (1) to investigate the effect of the addition of bovine serum albumin (BSA) to the cryopreservation medium (pre-freeze), and (2) the effect of the addition of caffeine to the activation medium (post-thaw), on post-thaw sperm characteristics in the critically endangered Booroolong frog (Litoria booroolongensis). Spermic urine samples were collected from 14 male frogs following hormonal induction of spermiation, and each sample was split among three cryopreservation treatments, where the cryopreservation medium contained either 0 (control), 0.5, or 1% BSA (w/v). Samples were cryopreserved and thawed, and sperm motility was then activated in one of two activation treatments: Milli-Q water (control) or Milli-Q water plus 4.5 mM caffeine. Sperm viability (proportion live/dead) was assessed using fluorescent microscopy, and sperm motility metrics were evaluated using computer-assisted sperm analysis (CASA). Results from this study showed that BSA concentration had no effect on post-thaw sperm viability. Additionally, neither BSA concentration nor activation in caffeine influenced post-thaw sperm motility characteristics (total motility, forward progressive motility, and velocity). Assessment time of sperm motility varied from 5 to 13 min post-activation and was significantly correlated with each motility measure, with motility and velocity metrics decreasing as time post-activation increased. The results reported herein provide no evidence for an effect of BSA or caffeine at the concentrations tested on post-thaw sperm characteristics in the Booroolong frog, but they highlight the time-sensitive nature of sperm assessment post-thaw and implications for the timing of sperm handling during assisted fertilisation efforts. Full article