Post-thaw Sperm Research Articles

Enhancement of cryopreserved rooster semen and fertility potential after oral administration of Thai ginger (Kaempferia parviflora) extract in Thai native chickens.

Semen cryopreservation is an effective method of preserving genetic material, particularly in native chicken breeds facing a substantial decline. In this study, we evaluated the quality of frozen/thawed rooster semen treated with different concentrations of oral administrations of black ginger (Kaempferia parviflora: KP) extract and determined its fertility. Thirty-two Thai native roosters (Pradu Hang Dum, 42 weeks old) were used in this study. The treatments were classified into four groups according to the concentration of KP extract administered to the roosters: 0, 100, 150, and 200 mg/kg body weight. The quality of fresh semen was analyzed before cryopreservation. Post-thaw sperm quality and fertility potential were determined. Also, lipid peroxidation was determined. The results showed that sperm concentration and movement increased in roosters treated with 200 mg/kg of KP extract (p<0.05). The malondialdehyde (MDA) in the roosters receiving 200 mg/kg KP extract was lower than that in the other but had an insignificant difference within the KP treatment groups (p>0.05). The highest MDA levels were observed in the control group (p<0.05). The percentage of motile sperm (total motility and progressive motility) after semen thawing was higher in roosters that received 150 and 200 mg/kg KP extract than in those that received 100 mg/kg KP extract and the control (p<0.05). MDA levels decreased significantly in roosters that received 150 and 200 mg/kg KP extract than in those that received 100 mg/kg KP extract and the control (p<0.05). Fertility and hatchability were greater in the KP150 and KP200 groups than in the KP100 and control groups (p<0.05). The optimal amount of KP extract influencing initial sperm quality was determined to be 200 mg/kg. However, 150 mg/kg was the optimal low dosage of KP extract administration that maintained sperm quality and fertility following semen cryopreservation.

The Effects on Post-Thaw Sperm Quality and Nuclear DNA Integrity of Supplementation of Low-Density Lipoprotein to Freezing Extender in the Mouse

Mice are an important research tool for genetic and molecular biology, allowing researchers to explore a variety of human illness models. Egg yolk is a common component of semen extenders for domestic animals and low-density lipoproteins (LDL) from egg yolk have some cryoprotective properties. This study aimed to investigate sperm quality characteristics and nuclear DNA integrity after post-thawing in an extender (18% raffinose + 3% skim milk) supplemented with different concentrations of LDL (2.5%, 5.0%, 7.5%, or 10%) in mice. 18% Raffinose+3% skim milk extender was used as a control group without LDL. CD-1 mice were used in the study, and semen was collected from the cauda epididymis and diluted with the extender. The straws were then frozen and thawed to evaluate progressive motility, viability, plasma membrane (HOST), acrosome, and nuclear DNA integrity parameters. Fresh sperm had the highest progressive motility, viability, plasma membrane integrity, and longevity (endurance) of progressive motility for 4 h in HTF solution. The greatest spermatologic results, including nuclear DNA integrity, were determined in fresh sperm (p

Protective Effects of Astaxanthin on Post-Thaw Sperm Quality in Normozoospermic Men

Fighting against free radical accumulation during the cryopreservation process with the help of exogenous antioxidants has become an effective strategy to augment post-thaw sperm quality. The present study explored the effects of various concentrations of astaxanthin, as a potent antioxidant, on human sperm parameters during the freeze–thaw process. Twenty-five normozoospermic specimens were included in this prospective study. Each sample was divided into five equal parts: fresh group, and frozen–thawed groups including 0, 1, 10, and 50 μM of astaxanthin. The prepared spermatozoa were cryopreserved by rapid freezing technique. Precryopreservation and post-thaw sperm motility parameters, sperm morphology, vitality, DNA fragmentation, reactive oxygen species (ROS) levels, and mitochondrial membrane potential (MMP) were investigated. All sperm parameters after thawing significantly decreased compared to before freezing. Treatment of spermatozoa with 50 μM of astaxanthin significantly increased their total and progressive motility, viability, DNA integrity, and MMP and decreased their intracellular ROS levels compared with the control group. In total, 10 μM of astaxanthin significantly improved total and progressive motility, DNA integrity, MMP, and decreased ROS levels, whereas, in the 1 µM group, there were significant differences only in ROS levels. As a result, we found that astaxanthin can improve sperm quality after freezing/thawing and decrease the detrimental effects of this process on sperm parameters.

Sperm characteristics of cryopreserved Prochilodus lineatus semen after adding cholesterol-loaded cyclodextrin

The experiment evaluated the effect of adding cholesterol-loaded cyclodextrin (CLC) to Prochilodus lineatus fish (Curimata) semen on post-thaw sperm quality. Twelve adult fish were used for sperm collection after induced spermiation with carp pituitary gland. The semen was diluted and treated with CLC in concentrations of 0 (control), 0.5, 1.0, 2.0, 3.0, and 4.0 mg for 120 × 106 spermatozoa/ml, loaded in 0.5 ml straws, packaged and placed in dry vapor vessel cylinders for 24 h before being submerged in liquid nitrogen for storage. The samples were thawed in a water bath at 60 °C for 8 s, and the sperm parameters evaluated were motility, activation duration, longevity, plasma membrane integrity, and morphology. Data were tested for normal distribution and ANOVA, followed by Friedman test (P < 0.05). Spermatozoa treated with CLC displayed higher motility than the control (P < 0.05). The duration of sperm activation was longer in sperm treated with 0.5, 1.0, and 2.0 mg of CLC than in control (P < 0.05). The membrane integrity was higher in sperm treated with 0.5, 1.0, 2.0, and 3.0 mg of CLC than in control and four mg-treated samples (P < 0.05). The sperm longevity and morphology alterations did not differ between treatments (P > 0.05). Adding 0.5, 1.0, or 2.0 mg of CLC in Prochilodus lineatus semen before cryopreservation improves sperm motility and membrane integrity.

In Vitro Effects of Charged and Zwitterionic Liposomes on Human Spermatozoa and Supplementation with Liposomes and Chlorogenic Acid during Sperm Freezing.

Semen handling and cryopreservation induce oxidative stress that should be minimized. In this study, human semen was supplemented during cryopreservation with formulations of handmade liposomes and chlorogenic acid (CGA), an antioxidant compound. Zwitterionic (ZL), anionic (AL), and cationic (CL) liposomes were synthesized and characterized. Three aliquots of swim-up-selected sperm were incubated with ZL, AL, and CL (1:10,000), respectively. The percentages of sperm with progressive motility, high mitochondrial membrane potential (MMP; JC-1), double-stranded DNA (dsDNA acridine orange), and acrosome integrity (Pisum sativum agglutinin) were assessed. Then, human semen was frozen using both 1:10,000 ZL and CGA as follows: freezing medium/empty ZL (EL), freezing medium/empty ZL/CGA in the medium (CGA + EL), freezing medium/CGA loaded ZL (CGA), freezing medium (CTR). The same sperm endpoints were evaluated. ZL were the most tolerated and used for semen cryopreservation protocols. All the supplemented samples showed better endpoints versus CTR (p < 0.001). In particular, spermatozoa from the CGA and CGA + EL A samples showed increased motility, dsDNA, and acrosome integrity versus CTR and EL (p < 0.001; motility EL vs. CGA + EL p < 0.05). ZL and CGA can improve post-thaw sperm quality, acting on both cold shock effect management and oxidative stress. These findings open new perspectives on human and animal reproduction.

Effect of Different Dilution Methods and Ratios of Ram Semen on Sperm Parameters after Cryopreservation.

The dilution method and ratio were tested to assess their effects on the Hu ram semen after cryopreservation. Experiment I aimed to explore the effect of various dilution ratios (1:1, 1:2, 1:3, 1:4) of diluent I (Tris-based and egg yolk) under the condition of 1:1 dilution of diluent II (diluent I and glycerol) on the Hu ram semen preserved in liquid nitrogen regarding spermatozoa motility and kinetic parameters. Experiment II aimed to investigate the effect of various dilution ratios (1:1, 1:2, 1:3, 1:4) of diluent I under the condition of 1:2 dilution of diluent II to the Hu ram semen for cryopreservation on spermatozoa motility and kinetic parameters. The purpose of experiment III is to assess the effect of various dilution methods and ratios on the cryopreservation of Hu ram semen by detecting spermatozoa motility, kinetic parameters, plasma membrane integrity, acrosome integrity and reactive oxygen species (ROS) level. Experiment III includes four groups: one-step dilution method and two-step dilution method. The two-step dilution method includes two groups: 1:2, 1:1 and 1:3, 1:2, and the one-step dilution method includes two groups: 1:5 and 1:11. The results indicated that the post-thawed spermatozoa total motility (TM), progressive motility (PM) and average motion degree (MAD) were highest in the 1:2 group and significantly higher (p < 0.05) than those in the 1:1 and 1:4 groups under the condition of 1:1 dilution of diluent II. The post-thawed spermatozoa TM and PM of the 1:3 group were significantly higher (p < 0.05) than those of the other groups under the condition of 1:2 dilution of diluent II. The post-thawed spermatozoa TM, PM, plasma membrane integrity and acrosome integrity of the two-step group (1:3, 1:2) were the highest and significantly higher (p < 0.05) than those in the other groups. Additionally, the post-thawed spermatozoa ROS level of the two-step group (1:3, 1:2) was significantly lower (p < 0.05) than that in the one-step groups (1:5 and 1:11). Therefore, a two-step dilution (1:3, 1:2) was found to be the most suitable method and ratio for diluting the Hu ram semen after cryopreservation.

Evaluation of Different Thawing Protocols on Iberian Boar Sperm Preserved for 10 Years at Different Liquid Nitrogen Levels.

The conservation of genetic resources in pig breeds, notably the Iberian pig, is crucial for genetic improvement and sustainable production. Prolonged storage in liquid nitrogen (LN2) is recognized for preserving genetic diversity, but potential adverse effects on seminal quality remain debated. This study aims to assess the impact of ten years of storage at different LN2 levels and to optimize thawing protocols for Iberian pig sperm. Sperm samples from 53 boars were cryopreserved and stored at varying LN2 levels and, a decade later, the samples were thawed at 37 °C for 20 s or at 70 °C for 8 s. Sperm motility, membrane integrity, acrosome status, and DNA fragmentation were evaluated in year 0 and year 10. Overall, no significant differences were observed in post-thaw sperm quality between storage levels in year 0 or year 10. But thawing at 70 °C 8 s showed significant improvements, particularly in samples that were always stored in LN2, in all analyzed parameters except fragmentation, which was not affected by cryostorage. This study suggests that the long-term preservation of Iberian pig sperm does not affect quality over time, regardless of whether the samples were fully submerged in LN2. Furthermore, it is determined that thawing at 70 °C for 8 s maximizes post-thaw sperm quality, especially in those samples stored constantly submerged in LN2.

Effects of apigenin and trans-ferulic acid on microscopic and oxidative stress parameters in the semen of water buffalo bulls during cryopreservation

Cryopreservation involves exposing sperm to stressful conditions that affect cell viability. The high quality of the Azerbaijani water buffalo's by-products, such as buffalo milk, makes it a species of significant importance. Our focus is on protecting its genetic resources by preserving its sperm, as their numbers will decrease in the coming years and they are at risk of extinction. This study's goal was to ascertain how apigenin (A) and trans-ferulic acid (t-FA) affected the semen quality of Azari water buffalo bulls under cryopreservation. Pooled buffalo sperm (n = 35 ejaculations) were diluted in a Tris-based diluent also containing varying amounts of apigenin (0.2, 0.4, 0.6, and 0.8 mM) and trans-ferulic acid (2.5, 5, 10 and 20 mM). Following a freeze-thaw procedure, samples were assayed for total antioxidant capacity (TAC), catalase (CAT), superoxide dismutase (SOD), malondialdehyde (MDA), glutathione activity (GSH), glutathione peroxidase (GPx), progressive motility and total motility, motility properties, plasma membrane functionality, and viability. Sixty days after insemination, the rectal examination was performed on 38 buffaloes that had undergone sexual breeding to confirm pregnancy. The results of the study show that the addition of A-0.2, A-0.4, and t-FA-10 to buffalo semen increases the percentage of intact plasma membrane, motility, and sperm viability, as well as the levels of GSH, GPx, CAT. and TAC. In addition, there is a decrease in MDA and DNA damage after cryopreservation. Furthermore, the results show that 0.4 mM apigenin significantly increases conception rates compared to the control group. The base extender of Tris supplemented with A (0.4 and 0.2 mM) and t-FA (10 mM) improves the antioxidant indices of both frozen and thawed buffalo sperm, which in turn improves post-thawing sperm quality and in vivo fertility improves buffalo sperm.

Cryopreservation of sperm from the gudgeon, Microphysogobio rapidus (Cyprinidae): Effects of cryoprotectant, diluents, and dilution ratio

We investigated methods for cryopreserving sperm from the endangered gudgeon, Microphysogobio rapidus, by examining the effects of cryoprotective agent (CPA) concentration, diluent, and dilution ratio on post-thaw sperm quality. The quality of frozen sperm was evaluated in terms of motility and kinematic parameters, viability, DNA damage, and fertilization rate. We evaluated methanol, glycerol, dimethyl sulfoxide (DMSO), and ethylene glycol as CPAs. Sperm motility, velocity, and viability were significantly higher when methanol was used as the CPA (p < 0.05). The diluents tested were Ringer's solution, Kurokura's Extender, Common Carp Sperm Extender (CCSE), and buffered sperm motility-inhibiting saline solution (BSMIS); post-thaw motility was highest when Ringer's solution was used as the diluent. Next, various quantities of methanol were combined with Ringer's solution to identify the optimal dose of methanol. The dilution ratios tested ranged from 1:1 to 1:7. Cryopreserved sperm was thawed at 20 °C for 15 s. The use of 10% methanol with Ringer's solution at a dilution ratio of 1:5 resulted in the highest post-thaw sperm motility, viability, and velocity including VAP, VCL, and VSL. Post-thaw sperm showed significantly greater DNA damage than the control (fresh sperm) (p < 0.05). The fertilization rate was highest with fresh sperm (p < 0.05), followed by sperm frozen with 10% methanol + Ringer's solution. We recommend that the best way to preserve sperm in the studied species is to use a combination of Ringer's solution and 10% methanol at a 1:5 dilution ratio. Our findings will facilitate the artificial fertilization of M. rapidus.

Post-thaw sperm morphokinetics and mitochondrial genes profile of buck semen supplemented with selenium in two different forms

Background The quality characteristics of semen is a determinant factor for improving fertility of rabbit. Objectives The current investigation was done to evaluate post-thaw sperm morphokinetics and mitochondrial genes expression profile of buck semen supplemented with selenium in two different forms. Materials and methods Sexually mature bucks (n=16) that aged three months on average were used for collecting two ejaculates which were evaluated using computer assisted semen analysis (CASA) program. The semen samples of all experimental animals which recorded progressive motility greater than or equal to 70% were pooled for cryopreservation. The pooled semen was divided into three groups that were supplemented with selenium nanoparticles and selenium in normal form in addition to the control group. Morphological characteristics as well as CASA parameters were assessed after freezing for 1 week. Moreover, enzymatic activity assays were performed to measure the antioxidant capacity of cryopreserved buck semen. Transcriptional profile of mitochondrial activity and antioxidant defense regulating genes was conducted using quantitative real-time polymerase chain reaction (PCR). Results and conclusion The addition of selenium in normal and Nano forms has significantly (P ≤0.05) enhanced some of CASA parameters such as DCL (µm), DAP (µm), DSL (µm), VCL (µm/s), VAP (µm/s), Amplitude of lateral head (ALH) (µ), BCF (Hz), and VSL (µm/s) during prefreezing period compared with control group. Moreover, the data presented in the present study indicated a significant (P ≤0.05) improvement of post-thaw total and progressive sperm motility in the two groups supplemented with normal and Nano compared with control group. The post-thaw level of total antioxidant capacity (TAC) and percentage of live sperm were higher in the two groups supplemented with selenium and Nano selenium than the control group. The expression profile of candidate genes regulating mitochondrial activity (ATP Synthase F1 Subunit Alpha (ATP5A1), NADH dehydrogenase subunit1 (ND1), NADH dehydrogenase subunit2 (ND2), and Carnitine palmitoyltransferase (CPT2)) was increased significantly (P ≤0.05) in semen supplemented with selenium in normal and Nano forms compared with the control group. In conclusion, the data of this investigation demonstrated enhancement of CASA parameters during pre-freezing post thaw total and progressive sperm motility in the two groups supplemented with normal and nano selenium. The proportion of sperm viability and the level of total antioxidant capacity were enhanced in the two groups supplemented with selenium which was coupled with up-regulation of mitochondrial transcripts. It seems the both two forms shared the same mechanism on improving post-thaw physical and molecular qualities of rabbit sperm.

Apigenin supplementation substantially improves rooster sperm freezability and post-thaw function

This pioneering research investigated apigenin potential to augment rooster sperm cryosurvival in an extender model. Apigenin is a natural antioxidant flavonoid showing promise for improved post-thaw sperm function. However, its effects on avian semen cryopreservation remain unexplored. This first study supplemented rooster sperm Lake extender with 0, 50, 100, 200, 400 μmol/L apigenin to determine the optimal concentrations for post-thaw quality. Supplementation with 100 μmol/L apigenin resulted in significant enhancements in total motility (from 41.5% up to 71.5%), progressive motility (18.1% to 29.1%) (p < 0.05), membrane integrity (40% to 68%), mitochondrial function (p < 0.001), viability (37% to 62%) and total antioxidant capacity (p < 0.001) compared to the control. It also substantially reduced percentages of abnormal morphology, reactive oxygen species and apoptosis (p < 0.001). Although 200 μmol/L apigenin significantly enhanced some attributes, effects were markedly lower than 100 μmol/L. Higher doses did not improve cryoprotective parameters. This indicates 100 μmol/L as the optimal apigenin concentration. This represents the first report of apigenin protecting rooster sperm from cryodamage. The natural antioxidant improved post-thaw sperm quality, likely by suppressing oxidative stress and apoptosis. Apigenin shows promise for enhancing rooster sperm cryosurvival.

Evaluation of the effects of glucose concentration in extender and sperm concentration in straw for cryopreservation of Atlantic sturgeon (Acipenser oxyrinchus) semen

The Atlantic sturgeon (Acipenser oxyrinchus) faces unique conservation challenges due to its extended maturation period and irregular spawning patterns, which restrict its reproductive capacity. Therefore, in order to overcome these difficulties we have made a first attempt to develop effective cryopreservation procedure for Atlantic sturgeon through evaluation of the effects of the glucose concentration in extender and final sperm concentration in straw on sperm motility parameters. A remarkably high (81 ± 8%) postthaw sperm motility was obtained. The other sperm velocity and trajectory parameters were differentially affected both by concentrations of glucose or sperm and cryopreservation process. Our results undoubtedly show that the post-thaw sperm motility of Atlantic sturgeon is highly influenced by the final glucose and sperm concentration. The recommended cryopreservation procedure includes 0.05 M glucose and 7.5% methanol at 1.0×109 spermatozoa ml−1. We have confirmed that our elaborated protocol can be used for cryopreservation of Atlantic sturgeon semen which can be subsequently used for long-term conservation of this endangered species, offering a valuable tool in efforts to safeguard its genetic diversity and contribute to its survival.

Cryopreservation of human semen by inherently-controlled icing probability: Or how the surface profile of superhydrophobic carbon soot coatings and the sperm volume affect the outcome of slow freezing?

The restoration of initial functionality of human spermatozoa subjected to cryopreservation is challenging, because the deleterious intracellular icing and the occurrence of osmotic shocks due to prolonged exposure to increased concentrations of intracellular solutes are oppositely dependent on the cooling rate. This longstanding problem could be overcome if using superhydrophobic soot coatings delaying the heat transfer rate, reducing the ice formation probability and triggering balanced and timely dehydration of the cells, but the effect of their surface profile and sperm volume on the success rate of slow freezing is unclear. Here, we show for the first time that the two-factor freezing injury is entirely avoidable by tailoring the solid-to-gas voids (pores) fraction in the soot, leading to increased nucleation free energy barrier, presumable incipiency of ice crystals with controllable shape and size and hence, fully (100 %) recovered post-thaw sperm motility. It is demonstrated that the reason for such a unique scientific result is the selection of soot coatings with appropriate morphochemical features, hypothetically (not directly proven yet) inducing equilibrium among the solution composition and ice crystals formation, retarding the undesirable compression of liquid-filled “slush ice” channels surrounding the cytoplasm and impeding the ice recrystallization. The novel insights introduced in this article open endless horizon for customizing and revolutionizing the technical protocols in cryobiology.

Camellia oil with its rich in fatty acids enhances post-thawed boar sperm quality

BackgroundBoar sperm are highly susceptible to specific conditions during cryopreservation, leading to a significant decrease in their fertilizing potential due to damage to their membranes. Camellia oil, known for its fatty acids with antioxidant and biological properties, has not been previously explored for the cryopreservation of boar semen. This study aimed to examine the effects of camellia oil on post-thawed boar sperm quality. Boar semen ejaculates (n = 9) were collected and divided into six equal aliquots based on camellia oil concentrations (0, 0.5, 1, 1.5, 2 and 2.5% v/v) in the freezing extender. Semen samples were processed and cryopreserved using the liquid nitrogen vapor method. Thereafter, frozen semen samples were thawed at 50 °C for 12 s and evaluated for sperm morphology by scanning electron microscope, sperm motility using a computer-assisted sperm analyzer, sperm viability, acrosome integrity, mitochondrial function, MDA level and total antioxidant capacity.ResultsThe results demonstrated that the supplementation of 1.5% (v/v) camellia oil showed superior post-thaw sperm qualities such as improved sperm morphology, motility, acrosome integrity and mitochondrial function by 14.3%, 14.3% and 11.7%, respectively, when compared to the control group. Camellia oil at a concentration of 1.5% (v/v) showed the lowest level of MDA (18.3 ± 2.1 µmol/L) compared to the other groups.ConclusionsIn conclusion, adding 1.5% (v/v) camellia oil in the freezing extender reduced the oxidative damage associated with cryopreservation and resulted in a higher post-thawed sperm quality.

Factors affecting the analysis and interpretation of sperm quality in frozen/thawed stallion semen

In the current study, we examined: 1) the agreement (bias) between fluorescence-based methods (NucleoCounter-SP100 [NC] vs. flow cytometry [FC]) for determining the viability (VIAB) of frozen/thawed stallion sperm; 2) the agreement between post-thaw sperm total motility (TMOT) and VIAB; 3) whether a difference between TMOT and VIAB [VIAB – TMOT] in frozen/thawed stallion sperm could be explained by the level of lipid peroxidation in viable sperm (VLPP); 4) the repeatability of post-thaw analysis of sperm quality; and 5) the effect of final post-thaw semen dilution (10, 30, or 50 x 106 sperm/mL) on sperm motion characteristics. Post-thaw VIAB was similar between NC and FC (P > 0.05), and the agreement between these two methods was high (bias: 1 to −3). The agreement between post-thaw TMOT and VIAB decreased as the pre-freeze percentages of morphologically normal sperm and DNA quality decreased: bias – 4 to – 25. The bias between [VIAB – TMOT] and VLPP ranged from – 5 to 7. Differences in post-thaw sperm quality (TMOT, PMOT, VIAB, and sperm concentration) were not observed when analyzing one or three straws per ejaculate (P > 0.05). There was no effect of post-thaw sperm concentration (i.e., 10 vs. 30 vs. 50 x 106 sperm/mL) on sperm motion characteristics (P > 0.05). This study reports factors other than post-thaw sperm motility that warrant further consideration when analyzing frozen/thawed stallion sperm.

Optimizing aquaculture-scale common carp artificial reproduction: a novel approach to sperm cryopreservation using large-volume containers and elevated thawing temperatures

IntroductionThe successful cryopreservation of common carp sperm is crucial for its application in aquaculture and selective breeding programs. This study investigates the efficacy of cryopreserving sperm in large containers (5 mL) with a low dilution rate (1:1) in three different cryoprotective media and thawing in different conditions.MethodsThe developed method utilizes a low-ionic (hypotonic) cryoprotective medium, freezing with a controlled cooling rate, and high-temperature sperm thawing (60°C). The investigation employs a detailed spermatozoon motility assessment.ResultsPost-thaw motility of 32.3% ± 14% and initial curvilinear velocity of 89 ± 20 μm/s across 30 males were observed. Principal component analysis of sperm kinematic characteristics revealed distinct populations of sperm cells exhibiting varying responses to cryopreservation. The developed method achieved successful fertilization comparable to that of the non-frozen control group using sperm from a single cryotube (2.5 mL, approximately 50 * 109 spermatozoa) to fertilize 200 g of eggs (1:120,000 egg:spz).DiscussionThis novel approach demonstrates an effective cryopreservation protocol for common carp sperm in large-volume cryo-containers in combination with low-ionic cryomedia and high thawing temperature, providing methods well-suited for fisheries practices and selective breeding programs. Future studies of the biological properties of different sperm subpopulations in post-thaw sperm samples can contribute to a deeper understanding of sperm biology, improve cryopreservation techniques, and enhance the success rates of assisted reproductive technologies.

Association of the polymorphism of the Pon-1 locus with the concentration of Paraoxonase-1 and post-thaw sperm viability in pig

The objective of the present work was to correlate the expression of the PON-1 locus polymorphism, the concentration of the enzyme paraoxonase 1 in seminal plasma and the effect on post-thawing sperm viability in the pig. Nine ejaculates from 3 boars were used, to obtain seminal plasma; part of the ejaculates was centrifuged at 800 g/10 min; the material obtained was stored at -20°C until use. To freeze the semen, the samples were diluted 1:1 in commercial diluent (Magapor®) at 37°C and the temperature was gradually lowered to 16°C. They were transported to the laboratory protected from light, and kept at the same temperature in the laboratory for 24 h. After this, they were frozen using the glycerolation technique and a portion was cryopreserved by adding 20% of seminal plasma to the conservation medium and another portion was made without seminal plasma. The concentration of PON-1 was determined by an enzyme-linked immunosorbent assay (ELISA). The functional status of the plasma membrane was evaluated by chlortetracycline staining. The amplification of a fragment of the PON-1 gene was carried out by PCR and the identification of allelic variability by the analysis of Restriction Fragment Length Polymorphism (RFLP), with the enzymes HinfI and FokI, from a DNA sample of each individual. The results were analyzed by analysis of variance (ANOVA) and Spearman correlation, with the use of statistical software (Statistica V10 for Windows). In electrophoresis of enzymatic digestion with the enzyme HinfI All the expected fragments were obtained, with the FokI enzyme a mutation was observed in one individual. No correlation was observed (R=-0.1, P&gt;0.05) between the concentration of the paraoxonase-1 enzyme in seminal plasma and the observed mutations. As well as the concentration of the enzyme paraoxonase-1 in the ejaculates was 1.8 to 2.7 ng/mL and this had no statistically significant difference in the seminal plasma of the ejaculates analyzed (P&gt;0.05). As well as with sperm viability in thawed semen (P&gt;0.05).

Effects of different ages on frozen semen quality and in vitro fertilization efficiency in Wannan black pigs.

According to previous studies, the quality and fertilization rate of fresh sperm from boars of different ages were significantly different. However, the difference of freeze-thaw sperm quality and fertility in boars of different ages is unclear. In this study, boars of a Chinese native breed were assigned into two groups. Each group consisted of five boars aged aged either 2-3 years (young boars = YB) or 5-6 years (aging boars = AB) A total of 60 ejaculates for each group were collected and cryopreserved. Semen quality and in vitro fertility of post-thaw sperm was evaluated. The results showed that the concentration and motility of fresh sperm collected from AB were similar to YB, but their semen volume was higher than that in YB (p < 0.05). Frozen-thawed sperm of AB had lower viability than YB, and higher abnormal rate and reactive oxygen species (ROS) levels of YB (p < 0.05). There was no effect of the age on post-thaw sperm motility and time survival. Functional assessments indicated that increasing age markedly compromises the integrity of the sperm plasma membrane and acrosome, as well as mitochondrial functionality post-thaw, albeit without affecting DNA integrity. Furthermore, increasing age of boars reduces the ability of sperm to bind to the oocyte zona pellucida after thawing, delaying the time of the first embryo cleavage after fertilization. Finally, the early developmental efficiency of in vitro fertilized embryos progressing from 4-cell to blastocyst derived from post-thaw sperm in AB significantly decreased compared to those from YB (p < 0.05). Taken together, these results suggest that increasing age in boars impairs the quality and in vitro fertility of frozen thawed sperm.

Quality and kinematic characteristics of Bali bulls frozen semen with purified green tea extract epigallocatechin-3-gallate antioxidant addition in diluent.

The preservation of semen quality and kinematic characteristics during cryopreservation is crucial for the reproductive success and genetic management of livestock, particularly in Bali bulls. This study aimed to investigate the effect of adding purified green tea extract antioxidant Epigallocatechin-3-gallate (EGCG) in tris egg yolk diluent on the quality and kinematic characteristics of frozen semen from Bali bulls. Fresh and frozen semen samples were obtained from Bali bull and divided into four different treatment groups. P0 contained semen samples + diluent, while P1 to P3 consisted of semen samples + diluent supplemented with EGCG levels of 0.1, 0.15, and 0.2 mg/100 ml, respectively. Data were analyzed using One-way ANOVA and followed by Duncan's test if significant differences were found (p<0.05). Parameters observed included the assessment of fresh semen quality, kinematic analysis, post-thawing sperm viability, and abnormality. The results indicated that the assessment of fresh semen quality showed macroscopic and microscopic semen quality according to SNI 4869-1:2021. Kinematic analysis revealed significant differences in DSL and STR parameters between P0 and P3 (p<0.05). EGCG supplementation also caused significant differences in motility between P0 and P3 (p<0.05). Viability and spermatozoa abnormality with EGCG supplementation did not show significant differences (p>0.05). The best results for motility, kinematics, and sperm morphology variables were found in P1 as it did not exhibit a decrease in motility, kinematics, and sperm morphology. Viability did not show significant differences between P1, P2, and P3, but the best results were found in P2 as it did not exhibit a decrease in viability with mean and standard deviation (66.84 ± 7.88). Abnormality variables also did not show significant differences between P1, P2, and P3, but the best results were found in P2 as it did not exhibit a decrease in abnormality with mean and standard deviation (23.80 ± 7.36).

Manila duck (Cairina moschata) frozen semen quality in lactated ringer's egg yolk-astaxanthin with different concentrations of DMSO.

This study was conducted to evaluate manila duck's (Cairina moschata) frozen semen quality after cryopreservation in lactated ringer's egg yolk-astaxanthin (LREY-A) with 5 different concentrations of dimethyl sulfoxide (DMSO). Methodology: Semen was collected from 3 manila ducks (Cairina moschata) using the cloaca massage technique twice a week. Fresh semen was evaluated macro and microscopically then polled and divided into 5 tubes of treatments. Each tube was diluted in DMSO4, DMSO6, DMSO8, DMSO10, and DMSO12. The semen of each treatment was loaded into a 0.25 mL straw and equilibrated at 5 °C for 2 h. Freeze above nitrogen vapor and stored a container of liquid nitrogen at -196 °C, then semen thawed in a water bath at 37 °C for 30 sec. Data were analyzed using One-Way ANOVA Analysis. Results of this showed that post-equilibration sperm motility and sperm viability have differed significantly (P<0.05) for each treatment, with the highest % sperm motility DMSO8 and DMSO6, this is also shown in post-thawing sperm motility and viability which have differed significantly (P<0.05) and the highest % sperm viability were DMSO8 and DMSO6. In conclusion, Frozen semen extender formulation of DMSO8 and DMSO6 which are used in manila duck semen cryopreservation was the best to other treatments to maintain % sperm motility and % sperm viability in post-equilibration and post-thawing. The highest sperm motility recovery rate was in DMSO8. The lowest sperm live and dead abnormality was in DMSO8. It is concluded that the combination of DMSO8 was the best in maintaining the quality of manila duck frozen semen.