Cyclodextrin-Cholesterol Alone or in Combination With Cyclodextrin-Vitamin E Improves Bull Sperm Cryopreservation in a Soybean Lecithin Extender.

Combining cholesterol-loaded methyl-ß-cyclodextrin (CD-CHL) with vitamin E-loaded methyl-ß-cyclodextrin (CD-Vit E) to combat cold shock and oxidative stress during sperm cryopreservation in soybean lecithin extenders remains unexplored. Thus, the current study aimed to investigate the effect of treating bull sperm with CD-CHL and CD-Vit E prior to cryopreservation in a soybean lecithin extender. Sperm collected from eight fertile bulls were pooled and split into six aliquots. Five aliquots were treated, in a Tris-based extender, with CD-CHL (2 mg/120 × 106 cells/mL) and either 0, 0.5, 1.0, 1.5 or 2 mg CD-Vit E/120 × 106 cells/mL. The control aliquot was diluted in a Tris-based extender without further supplementation. After incubation at 22°C for 15 min and addition of a soybean lecithin extender, all aliquots were equilibrated for 2 h at 4°C and then cryopreserved in liquid nitrogen. Computer-assisted sperm analysis (CASA) was used to explore the different sperm motility parameters, hypo-osmotic swelling test to determine membrane functionality and fluorescein isothiocyanate-conjugated Aeachis hypogaea (peanut) agglutinin (FITC-PNA) to quantify acrosome integrity. The effect of oxidative stress on the sperm membrane was assessed through lipid peroxidation measurement. Compared to control, CD-CHL alone improved significantly (p < 0.05) all CASA motility parameters, membrane functionality and acrosome integrity of thawed sperm. The membrane functionality was more significantly (p < 0.05) improved when 0.5 mg CD-Vit E was combined with CD-CHL. Concerning lipid peroxidation, no significant differences (p > 0.05) in malondialdehyde (MDA) levels were registered between groups. In conclusion, the combination of CD-CHL and CD-Vit E demonstrated a significant positive effect on the cryopreservation of bull sperm in a soybean lecithin extender.

Enhancing canine semen quality through a second centrifugation after 48 hours of storage: a comparative study.

BACKGROUND: Centrifugation is a common procedure to improve the quality of chilled and frozen canine semen by removing debris and seminal plasma and adding semen extenders. The aim of this study was to evaluate the efficacy and influence of a second centrifugation after 48 h of storage at 5 °C on the sperm quality of canine semen. The ejaculates of 45 healthy male dogs, divided into three groups according to body weight, were analyzed for macro- and microparameters such as ejaculate volume, sperm concentration, kinematic parameters, morphology, and integrity of plasma membrane. Samples were analyzed at baseline conditions (T0), after 24 h (T24) and after 48 h (T48) to assess the effects of the different treatments on sperm quality. RESULTS: The results showed a significant effect of a second centrifugation on the improvement of chilled sperm quality compared to the other techniques, especially up to 48 h. CONCLUSIONS: Analysis of the data showed that the semen samples centrifuged and then cooled at 5 °C had acceptable semen parameters, especially in terms of motility, with a gradual decrease in serial evaluations after 24 and 48 h. A second centrifugation after 48 h of storage may lead to better semen quality and improve the kinetics of sperm parameters, the percentage of morphologically normal sperm and the percentage of sperm with intact membranes.

Pre- and Post-Thaw Addition of L-Carnitine and Pyruvate: Effect on Stallion Sperm Parameters.

The addition of antioxidants to cryopreservation media reportedly improves sperm post-thaw quality and reproductive performance after artificial insemination. Therefore, the objectives of this study were to evaluate if the addition of L-carnitine and pyruvate to freezing media, or their addition to samples after thawing, improves the post-thaw quality of equine spermatozoa. Thus, in Experiment 1, stallion semen samples were cryopreserved in: (1) EDTA-glucose-based extender with 20% egg yolk and 5% dimethylformamide (EDTA control); (2) skim milk-based extender with 20% egg yolk and 5% dimethylformamide (milk control); (3) Extender 1 supplemented with 50 mM L-carnitine and 10 mM pyruvate (EDTA-carnitine-pyruvate); and (4) Extender 2 supplemented with 50 mM L-carnitine and 10 mM pyruvate (milk-carnitine-pyruvate). In Experiment 2, 50 mM L-carnitine and 10 mM pyruvate were added post-thaw to samples cryopreserved with extenders 1 and 2 (EDTA control and milk control). Sperm kinematic parameters, DNA fragmentation, membrane lipid peroxidation, acrosome status and viability were evaluated after thawing. No significant differences (p > 0.05) were observed for most of the kinematic parameters, DNA fragmentation, membrane lipid peroxidation, acrosome status and viability of spermatozoa, between the samples frozen in the presence or absence of L-carnitine and pyruvate, nor between the samples after the post-thaw addition of these components. A higher (p < 0.05) mean velocity and higher (p < 0.05) amplitude of lateral head displacement were observed in the samples frozen in the milk-based extender with the addition of L-carnitine and pyruvate after thawing. The addition of 50 mM L-carnitine and 10 mM pyruvate, either to the freezing extenders or after thawing, was not deleterious for sperm; however, it did not improve equine sperm motility, viability, acrosome and DNA integrity, nor decrease membrane lipid peroxidation after thawing.

Trends and challenges in liquid-preserved boar semen production: From boar to product.

Boar semen production plays a pivotal role in modern swine breeding programmes, influencing the genetic progress and overall efficiency of the pork industry. This review explores the current challenges and emerging trends in liquid-preserved boar semen production, addressing key issues that impact the quality and quantity of boar semen. Advances in new reproductive technologies, boar selection, housing, semen processing, storage and transport, and the need for sustainable practices including the use of artificial intelligence are discussed to provide a comprehensive overview of the field.

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

Background: 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. Aim: 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. Methods: 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. Results: 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). Conclusion: 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).

Exploratory Metabolomics and Lipidomics Profiling Contributes to Understanding How Curcumin Improves Quality of Goat Semen Stored at 16 °C in Tropical Areas.

Reactive oxygen species (ROS) exert a vital role in sperm quality during semen preservation, where excessive ROS leads to oxidative damage and undermines sperm integrity. Curcumin, a botanical extract, is capable of neutralizing ROS and enhancing the activity of antioxidant enzymes. This study was aimed at evaluating the effects of curcumin on sperm viability, acrosome integrity, and antioxidant levels, as well as metabolomic and lipidomic profiles. The results demonstrated that curcumin at 25 µmol/L significantly enhanced sperm motility, plasma membrane, and acrosome integrity, elevated the levels of antioxidant enzymes (T-AOC, CAT, SOD), and decreased ROS production (p < 0.05). Metabolomic analysis identified 93 distinct metabolites that showed significant differences between the control and curcumin-treated groups. KEGG pathways emphasized the participation of these metabolites in key metabolic processes such as the citric acid cycle, cholesterol metabolism, and fatty acid metabolism. Curcumin treatment brought about notable variations in lipid profiles, including increased levels of phosphatidylcholine, acylcarnitine, and triglyceride over the storage time, suggesting enhanced lipid anabolic activity. Overall, the supplementation of curcumin at 25 µmol/L effectively mitigates oxidative stress and prolongs the viability of semen storage at 16 °C by modulating specific metabolic and lipid profiles.

Improvement of nonsurgical artificial insemination with cryopreserved spermatozoa in Alexandromys montebelli, Japanese grass vole.

Microtus genus is the herbivorous animal with multiple stomachs, and some of them possess a mating system similar to human and thereby has been expected as a model animal for the large herbivory and human mating system model, respectively. Thus, it is significant to maintain Microtus as an animal genetic resource. We have studied the establishment of assisted reproductive technologies in Alexandromys. montebelli (formerly as Microtus motebelli: A. motebelli), and here, we investigated the effects of hypotaurine treatment to frozen-thawed (FT) spermatozoa and modified timing of nonsurgical artificial insemination (AI) on the number of offspring. As the results, regardless of without or with hypotaurine treatment, when the timing of nonsurgical AI was made closer to the estimated ovulation time (at 7-9 h post coitus), the total number of offspring derived from FT spermatozoa (27 and 28 pups, respectively) increased compared with AI at 4-6 h (five and six pups, respectively) and was equivalent to those of fresh spermatozoa (43 pups) or natural mating (33 pups). These results will lead to further dissemination of nonsurgical AI and could support the "3R principle," which is the standard philosophy of animal experiment because the procedure declines the stress and the recipient can be used repeatedly.

Treatment of cryopreserved bovine sperm with calcium ionophore A23187 increases in vitro embryo production.

After ejaculation, mammalian sperm undergo a series of molecular events conducive to the acquisition of fertilizing competence. These events are collectively known as capacitation and involve acrosomal responsiveness and a vigorous sperm motility called hyperactivation. When mimicked in the laboratory, capacitating bovine sperm medium contains bicarbonate, calcium, albumin and heparin, among other components. In this study, we aimed at establishing a new capacitation protocol for bovine sperm, using calcium ionophore. Similar to our findings using mouse sperm, bovine sperm treated with Ca2+ ionophore A23187 were quickly immobilized. However, these sperm initiated capacitation after ionophore removal in fresh medium without heparin, and independent of the Protein Kinase A. When A23187-treated sperm were used on in vitro fertilization (IVF) procedures without heparin, eggs showed cleavage rates similar to standardized IVF protocols using heparin containg synthetic oviduct fluid (IVF-SOF). However, when A23187 pre-treated sperm were further used for inseminating eggs in complete IVF-SOF-heparin, a significantly higher percentage of embryo development was observed, suggesting a synergism between two different signaling pathways during bovine sperm capacitation. These results have the potential to improve current protocols for bovine IVF that could also be applied in other species of commercial interest.

Seasonal seminal quality variations and vitrification prospects in black flounder Paralichthys orbignyanus.

Long-term preservation of gametes has been identified as a tool to improve broodstock management and increase the number of juveniles produced by artificial fertilization. Paralichthys orbignyanus is an important commercial and recreational species distributed in marine and estuarine waters from Rio de Janeiro (Brazil) to the San Matías Gulf (Argentina). This work focused on studying the seminal quality of tank-reared P. orbignyanus, demonstrating that males are fluent year-round, with the highest yields at the early reproductive season. Fresh sperm exhibited good forward swimming, and samples could be refrigerated up to 48 h while retaining their motility after activation. The optimal conditions for P. orbignyanus sperm motility activation were established as 950 mOsmol/Kg and pH values between 7 and 7.9. Additionally, a well-defined protocol for semen vitrification was developed to assess the cryotolerance of this species' sperm. We successfully produced high-quality sperm samples, using two vitrification formulations containing trehalose and both z-1000 and x-1000 polymers, that can be used in a near-future in vitro embryo production program.

Effect of seminal plasma cholesterol and triacylglycerides concentrations and sperm morphology on semen freezability in domestic cats (Felis silvestris catus).

There is scarce information about the effect of sperm morphology and seminal plasma composition on cat semen freezability. Thus, this study aims to assess the effect of cat sperm morphology and seminal plasma cholesterol (CHOL) and triacylglyceride (TAG) concentrations on sperm post-thaw survival. Ejaculates (n = 49) were evaluated, and seminal plasma was separated and frozen until CHOL and TAG concentrations were measured. The sperm pellet was diluted in a tris-based egg yolk extender, frozen (n = 38), or processed for sperm ultrastructure study (n = 11). Abnormalities recorded were abnormal head shape and size, detached heads, knobbed or ruffled acrosomes, eccentric mid-piece insertion, proximal and distal cytoplasmic droplets, folded and coiled tails, and Dag defect. Ultramicroscopic evaluation detected several sperm abnormalities in fresh semen and some sperm damage in frozen semen. Seminal plasma lipids components were positively correlated with post-thaw motility and acrosome integrity. Higher freezability indices for motility and acrosome integrity were observed in frozen-thawed semen with high seminal plasma CHOL and TAG concentrations. No freezability differences were observed between teratozoospermic and normozoospermic ejaculates. Our results showed that even when seminal plasma was removed before cryopreservation, sperm survival after thawing was significantly higher in samples with high seminal plasma CHOL and TAG concentrations, indicating a rapid adherence to these compounds to the sperm plasma membrane, protecting sperm cells from temperature changes. Nevertheless, there were no differences in sperm freezability by sperm morphology.

Incubating frozen-thawed buffalo sperm with olive fruit extracts counteracts thawing-induced oxidative stress and improves semen quality.

Freezing-thawing procedures and semen manipulation for in vitro fertilization induce oxidative stress, which in turn leads to impaired sperm quality. The aim of this study was to evaluate whether incubation of frozen-thawed buffalo semen with olive fruit extracts (OFE), known to contain a high concentration of phenolic antioxidants, would improve semen quality by reducing oxidative stress. Frozen sperm (4 ejaculates/4 bulls/3 replicates) were thawed and diluted to 30 × 106/mL in IVF medium with 0, 72, 143, and 214 µL/mL of OFE, corresponding to 0 (D0-control), 50 (D50), 100 (D100), and 150 (D150) µM hydroxytyrosol. Sperm viability, acrosome integrity, membrane functionality, motility, and sperm kinetics were evaluated immediately after thawing (T0) and after 1 (T1) and 2 h (T2) of incubation at 38.7 °C. Based on the results, sperm biological antioxidant potential (BAP) and ROS levels (ROMs) were assessed in D0 and D100 groups at T1 and T2. To assess the effect of OFE on fertilizing ability, heterologous penetration rates were also evaluated, using bovine abattoir-derived oocytes. The treatment with OFE at all concentrations tested increased (P < 0.05) the percentage of acrosome intact spermatozoa compared to the D0-control at T1, but the effect was more evident (P < 0.01) with D100 (54.5 ± 3.0, 60.5 ± 1.5, 65.2 ± 3.3, and 62.5 ± 1.7, with D0, D50, D100, and D150 OFE, respectively). Total motility, progressive motility, rapid velocity, and progressive velocity decreased (P < 0.05) at T2 only in the D0-control group. The percentage of rapidly progressive sperm and the progressive motility tended to increase (P < 0.10) at T1 and T2, respectively, in D100 compared to D0 (24.7 ± 4.1 vs 16.4 ± 1.6 and 22.8 ± 2.7 vs 17.0 ± 1.2, respectively). The treatment with D100 OFE of frozen-thawed sperm increased (P < 0.05) some kinetic parameters (VAP and WOB). Spermatozoa incubated with D100 OFE exhibited higher (P < 0.01) total and normospermic oocyte penetration rates compared to D0 (86.5 ± 1.4 vs 78.5 ± 0.7, and 70.6 ± 1.5 vs 63.8 ± 1.1, respectively). Additionally, D100 OFE increased sperm BAP concentrations at both T1 and T2, while ROS levels were unaffected. These results suggest that incubating frozen-thawed buffalo semen with OFE is an effective strategy for preserving semen quality and in vitro fertilization ability by enhancing sperm antioxidant capacity.

In vitro, the aging of stallion spermatozoa at 22 °C is linked to alteration in Ca2+ and redox homeostasis and may be slowed by regulating metabolism.

BACKGROUND: Conservation of equine semen in the liquid state is a central procedure in horse breeding and constitutes the basis of associated reproductive technologies. The intense mitochondrial activity of the stallion spermatozoa increases oxidative stress along the storage period, leading to sperm demise within 24-48 h of storage, particularly when maintained at room temperature. Recently, the relationship between metabolism and oxidative stress has been revealed. The study aimed to extend the period of conservation of equine semen, at room temperature through modification of the metabolites present in the media. MATERIAL AND METHODS: Processed ejaculates (n = 9) by single-layer colloid centrifugation were split in different aliquots and extended in Tyrode's basal media, or modified Tyrode's consisting of 1 mM glucose, 1 mM glucose 10 mM pyruvate, 40 mM glucose, 40 mM Glucose 10 mM pyruvate, 67 mM glucose and 67 mM glucose 10 mM pyruvate. At time 0h, and after 24 and 96 h of storage, motility was evaluated by CASA, while mitochondrial production of Reactive oxygen species (ROS), and intracellular Ca2+ concentrations were determined via flow cytometry using Mitosox Red and Fluo-4 respectively. ROS and Ca2+ were estimated as Relative Fluorescence Units (RFU) in compensated, arcsin-transformed data in the live sperm population. RESULTS: After 48 h of incubation, motility was greater in all the 10 mM pyruvate-based media, with the poorest result in the 40 mM glucose (41 ± 1.1 %) while the highest motility was yielded in the 40 mM glucose 10 mM pyruvate aliquot (60.3 ± 3.5 %; P < 0.001); after 96 h of storage highest motility values were observed in the 40 mM glucose 10 mM pyruvate media (23.0 ± 6.2 %) while the lowest was observed in the 1 mM glucose media was 9.2 ± 2.0 % (P < 0.05). Mitochondrial ROS was lower in the 40 mM glucose 10 mM pyruvate group compared to the 40 mM glucose (P < 0.01). Over time Ca2+ increased in all treatment groups compared to time 0h. DISCUSSION AND CONCLUSION: Viable spermatozoa may experience oxidative stress and alterations in Ca2+ homeostasis during prolonged storage, however, these effects can be reduced by regulating metabolism. The 40 mM glucose- 10 mM pyruvate group yielded the highest sperm quality parameters.

Thawing of cryopreserved sperm from domestic animals: Impact of temperature, time, and addition of molecules to thawing/insemination medium.

In recent decades, there has been a growing interest in optimizing the protocols intended to sperm cryopreservation in domestic animals. These protocols include initial cooling, freezing, and thawing. While different attempts have been devised to improve sperm cryopreservation, the efficiency of this reproductive biotechnology is still far from being optimal. Furthermore, while much attention in improving cooling/freezing, less emphasis has been made in how thawing can be ameliorated. Despite this, the conditions through which, upon thawing, sperm return to physiological temperatures are much relevant, given that these cells must travel throughout the female genital tract until they reach the utero-tubal junction. Moreover, the composition of the media used for artificial insemination (AI) may also affect sperm survival, which is again something that one should bear because of the long journey that sperm must make. Furthermore, sperm quality and functionality decrease dramatically during post-thawing incubation time. Added to that, the deposition of the thawed sperm suspension devoid of seminal plasma in some species during an AI is accompanied by a leukocyte migration to the uterine lumen and with it the activation of immune mechanisms. Because few reviews have focused on the evidence gathered after sperm thawing, the present one aims to compile and discuss the available information concerning ruminants, pigs and horses.

The effect of supplementing freezing extender with Mn2+-, Zn2+- or Cu2+-nanosuccinate on select post-thaw characteristics of ram semen.

The effects of Mn2+-, Zn2+- or Cu2+-nanosuccinate added to freezing extender on select post-thaw semen characteristics were determined in six Texel rams (aged 2-4 years) during seasonal anestrus (April-May). Ejaculates (n = 6 per ram) collected into an artificial vagina were divided into ten isovolumetric fractions each. Semen was diluted in lactose-yolk-tris-citrate-glycerin medium and nanosuccinates (Mn2+- and Zn2+-nanosuccinate: 0.0 (control), 2.5, 5.0 and 7.5 µg/l; Cu2+-nanosuccinate: 0.0 (control), 1.25, 2.5 and 3.75 µg/l) were added to semen extender. Extended semen was loaded into 0.25-ml straws and frozen in liquid nitrogen. After thawing, sperm motility parameters were determined with computer assisted semen analysis (CASA), and the activity of superoxide dismutase (SOD), glutathione peroxidase (GPx) and catalase (CAT) was measured with a spectrophotometric technique. The addition of 5.0 µg/l of Mn2+- and Zn2+-nanosuccinate significantly increased the sperm progressive motility and both 2.5 and 5.0 µg/l improved sperm motion kinetics. Further, both nanosuccinates at a dose of 5.0 µg/l significantly decreased SOD activity and stimulated an increase in GPx and CAT activity in semen samples. Alternatively, the addition of Cu2+-nanosuccinate (highest dose) significantly reduced the progressive motility and velocity of ram spermatozoa, increased the percentage of sperm with acrosomal/head defects and seminal SOD activity, and depressed CAT (highest dose) and GPx (all doses) activity. In summary, the addition of Mn2+- and Zn2+-nanosuccinate to semen extender had beneficial effects on sperm motility/motion kinetics and structural integrity, whereas Cu2+-nanosuccinate generally had debilitating effects on the post-thaw semen characteristics in rams.

Effect of different concentrations of inulin on ram sperm quality during cryopreservation.

BACKGROUND: In reproductive biotechnology, sperm cryopreservation has a vital role to play. Cryopreservation of sperm produces reactive oxygen species (ROS), which disrupt sperm function and structural competence. Numerous protective chemicals, including fructans, have been used during sperm cryopreservation. OBJECTIVES: To evaluate the effect of different concentrations of the fructosan inulin on ram sperm quality parameters, superoxide dismutase (SOD) activity, and malondialdehyde (MDA) production after freezing and thawing. MATERIALS AND METHODS: The pooled samples from four healthy rams were divided into seven equal aliquots and diluted in a Tris-base extender supplemented with 1, 2, 4, 8, 16, and 28 mM of inulin or without inulin supplementation (control). By using liquid nitrogen vapor, the semen was frozen and stored at 196 degree C. RESULTS: The total motility, viability, and DNA integrity were significantly improved after freeze-thawing with 28 mM inulin, compared to other treatment groups (P < 0.05). A Tris-based extender containing 16 and 28 mM of inulin displayed the highest levels of ram sperm membrane integrity when compared with the control (p <0.05). The abnormality of ram sperm was increased during freeze-thawing at control and 1 mM of inulin, compared to 16 and 28 mM of inulin (P < 0.05). Additionally, 28 mM of inulin decreased MDA and increased SOD activity in ram sperm in comparison with the other treatments (P < 0.05). CONCLUSION: As a result, 28 mM of inulin could be beneficial for the cryopreservation industry and reduce the harmful effects of freeze-thawing on ram sperm. Doi.org/10.54680/fr24510110512.

In vitro dissolution of encapsulated or dispersed Vitamin e and cholesterol is correlated with preservation of refrigerated ovine sperm.

BACKGROUND: Vitamin E ( -tocopherol) and cholesterol are crucial components in cellular protection and physiological processes. Their uses in biological media face challenges due to their poor solubility and stability. OBJECTIVE: The study investigated the complex interactions of these bioactive compounds in various encapsulation systems of cyclodextrin and liposome, as well as dispersion in PEG-6000, in an attempt to improve the viability, motility, and preservation of ovine sperm cells. MATERIALS AND METHODS: The work explored the in vitro dissolution kinetics of vitamin E (d-tocopherol) and cholesterol using semi-empirical models. RESULTS: The release profiles of VitE and Chl varied considerably, depending on the specific carrier systems. For liposome-loaded VitE and Chl, the Korsmeyer-Peppas model gave the best fit; for CD/VitE and CD/Chl, the Higuchi model provided the best fit, whereas for PEG-6000 dispersions (VitE and Chl) both the Higuchi and Korsmeyer-Peppas models demonstrated the excellent fit. All systems indicated a Fickian diffusion mechanism dictated by the concentration gradient. The delivery of VitE and Chl with CD, liposome and PEG dispersion significantly increased sperm mobility and motility. The effect on the VCL parameter was the greatest by liposome-loaded VitE and Chl, followed by CD encapsulation and PEG-6000 dispersion. CONCLUSION: The dynamics of vitamin E and cholesterol within innovative delivery systems offers valuable insights into the development of advanced solutions in reproductive health, particularly on improving the viability, motility of refrigerated ovine sperm cells. Doi.org/10.54680/fr24510110712.

Immunofluorescence and biochemical investigation of the protective effects of naringin and diosmin on the freezability of merino ram semen.

BACKGROUND: Various antioxidant substances are added to sperm extenders to protect spermatozoa against oxidative stress and cryodamage. OBJECTIVE: To investigate the effects of the flavonoid diosmin (DIO) and a flavanone glycoside naringin (NAR) on the freezability of ram semen. MATERIALS AND METHODS: In this study, six Merino rams were used during the breeding season. The ejaculates were pooled after collection from the rams. Pooled ejaculates were divided into six groups: control, NAR 1 mM, NAR 2 mM, NAR 4 mM, DIO 2 mM, and DIO 4 mM, and then diluted with a TRIS-based diluent. The pooled semen was equilibrated, placed in 0.25 mL pipettes with 10 × 10 7 sperm cells in each pipette, and frozen in liquid nitrogen vapor. After 24 h, the pipettes were thawed at 37 degree C for 25 s and analyzed in terms of spermatological parameters. RESULTS: The highest plasma membrane integrity ratio was found in the DIO 4 mM group, whereas a statistically significant difference was found between the NAR 1 mM and NAR 2 mM groups (p < 0.05). While the DIO 4 mM group had the highest acrosome integrity rate, a statistically significant difference was found between the other groups (p < 0.05). Mitochondrial activity was the highest in the NAR 4 mM, DIO 4 mM and DIO 2 mM groups (p < 0.05). In the analysis of the sperm membrane lipid profile, it was observed that the DIO group had the highest lipid-phospholipid ratio. In sperm membrane protein profile analysis, it was found that both additives exerted protective effects at different levels. The highest total protein content was seen in the DIO 4 mM and NAR 4 mM groups. 8-hydroxydeoxyguanosine (8-OhDG) positivity was more common in the control group than in the DIO and NAR groups. Cu-Zn superoxide dismutase (SOD) expression was lower in the control group and more intense in all other groups. Positive results were especially observed in the acrosome of the sperm cells. CONCLUSION: The addition of NAR and DIO to the ram semen extender increased the quality of sperm parameters after the freeze-thaw process. Doi.org/10.54680/fr24510110412.

Effect of cryoprotectant and concentration on the sperm quality of walking catfish, Clarias batrachus, post-cryopreservation.

BACKGROUND: Walking catfish, Clarias batrachus is one of the native and most popular freshwater catfish species in Indonesia. However, cultivation faces challenges, particularly due to the scarcity of larvae resulting from underdeveloped breeding technologies. Cryopreservation is a method of storing sperm to maintain viability for a long period and support the breeding technology of the fish. Cryoprotectant, in this context, plays an important role in determining the success of sperm cryopreservation. OBJECTIVE: To determine the best type and concentration of cryoprotectant for cryopreservation of walking catfish sperm. MATERIALS AND METHODS: A total of five different types of cryoprotectants, namely DMSO, glycerol, ethyl glycol, ethanol, and methanol, were tested at four concentration levels namely 0%, 5%, 10%, 15%, and 20%, each with four replications. RESULTS: The type and concentration of cryoprotectant had a significant effect on sperm motility and viability (P < 0.05). The best outcomes were obtained with 5% DMSO and ethyl glycol, 10% glycerol and methanol, as well as 15% ethanol. CONCLUSION: The highest motility and viability values were obtained with 5% DMSO, resulting in its recommendation for cryopreservation of walking catfish sperm. Doi.org/10.54680/fr24510110612.

Revisiting the Injury Mechanism of Goat Sperm Caused by the Cryopreservation Process from a Perspective of Sperm Metabolite Profiles.

Semen cryopreservation results in the differential remodeling of the molecules presented in sperm, and these alterations related to reductions in sperm quality and its physiological function have not been fully understood. Given this, this study aimed to investigate the cryoinjury mechanism of goat sperm by analyzing changes of the metabolic characteristics in sperm during the cryopreservation process. The ultra-high-performance liquid chromatography-quadrupole time-of-flight mass spectrometry (UHPLC-QTOF-MS) technique was performed to explore metabolite profiles of fresh sperm (C group), equilibrated sperm (E group), and frozen-thawed sperm (F group). In total, 2570 metabolites in positive mode and 2306 metabolites in negative mode were identified, respectively. After comparative analyses among these three groups, 374 differentially abundant metabolites (DAMs) in C vs. E, 291 DAMs in C vs. F, and 189 DAMs in E vs. F were obtained in the positive mode; concurrently, 530 DAMs in C vs. E, 405 DAMs in C vs. F, and 193 DAMs in E vs. F were obtained in the negative mode, respectively. The DAMs were significantly enriched in various metabolic pathways, including 31 pathways in C vs. E, 25 pathways in C vs. F, and 28 pathways in E vs. F, respectively. Among them, 65 DAMs and 25 significantly enriched pathways across the three comparisons were discovered, which may be tightly associated with sperm characteristics and function. Particularly, the functional terms such as TCA cycle, biosynthesis of unsaturated fatty acids, sphingolipid metabolism, glycine, serine and threonine metabolism, alpha-linolenic acid metabolism, and pyruvate metabolism, as well as associated pivotal metabolites like ceramide, betaine, choline, fumaric acid, L-malic acid and L-lactic acid, were focused on. In conclusion, our research characterizes the composition of metabolites in goat sperm and their alterations induced by the cryopreservation process, offering a critical foundation for further exploring the molecular mechanisms of metabolism influencing the quality and freezing tolerance of goat sperm. Additionally, the impacts of equilibration at low temperature on sperm quality may need more attentions as compared to the freezing and thawing process.

Effect of naringenin on post-thaw quality, fertility-associated gene expression and fertilization potential of buffalo (Bubalus bubalis) bull sperm.

Our objectives were to explore the effect of naringenin addition in the semen extender on the post-thaw 1) sperm quality, 2) fertility-associated gene expression, and 3) fertilization potential of buffalo bull sperm. In experiment 1, semen samples (n = 32) from four Nili-Ravi buffalo bulls were pooled (n = 8) and diluted with the tris-citric acid (TCF-EY) extender containing different concentrations of naringenin, i.e., placebo (DMSO), 0 (control), 50, 100, 150 and 200 µM naringenin. After dilution, semen samples were packed in 0.5 mL French straws, cryopreserved and analyzed for post-thawed sperm quality and gene expression. Computer-assisted Semen Analysis, Hypo-osmotic Swelling test, Normal Apical Ridge assay, Rhodamine 123, Acridine orange, Propidium iodide staining and Thiobarbituric Acid Reactive Substances assay were performed to assess sperm motility parameters, plasma membrane functionality, acrosome integrity, mitochondrial membrane potential, DNA integrity, viability and lipid peroxidation, respectively. Expression levels of sperm acrosome-associated SPACA3, DNA condensation-related PRM1, anti-apoptotic BCL2, pro-apoptotic BAX, and oxidative stress-associated ROMO1 genes were evaluated through qPCR. Results revealed that total and progressive motility, plasma membrane functionality, acrosome integrity, mitochondrial membrane potential, DNA integrity and viability were higher (P < 0.05) with 50, 100 and 150 µM naringenin compared to 200 µM naringenin, placebo and control groups. Moreover, all naringenin-treated groups improved catalase activity, and reduced lipid peroxidation compared to placebo and control groups (P < 0.05). Relative expression levels of SPACA3 and PRM1 genes were higher (P < 0.05) with 150 µM naringenin compared to all groups except 100 µM (P > 0.05). No difference (P > 0.05) in the expression level of BCL2 gene was observed among all groups. Furthermore, BAX gene was expressed higher (P < 0.05) in the 200 µM naringenin group, whereas no difference (P > 0.05) in expression was noticed among the remaining groups. In addition, ROMO1 gene was expressed lower (P < 0.05) in all naringenin-treated groups compared to the control. In experiment 2, the in vivo fertility of semen doses (n = 400; 200/group) containing optimum concentration of naringenin (150 µM; depicted better in vitro sperm quality in experiment 1) was compared with control during the breeding season. Buffaloes were inseminated 24 h after the onset of natural estrus and palpated transrectal for pregnancy at least 60 days post-insemination. The fertility rate of 150 µM naringenin group was higher (P = 0.0366) compared to the control [57.00 ± 0.03 % (114/200) vs. 46.50 ± 0.04 % (93/200), respectively]. Taken together, it is concluded that naringenin supplementation in semen extender improves post-thaw quality, fertility-associated gene expression and fertilization potential of buffalo bull sperm, more apparently at 150 µM concentration.