Influence of nano-formulations of clove bud (Syzygium aromaticum) extract on freezing ability, antioxidant capacity, caspase-3 activity, acrosome reaction and fertility of frozen rabbit semen.

The current study aimed to investigate the impact of nano-formulations of clove bud ethanolic extract (CBENF) in the extender on sperm characteristics, antioxidant capacity, oxidative biomarkers, enzymatic activity, apoptosis and fertility of post-thawed rabbit semen. Twelve mature male rabbits semen samples were pooled and cryopreserved in a Tris-egg yolk-based extender containing varying concentrations of CBENF (0, 25, 50, 75 and 100 µg/mL). After the equilibration and freezing-thawing process, CBENF (100 µg /mL) significantly enhanced progressive motility, viability and membrane integrity. Conversely, sperm abnormality was significantly reduced by CBENF supplementation. Total antioxidant capacity was increased in the post-thawed sperm medium, while nitric oxide and malondialdehyde were decreased in all CBENF concentrations. The lactic dehydrogenase and caspase-3 activities were decreased, whereas the number of live spermatozoa with an intact acrosome was increased in all CBENF concentrations. Conception rate and litter size per doe were higher in doe rabbits inseminated with semen supplemented with 100 µg CBENF/mL than un-supplemented group (76% vs. 52% and 8.4 vs. 7.7/doe), with no statistical differences. These findings suggest that supplementing rabbit extenders with 100 µg of CBENF/mL could be an effective strategy for enhancing freeze-thawing rabbit sperm attributes and fertility.

Recent approaches in the use of antioxidants and proteomic modifications in ram semen preservation.

Sperm preservation is a well-established technique in reproductive biotechnology that is widely used to maintain the genetic quality of male individuals. However, there are several factors during the preservation process that can affect the vitality, functionality, and quality of sperm, thereby reducing their fertility potential after thawing. One of these factors is the synthesis of high levels of oxidative stress (OS) during semen preservation, which can have detrimental effects on sperm health and functionality. To counter the negative impact of OS on sperm, researchers have explored the supplementation of several exogenous antioxidants in the extenders used to preserve ram sperm. This approach has shown promising results in improving sperm health, functionality, and fertility potential in ram. Additionally, the preservation process can induce modifications in the ram sperm proteome. By employing targeted proteomics techniques, researchers have been able to identify and modify specific proteins in cryopreserved ram sperm, potentially offering further improvements in the quality of the cryopreserved ram sperm. In summary, this review provides a comprehensive overview of the antioxidants and targeted proteomics modifications that have been investigated for enhancing ram sperm preservation. These advancements aim to mitigate the negative effects of OS and optimize the techniques used in preserving ram sperm.

Influence of adding zeolite loaded with different charges to semen extender on sperm quality in rabbits after cryopreservation.

The aim of the present study was to investigate the effect of supplementing rabbit semen extender with zeolite loaded with different charges (Z+ or Z-, Z±) on sperm cryopreservation. Semen was collected from six healthy, fertile New Zealand rabbit bucks using an artificial vagina. The collected ejaculates were pooled and diluted with a tris-yolk fructose (TYF) extender supplemented with Z± (+16, +12, +8, -16, -12, and -8) at a concentration of 1% for a final sperm concentration of 25 × 106 sperm cells/mL. The diluted semen samples were then cryopreserved in 0.25 mL straws and stored in liquid nitrogen for 1 month. To evaluate sperm quality, we examined sperm progressive motility, vitality, morphological abnormalities, and plasma membrane integrity. In addition, apoptotic rates were determined using flow cytometry and by examining sperm ultrastructure under a transmission electron microscope (TEM). Moreover, total antioxidant capacity and markers of lipid peroxidation were measured in the extender after thawing. Addition of Z± had a positive effect on progressive motility, vitality, and membrane integrity after an equilibration period and post-thawing as compared with the controls (P < 0.05). Z± supplementation, particularly with a strong negative charge, also decreased the percentages of apoptotic and necrotic sperm cells compared to controls (P < 0.05), as shown both by flow cytometry and TEM. This was not associated with any marked effects on the oxidative biomarkers in the extender. In conclusion, addition of Z± to semen extender improved post-thawing sperm quality by improving sperm characteristics, decreasing apoptosis, and minimizing sperm damage during cryopreservation.

Effect of zinc chloride and sodium selenite supplementation on in vitro maturation, oxidative biomarkers, and gene expression in buffalo (Bubalus bubalis) oocytes.

This study examined the effects of zinc chloride (ZnCl2) and sodium selenite (Na2SeO3) supplementation in maturation medium on in vitro maturation (IVM) rate, oxidative biomarkers and gene expression in buffalo oocytes. Ovaries from a slaughterhouse were aspirated and good quality cumulus-oocyte complexes (COCs) with at least four layers of compact cumulus cells and evenly granulated dark ooplasm were selected. COCs were randomly allocated during IVM (22 h) to one of four treatment groups: (1) control maturation medium (basic medium), or basic medium supplemented with (2) ZnCl2 (1.5 µg/ml), (3) Na2SeO3 (5 µg/l), or (4) ZnCl2 + Na2SeO3 (1.5 µg/ml + 5 µg/l, respectively). Oocytes were denuded after 22 h of IVM in the first four replicates. Specimens were fixed and stained to evaluate the stage of nuclear maturation. The spent medium was collected for biochemical assays of total antioxidant capacity (TAC), malondialdehyde (MDA) and hydrogen peroxide concentrations. A second four replicates were used for COCs for RNA extraction. The expression levels of antioxidant (SOD1, GPX4, CAT and PRDX1), antiapoptotic (BCL2 and BCL-XL) and proapoptotic (BAX and BID) genes were measured. Supplementation with ZnCl2 and Na2SeO3 during IVM increased the ratio of oocytes reaching metaphase II at 22 h, increased TAC and decreased MDA and H2O2 concentrations in the maturation medium (P < 0.05). Moreover, beneficial effects were associated with complementary changes in expression patterns of antioxidative, antiapoptotic and proapoptotic genes, suggesting lower oxidative stress and apoptosis. Supplementation medium with zinc chloride and sodium selenite improves the maturation rate, reduces oxidative stress and increases expression levels of antioxidative and antiapoptotic genes.

Effects of vitamin C, vitamin E, selenium, zinc, or their nanoparticles on camel epididymal spermatozoa stored at 4 °C.

This study determined the effects of antioxidant supplementation and storage time at cool temperatures on the characteristics of epididymal camel spermatozoa. Camel testes were collected at the abattoir after animal slaughtering and kept at 4 °C during transportation and until processing (max 6 h). Spermatozoa were retrieved and diluted with SHOTOR extender, split in aliquots, supplemented with the following antioxidants: 200 µm/mL vitamin E, 1.0 g/L vitamin C, 1 µg/mL selenium nanoparticles, 50 µg/mL zinc nanoparticles, 2 µg/mL sodium selenite, and 100 µg/mL zinc sulfate, and stored at 4 °C for 2, 48, 96, and 144 h. The storage time significantly affected (P < 0.05) the sperms' motility and livability, the sperms' membrane integrity, and the percentages of cytoplasmic droplets as well as the percentage of morphologically normal spermatozoa. Epididymal sperm characteristics (progressive motility, livability, membrane integrity, and abnormalities) were significantly improved (P < 0.05) when the spermatozoa were diluted with antioxidants as compared with the control group, and the best additives were identified as nano-selenium, sodium selenite, nano-zinc, and zinc sulfate. In conclusion, adding nano-sized minerals or inorganic trace elements and vitamins maintained the progressive motility, livability, and membrane integrity, and decreased abnormalities and cytoplasmic droplet percentages of epididymal camel spermatozoa stored at 4 °C up to 144 h.

Effects of mint, thyme, and curcumin extract nanoformulations on the sperm quality, apoptosis, chromatin decondensation, enzyme activity, and oxidative status of cryopreserved goat semen.

Goat semen cryopreservation is a challenging process as it results in reduced motility, vitality, and fertility of spermatozoa after freezing. In this study, we evaluated the effects of different herbal extract nanoformulations (NFs) [mint (MENFs), thyme (TENFs), and curcumin (CENFs)], supplemented at either 50 or 100 µg into Tris-extender on the cryopreserved goat semen quality. The hydrothermal squeezing method was used for the preparation of the NFs extracts. The morphological evaluation of the NFs extracts was conducted by transmission electron microscopy. All NFs supplements improved (p < 0.05) the progressive motility, vitality, and plasma membrane integrity of sperm compared with the control extender after equilibration (5 °C for 2 h) and thawing (37 °C for 30 s), but had no effect on sperm abnormality and acrosome integrity. All NFs supplements decreased (p < 0.05) the apoptosis, malondialdehyde level, and chromatin decondensation of sperm cells, while increased (p < 0.05) the total antioxidant capacity and catalase activity in the frozen/thawed extender. Particularly, CENFs at a level of 100 µg showed improvement of sperm parameters and antioxidant status during cryopreservation of goat semen more than TENFs and MENFs. The CENFs improved the quality of goat spermatozoa in post-thawed semen in terms of preventing cryodamage and promoting the cryotolerance of spermatozoa when compared with TENFs and MENFs. Therefore, supplementation of Tris-extender with CENFs could enhance goat semen processing during cryopreservation.

Antioxidant response element activator, BTZO-1, improves the developmental competence of bovine oocytes.

Increased reactive oxygen species (ROS) generation may disrupt the oocytes function and their competence. In this study, we introduced BTZO-1, a new supplement that can regulate the oxidative stress. Addition of BTZO-1 during IVM of bovine oocytes improved their developmental competence in the term of improvement of blastocyst rates. In addition, the quality of the produced embryos was improved by decreasing the apoptosis level by showing a decreased number of TUNNEL positive cells.

Polyphenolics from Albizia harveyi Exhibit Antioxidant Activities and Counteract Oxidative Damage and Ultra-Structural Changes of Cryopreserved Bull Semen.

Albizia harveyi is a tropical deciduous tree, found across South and Eastern Africa and widely used in traditional medicine. The leaf extract ameliorated the damaging effects of the frozen-thawing process in cryopreserved bull semen. In a dose-dependent pattern, sperm motility, viability, and membrane integrity were improved compared to the untreated control. Furthermore, the extract increased the percentage of viable sperm cells and reduced the percentages of early apoptotic and apoptotic sperm cells as well as the damage in sperm ultra-structure. These activities are in agreement with the robust antioxidant properties in vitro and in the seminal fluid as observed in the total antioxidant capacity and the lipid peroxidation parameter malondialdehyde. LC-MS yielded 35 compounds. The extract was dominated by quercetin-O-galloyl-hexoside and quercetin-O-pentoside, along with other flavonoid glycosides. The polyphenols are probably responsible for the observed activities. In conclusion, the current findings show that A. harveyi leaves are rich in bioactive polyphenols with functional properties, validating its traditional use.

L-Proline: A Promising Tool for Boosting Cryotolerance and Fertilizing Ability of Cryopreserved Sperm in Animals.

Sperm cryopreservation technology significantly contributes to the safeguarding of genetic resources, particularly for endangered species, and supports the use of artificial insemination in domestic animals. Therefore, cryopreservation can negatively affect sperm health and function leading to reduce the freezing ability and fertility potential. Therefore, it is essential to prioritize the improvement of cryotolerance in cryopreserved sperm to enhance reproductive efficiency and ensure sustainability in livestock herds. The main reason for sperm dysfunction after thawing may be related to the excessive amount of oxidative stress (OS) produced during cryopreservation. Scientists have different ways for counteracting this OS including the use of plant extracts, enzymes, minerals, anti-freezing proteins, and amino acids. Recently, one such amino acid is L-proline (LP), which has multiple roles such as osmotic and OS defense, nitrogen, and carbon metabolism, as well as cell survival and signaling. LP has been found in seminal plasma and has recently been added to the freezing extender to improve the various post-thaw parameters of sperm. This improvement is related to the ability of LP to reduce the OS, sustain the plasma membrane and to act as an osmoregulatory agent. Moreover, LP can suppress cell apoptosis by modulating intracellular redox in sperm. This review addresses the ongoing research on the addition of L-proline as an osmoregulatory agent in freezing extenders to increase the cryotolerance of animal spermatozoa to freeze-thaw.

Effect of interleukin 6 (IL-6) on sperm quality, kinematic parameters, acrosome integrity, apoptosis, ultrastructure, and molecular docking in cryopreserved ram spermatozoa.

Sperm cryopreservation can lead to subfertility due to potential damage to sperm DNA, membranes, and overall motility caused by the freeze-thaw process. Interleukin-6 (IL-6) is a versatile cytokine with various roles in reproductive processes. However, the impacts of IL-6 supplementation on cryopreserved ram sperm have not been thoroughly investigated. Therefore, this study aims to assess the influence of IL-6 on the sperm quality of cryopreserved ram sperm. Ram semen was collected, pooled, and extended with tris-citrate soybean lecithin extender supplemented with 0, 50, 100, and 200 ng/mL of IL-6. The samples experienced a standard freezing protocol, and sperm quality, kinematic parameters, ultrastructure, and molecular docking of cryopreserved ram spermatozoa were evaluated. The results showed that sperm kinematics, viability, progressive motility, and membrane integrity were significantly enhanced by the addition of 100 or 200 ng of IL-6/mL (p < 0.05). Semen supplemented with 100 or 200 ng/mL of IL-6 also exhibited higher percentages of sperm kinematics, including DAP, DCL, DSL, VSL, VAP, VCL, and ALH, compared to other groups (p < 0.05). IL-6 supplementation enhanced acrosome integrity, and reduced caspase-3 activity in post-thawed ram spermatozoa (p < 0.05) compared to untreated group. Supplementation with IL-6 (200 ng/mL) significantly decreased oxidative biomarkers (NO, MDA, and H2O2) (p < 0.001) and improved total antioxidant capacity (p < 0.05). The percentage of sperm damage (tail, head, and midpiece) was significantly reduced by IL-6 supplementation (p < 0.05). Electron micrographs showed that supplementation with 100 or 200 ng/mL IL-6 protected acrosome stability, plasma membrane integrity, and sustained the ultrastructure integrity of cryopreserved ram spermatozoa. The docking exploration indicates a higher binding affinity with sperm function biomarkers, including caspase 3, BCL2, and PSMA6, with binding energies of - 52.30 kcal/mol, - 56.04 kcal/mol, and - 57.06 kcal/mol, respectively. In conclusion, the addition of IL-6 to the freezing extender can enhance the post-thaw quality of cryopreserved ram spermatozoa.

Spatial local expressions of kisspeptin in the uterus and uterine tubes and its relationship to the reproductive potential in goats.

Kisspeptins are neuropeptides encoded by the Kiss1 gene that was discovered as a metastasis suppressor gene in melanoma and breast cancer. Kisspeptin has pivotal functions for gonadotropin-releasing hormone secretion and plays integrated roles in the hypothalamic-pituitary-gonadal axis. However, little is known about the peripheral expression of kisspeptin in ruminants, especially in the female reproductive tract. Here, the objectives of the current study were to investigate the spatial localization of kisspeptin and mRNA expression of Kiss1 and its receptor (Kiss1r) in the fallopian tubes (FT) and uterus of goats at varied reproductive activity (cyclic versus true anoestrous goats, n=6, each). Specimens of the uterus and FT were collected and fixed using paraformaldehyde to investigate the localizations of kisspeptin in the selected tissues by immunohistochemistry. Another set of samples was snape-frozen to identify the expressions of mRNAs encoding Kiss1 and Kiss1r using real-time PCR. Results revealed immunolocalizations of kisspeptin in the uterus and the FT. The staining of kisspeptin was found mainly in the mucosal epithelium of the uterus the FT, and the endometrial glands. Very intense staining of kisspeptin was found in the uterine and FT specimens in the true anoestrous goats compared to that in cyclic ones. The expression of mRNA encoding Kiss1 gene was significantly higher in the uterine specimen of cyclic goats (1.00±0.09) compared to that in the true anoestrous goats (0.62±0.08) (P ˂0.05), while the expression of mRNA encoding Kiss1r was significantly (P ˂0.001) higher in the uterine tissues of true anoestrous goats (1.78±0.17) compared to that in cyclic ones (1.00±0.11). In conclusion, immunohistochemical localization of kisspeptin and the expression of mRNA encoding Kiss1/Kiss1r revealed spatial changes in the uterus and FT of goats according to the reproductive potential of goats (cyclic versus true anoestrous goats). However, the definitive local role of kisspeptin in the uterus and FT need further investigation.

The Effect of Metallic Nanoparticles Supplementation in Semen Extender on Post-thaw Quality and Fertilizing Ability of Egyptian Buffalo (Bubalus bubalis) Spermatozoa.

Nanomaterials offer several promising prospects in the field of farm animal reproduction, encompassing a broad range of applications such as transgenesis and the precise delivery of substances to sperm cells, antimicrobial, antioxidants properties as well as their potent role in improving cryopreservation methods. The aim of the present study is to explore the effect of supplementing the semen extender with 10 µg/mL nano gold (Au-NPs10), 10 µg/mL nano silver (Ag-NPs10), 1 µg/mL nano selenium (Se-NPs1), and 100 µg/mL nano zinc oxide (ZnO-NPs100) on sperm characteristics and kinematics parameters, acrosome integrity, oxidative biomarkers, morphological and apoptosis-like changes of frozen-thawed buffalo bull sperm, and, ultimately, their fertilizing capacity. The results revealed that all aforementioned nano materials significantly improved viability, progressive motility, membrane integrity, acrosome integrity, and kinematic parameters as well as apoptosis-like changes of post-thawed buffalo bull sperm compared to the control (p < 0.05). No discernible effects were observed on sperm ultrastructure morphology measures as a response to the addition of these metallic nanoparticles to the extender. The values of caspase 3 significantly decreased by 64.22, 45.99, 75.59, and 49.39% in Au-NPs10, Ag-NPs10, Se-NPs1, and ZnO-NPs100 treated groups, respectively, compared to the control. The addition of 100 µg ZnO-NPs to the extender significantly decreased the total count of bacteria, fungi, and yeast compared to the control (p < 0.05). The AuNPs10 and SeNPs1 treated groups showed lower content of malondialdehyde, hydrogen peroxide, and nitric oxide concentrations and higher values of total antioxidant capacity of post-thawed extended semen (p < 0.05). Pregnancy rates increased by 17.5, 20, and 30% in buffalo cows inseminated with sperm treated with ZnO-NPs100, Se-NPs1, and Au-NPs10, respectively, compared to the control group. The present results indicate that the freezing extender supplemented with metallic nanoparticles can be an effective strategy to enhance the cryotolerance and fertility potential of buffalo bull sperm.

The beneficial effects of quinoa seed extract supplementation on ram sperm quality following cryopreservation.

Although cryopreservation is a reliable method used in assisted reproduction to preserve genetic materials, it can stimulate the occurrence of oxidative stress, which affects sperm structure and function. This research was conducted to explore the effects of quinoa seed extracts (QSE) on ram sperm quality, oxidative biomarkers, and the gene expression of frozen-thawed ram sperm. Semen samples were diluted in extenders supplemented with 0 (QSE0), 250 (QSE1), 500 (QSE2), 750 (QSE3), and 1000 (QSE4) µg of QSE /mL, and then frozen according to the typical procedure. The findings indicate that the QSE3 and QSE4 groups provided the optimal results in terms of sperm viability and progressive motility. Sperm kinematics were considerably enhanced in the QSE3 group compared to the other groups (P<0.01). QSE (500-1000 µg/mL) significantly decreased the apoptosis-like changes (higher viable and lower apoptotic sperm) in ram sperm (P<0.001). The percentage of live sperm with intact acrosomes was significantly increased, while the percentage of detached and intact acrosomes in live and dead sperm were significantly decreased respectively by the QSE addition (P<0.001). All QSE groups had higher TAC and lower MDA and H2O2 levels than the control group (P<0.001). The expressions of SOD1, CAT, GABPB1, and GPX1 genes in sperm samples were significantly increased, while the CASP3 gene was significantly decreased in all QSE-supplemented samples. Our data suggest that QSE has beneficial effects on sperm quality of cryopreserved ram semen, which are achieved by promoting sperm antioxidant-related genes and reducing apoptosis-related gene.

Supplementation of Thymoquinone Nanoparticles to Semen Extender Boosts Cryotolerance and Fertilizing Ability of Buffalo Bull Spermatozoa.

Thymoquinone nanoparticles (TQNPs) are broadly utilized in numerous pharmaceutical applications. In the present study, we tested the effects of TQNP supplementation on sperm quality and kinematics, acrosome exocytosis, oxidative biomarkers, apoptosis-like and morphological changes of frozen-thawed buffalo sperm, as well as the fertilizing capacity. Semen was collected from buffalo bulls, diluted (1:10; semen/extender), and divided into five aliquots comprising various concentrations of TQNP 0 (CON), 12.5 (TQNP12.5), 25 (TQNP25), 37.5 (TQNP37.5), and 50 (TQNP50) µg/mL, and then cryopreserved and stored in liquid nitrogen (-196 °C). The results revealed that TQNPs (25 to 50 µg/mL) provided the most optimal results in terms of membrane integrity (p < 0.001) and progressive motility (p < 0.01). In contrast, TQNP50 resulted in a greater post-thawed sperm viability (p = 0.02) compared with other groups. The addition of TQNPs to the extender had no discernible effects on sperm morphology measures. Sperm kinematic motion was significantly improved in the TQNP50 group compared to the control group (p < 0.01). TQNPs effectively reduced the content of H2O2 and MDA levels and improved the total antioxidant capacity of post-thawed extended semen (p < 0.01). The addition of TQNP significantly increased the number of intact acrosomes (p < 0.0001) and decreased the number of exocytosed acrosomes (p < 0.0001). A significant reduction in apoptosis-like changes was observed in TQNP groups. The non-return rates of buffalo cows inseminated with TQNP50-treated spermatozoa were higher than those in the control group (p < 0.05; 88% vs. 72%). These findings suggested that the freezing extender supplemented with TQNPs could effectively enhance the cryotolerance and fertility of buffalo sperm.

Effects of propolis-loaded nanoliposomes fortification in extender on buffalo semen cryopreservation.

Buffalo sperm is sensitive to cryoinjuries, thus improving sperm cryoresistance is a critical approach for wide spreading the assisted reproductive technologies in buffalo. The intention of this work was to assess the effect of propolis-loaded in nanoliposomes (PRNL) supplementation of semen extender on semen quality, antioxidant status and some apoptotic genes of cryopreserved buffalo semen. PRNL were prepared using cholesterol (Chol) as well as soybean lecithin and their physicochemical properties were characterized. Egyptian buffalo bulls (4-6 years) were involved, and the semen samples were collected using the artificial vagina method. Buffalo semen was pooled (n = 25 ejaculates) and cryopreserved in tris extender containing PRNL at 0 (PRNL0), 2 (PRNL2), 4 (PRNL4) and 6 µg/mL (PRNL6), respectively. The PRNL had a size of 113.13 nm and a negative zeta potential (- 56.83 mV). Sperm progressive motility, viability, membrane integrity, abnormalities, chromatin damage, redox status, apoptosis status, and apoptotic genes were investigated after post-thawed buffalo semen. Using 2 or 4 µg/mL PRNL significantly increased sperm progressive motility, viability, and membrane integrity, while sperm abnormalities and the percentage of chromatin damages were the lowest in PRNL2 group. Moreover, the PRNL2 group exhibited the best results for all antioxidative activities (TAC, SOD, GPx and CAT) with significantly higher levels than the other groups (P < 0.05). The levels of ROS and MDA were significantly lower in the PRLN2 compared with other groups. The sperm caspase 3 enzyme activities showed the lowest values in PRNL2 groups followed by PRNL4 and PRNL6 groups with significant differences compared with the control. Adding 2 µg/mL PRNL to freezing media significantly reduced apoptotic genes such as Bax and Caspase 3 in sperm, while significantly increase in Bcl2 expression compared with the control (P < 0.001). The expression of Bcl2, Caspase 3 and Bax genes in sperm were not affected by the 6 µg/mL PRNL addition (P > 0.05). The electron micrography descriptions exemplified that the fortification of 2 or 4 µg/mL PRNL maintained the acrosomal and plasma membrane integrities as well as sustained the ultrastructure integrity of the cryopreserved buffalo spermatozoa when compared with control group, whereas the 6 µg/mL of PRNL demonstrated highest injury to the acrosome and plasma membranes. Results show supplementation of the buffalo freezing extender with 2 or 4 µg/mL of PRNL enhanced post-thawed sperm quality via boosting the antioxidant indices, diminishing the oxidative stress and apoptosis as well as maintained the ultrastructure integrity of frozen-thawed buffalo sperm.

Effect of olive, flaxseed, and grape seed nano-emulsion essential oils on semen buffalo freezability.

The existing treatise targeted to compare the effects of adding different nano-emulsions essential oils (olive, flaxseed, and grapeseed oils) in freezing extender on semen quality and freezability in buffalo. Nano-emulsions were prepared from olive, flaxseed, and grapeseed oils and characterized for their sizes and shapes. Semen extended in four tubes were supplemented with 0 (control) and 3.5% nanoemulsion oils, including olive (NEO), flaxseed (NEFO) and grape seed oils (NEGSO) respectively. NEGSO resulted in the highest (p < 0.05) membrane integrity, vitality, progressive motility (P-motility) of sperm compared to the other groups in post-thawed buffalo bull semen (at 37 °C for 30 s). The addition of NEGSO had the best results for membrane integrity, progressive motility, and vitality of sperm after incubation (at 37 °C and 5% CO2 for 2 h). A superior (p < 0.05) value of total antioxidant capacity in frozen-thawed spermatozoa was monitored in all supplemented groups as relative to the control. The values of malondialdehyde (MDA) and nitric oxide (NO) were lower (p < 0.05) in NEGSO group compared with other groups. Both NEO and NEFO exhibited the same results for MDA, and NO levels (p > 0.05). All supplemented groups exhibited lower hydrogen peroxide levels (p < 0.05) as relative to the un-treated group. The lowest (p < 0.05) caspase 3 levels were verified in NEGSO treatment, followed by NEFO and NEO treatments. Post-thawed sperm showed ultrastructural damages in the control group, and theses damages were attenuated or resorted by the NEGSO, NEFO and NEO supplemented to freezing extender. In consequences with in vitro results regarding the sperm attribute, a greater pregnancy rate (92%) was observed in NEGSO group as compared with NEFO (88%), NEO (76%) and CON (68%) groups. Our findings demonstrate that NEGSO (3.5%) could be used as a new strategy in enhancing sperm functionality, potential fertility and reducing the oxidative damage and apoptosis markers. This could be significantly applicable for sperm physiology cryopreservation in the milieu of assisted reproduction systems.

Supplementation of Alpha-lipoic acid-loaded nanoliposomes in semen extender improves freezability of buffalo spermatozoa.

This research was designed to explore the protective effect of alpha-lipoic acid-loaded nanoliposomes (ALAN) during cryopreservation of buffalo sperm. Buffalo semen was cryopreserved in a tris-citrate egg yolk extender without any supplement (ALAN0, control group) or with ALAN at levels of 25, 50, 75 or 150 µg (ALAN25, ALAN50, ALAN75 and ALAN150, respectively). The ALAN had a size of 171.80 nm and a negative zeta potential (- 43.40 mV). The progressive motility, vitality and membrane integrity significantly improved in all ALAN groups (except ALAN25 for membrane integrity). ALAN150 group exhibited the best values of progressive sperm motility, vitality and membrane integrity after thawing at 37 °C for 30 s or incubated for 2 h at 37 °C and 5% CO2 compared with those in other groups. Both ALAN75 and ALAN150 groups significantly improved the TAC, GR and catalase, while lipid peroxidation and early apoptotic spermatozoa significantly decreased in ALAN150 group followed by ALAN75 group. Collectively, the adding ALAN to buffalo semen freezing extender plays a substantial shielding function against cryodamage by preserving the sperm functional parameters.

The Supplementation of Bee Bread Methanolic Extract to Egg Yolk or Soybean Lecithin Extenders Can Improve the Quality of Cryopreserved Ram Semen.

Bee bread has numerous nutritional benefits and bioactive compounds. Other bee byproducts have been used as extender additives to improve semen cryopreservation. Here, we examined the effects of supplementing egg yolk extender (EYE) or soybean lecithin extender (SBLE) with bee bread extract (BBE) on the quality of cryopreserved ram semen. Semen was collected from five adult Rahmani rams once a week for 7 weeks. EYE and SBLE were supplemented with BBE. Antioxidant capacity and total phenolic compound, total flavonoid compound, and total soluble carbohydrate levels of BBE were measured. Sperm characteristics, including progressive motility, viability, abnormalities, membrane integrity, and acrosome integrity, were analyzed after equilibration, thawing, and thawing followed by a 2-h incubation. The total antioxidant capacity and malondialdehyde, hydrogen peroxide, aspartate transaminase, alanine transaminase, alkaline phosphatase, and total acid phosphatase levels in extenders were determined after thawing. Sperm apoptosis was analyzed using annexin V assays. SBLE was more effective than EYE for cryopreserving ram semen. Extender supplementation with BBE improved ram semen quality during freezing in a concentration-dependent pattern. Motility, vitality, and membrane integrity were particularly enhanced in BBE-treated semen. Additionally, BBE promoted antioxidant and enzymatic activities and reduced apoptosis in semen. Thus, extender supplementation with BBE improved sperm cryopreservation.

Antioxidant and Antiapoptotic Effects of a Turraea fischeri Leaf Extract on Cryopreserved Goat Sperm.

This study evaluated the efficacy of Turraea fischeri leaf extract for maintaining the viability of cryopreserved goat sperm. Ejaculated semen was collected from 5 mature Baladi bucks (50-60 kg, 2-4 years of age) and those samples with mass motility ≥ 70% and sperm concentration ≥ 2.5 × 109/mL were selected, pooled, and divided into 4 aliquots. Each aliquot was diluted in Tris-citric-soybean lecithin extender containing a different concentration of T. fischeri leaf extract (0, 125, 250, or 375 µg/mL). Treated semen samples were cooled to 5 °C, transferred to 0.25-mL French straws, and stored in liquid nitrogen (LN2) at -196 °C. After thawing, membrane integrity was examined by transmission electron microscopy, apoptotic activity by Annexin/propidium iodide staining and flow cytometry, and both enzyme activities and antioxidant capacity by spectroscopic assays. The leaf extract at 375 µg/mL significantly improved semen quality as indicated by enhanced total antioxidant capacity, reduced H2O2 concentration, a greater proportion of structurally intact motile sperm, and concomitant reductions in apoptosis and necrosis. The extract also significantly increased the proportion of sperm with a contiguous plasma membrane and intact acrosome (p < 0.05). Furthermore, LC-MS revealed numerous secondary metabolites in the extract that may contribute to sperm cryopreservation.

The Current Trends in Using Nanoparticles, Liposomes, and Exosomes for Semen Cryopreservation.

Cryopreservation is an essential tool to preserve sperm cells for zootechnical management and artificial insemination purposes. Cryopreservation is associated with sperm damage via different levels of plasma membrane injury and oxidative stress. Nanoparticles are often used to defend against free radicals and oxidative stress generated through the entire process of cryopreservation. Recently, artificial or natural nanovesicles including liposomes and exosomes, respectively, have shown regenerative capabilities to repair damaged sperm during the freeze-thaw process. Exosomes possess a potential pleiotropic effect because they contain antioxidants, lipids, and other bioactive molecules regulating and repairing spermatozoa. In this review, we highlight the current strategies of using nanoparticles and nanovesicles (liposomes and exosomes) to combat the cryoinjuries associated with semen cryopreservation.