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.

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.

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 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.

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.

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.

Comparison between the Effects of Adding Vitamins, Trace Elements, and Nanoparticles to SHOTOR Extender on the Cryopreservation of Dromedary Camel Epididymal Spermatozoa.

There are several obstacles in camel semen cryopreservation; such as increasing semen viscosity and the reduction in motile spermatozoa after ejaculation. Epididymal spermatozoa offer an efficient alternative to overcome these problems and are well-suited for artificial insemination in camels. In the current study, we compared the effects of supplementation with vitamin C, E, inorganic trace elements of selenium (Na2SeO3) and zinc (ZnSO4), and zinc and selenium nanoparticles (ZnONPs and SeNPs, respectively) on the cryopreservation of dromedary camel epididymal spermatozoa. When the SHOTOR extender was supplemented with ZnONPs and SeNPs; the sperm showed increased progressive motility; vitality; and membrane integrity after cooling at 5 °C for 2 h; when compared to the control and vitamin-supplemented groups. Moreover, the ZnONPs and SeNPs supplementation improved the progressive motility, vitality, sperm membrane integrity, ultrastructural morphology, and decreased apoptosis when frozen and thawed. SeNPs significantly increased reduced glutathione (GSH), superoxide dismutase (SOD), and decreased lipid peroxide malondialdehyde (MDA) levels. The advantageous effects of the trace elements were potentiated by reduction into a nano-sized particle, which could increase bioavailability and reduce the undesired liberation of toxic concentrations. We recommend the inclusion of SeNPs or ZnONPs to SHOTOR extenders to improve the cryotolerance of camel epididymal spermatozoa.

Impact of selenium nano-particles in semen extender on bull sperm quality after cryopreservation.

The aim of this study was to evaluate the effect of adding different concentrations of selenium nano-particles (Se-NPs) in semen extender on bull sperm cryopreservation. Five healthy, fertile Friesian bulls were used, and the ejaculates were obtained using an artificial vagina. Semen of all bulls were pooled and diluted in a tris-yolk fructose (TYF) extender supplemented with Se-NPs at concentrations of 0 (T1, control), 0.5 (T2), 1.0 (T3) and 1.5 (T4) µg/ml for a final sperm concentration of 80 × 106 sperm cells/ml. Diluted semen was packed in straws (0.25 ml) and stored in liquid nitrogen (-196 °C) for one month. After thawing, semen of each treatment was evaluated for sperm quality parameters, including sperm progressive motility, livability, morphological abnormalities, plasma membrane integrity and chromatin integrity. Apoptosis and sperm ultrastructure were also examined. Total antioxidant capacity and lipid peroxidation markers were determined in seminal plasma of semen in each treatment. Finally, the effect of Se-NPs on fertilization capacity was checked in vivo using n = 81cows. Results showed that T2 and T3 had a positive effect (P < 0.05) on post-thawing sperm progressive motility, livability and membrane integrity as compared with the control. Percentage of viable sperm increased (P < 0.05), while percentages of early apoptotic, apoptotic and necrotic sperm cells decreased (P < 0.05) in T3 as compared to T1. Total antioxidants capacity (TAC) in seminal plasma increased (P < 0.05) and malondialdhyde (MDA) concentration decreased (P < 0.01) in T3 as compared to T1, but did not differ in T4 from that in T1. In vivo fertility rate was higher in T3 (90%) than in T1 (59%). In conclusion, enrichment of semen extender with Se-NPs at a concentration of 1.0 µg/ml improved post-thaw sperm quality of Holstein bulls, and consequently in vivo fertility rate by reducing apoptosis, lipid peroxidation and sperm damage occurring by cryopreservation.

Polyunsaturated fatty acids influence offspring sex ratio in cows.

Dietary polyunsaturated fatty acids (PUFAs) can influence fertility in farm animals. Some evidence in mice and sheep have suggested that PUFAs may influence offspring sex ratio, which may have significant value for cattle production. To test this hypothesis, three groups of Holstein cows were supplemented with either 0%, 3% or 5% protected fat (PF) in the form of calcium salt of fatty acids (rich in omega-6) from 14-21 days pre-partum until conception. Proven-fertile frozen semen from the same ejaculate was used for insemination. Calf sex recorded at birth was 8/19 (42.1%) male offspring in the control group, increasing to 14/20 (70%, P > 0.05) and 17/20 (85%, P < 0.05) in 3% and 5% PF, respectively. To test if this effect was caused by a direct influence on the oocyte, we supplemented bovine cumulus oocyte complexes during in vitro maturation with either omega-3 alpha-linolenic acid (ALA), omega-6 linoleic acid (LA) or trans-10, cis-12 conjugated linoleic acid (CLA). Sex ratio of the produced transferable embryos was determined using PCR of SRY gene. Similar to the in vivo results, sex ratio was skewed to the male side in the embryos derived from LA- and CLA-treated oocytes (79% and 71%) compared to control and ALA-treated oocytes (44% and 54%, respectively). These results indicate that both dietary and in vitro supplementation of omega-6 PUFAs can skew the sex ratio towards the male side in cattle. Further experiments are required to confirm this effect on a larger scale and to study the mechanisms of action that might be involved.

Evaluation of bull spermatozoa during and after cryopreservation: Structural and ultrastructural insights.

Semen cryopreservation is a well-established procedure used in veterinary assisted reproduction technology applications. We investigated damaging effects of cryopreservation on the structural and ultrastructural characteristics of bull sperm induced at different temperatures and steps during standard cryopreservation procedure using transmission (TEM) and scanning electron microscopy. We also examined the effect of cryopreservation on sperm DNA and chromatin integrity. Five healthy, fertile Friesian bulls were used, and the ejaculates were obtained using an artificial vagina method. The semen samples were pooled and diluted in a tris-yolk fructose (TYF) for a final concentration of 80 × 106 spermatozoa/ml. The semen samples were packed in straws (0.25 ml), and stored in liquid nitrogen (-196°C). Samples were evaluated before dilution, just after dilution (at 37°C), at 2 h and 4 h during equilibration, and after thawing (37°C for 30 s in water bath). In association with step-wise decline in motility and viability, our results showed that the plasma membrane surrounding the sperm head was the most vulnerable structure to cryo-damage with various degrees of swelling, undulation, or loss affecting about 50% of the total sperm population after equilibration and freezing. Typical acrosome reaction was limited to 10% of the spermatozoa after freezing. We also observed increased number of mitochondria with distorted cristae (15%). Chromatin damage was significantly increased by cryopreservation as evident by TEM (9%). This was mainly due to DNA breaks as confirmed by Sperm Chromatin Structure Assay (SCSA) (8.4%) whereas the chromatin structure was less affected as evaluated microscopically by toluidine blue staining. We concluded that, using standard cryopreservation protocol, the most pronounced damage induced by cryopreservation is observed in the plasma membrane. Further improvement of cryopreservation protocols should thus be targeted at reducing plasma membrane damage. Acrosomal, mitochondrial and chromatin damage are also evident but appear to be within acceptable limits as discussed.