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.

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.

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.

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.

A Polyphenol-Rich Extract From Entada abyssinica Reduces Oxidative Damage in Cryopreserved Ram Semen.

The Splinter bean, Entada abyssinica, is widely used in folk medicine. In the current work, we profiled the secondary metabolites from E. abyssinica bark extract using LC-MS and investigated its effect on cryopreserved ram semen. Twenty-eight compounds, including tannins and gallic acid derivatives that prevailed in the extract, were tentatively identified. Results showed that the quality of the post-thawed semen showed a significant improvement when the extract was added to the extender at a concentration of 375 µg/mL. The progressive motility and plasma membrane integrity of sperm cells were significantly increased in the post-thawed semen; however, the total antioxidant capacity (TAC) was insignificantly increased. A significant decrease in the concentration of hydrogen peroxide was detected as well. No significant changes were observed in activities of lactate dehydrogenase (LDH), alanine aminotransaminase (ALT), and aspartate transaminase (AST) within the treated samples. Intact sperm percentage was significantly increased, while apoptotic and necrotic sperm percentages were reduced significantly. Molecular docking of some individual components from the extract revealed their potential to interfere with the apoptosis cascade in which Bcl-2 is involved. In conclusion, Entada abyssinica appears to be useful for cryopreservation presumably owing to its polyphenol content that has potent antioxidant capacity scavenging reactive oxygen species (ROS), enhancing the endogenous antioxidant system and inhibiting lipid peroxidation.

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.

Possible mechanisms for the renoprotective effects of date palm fruits and seeds extracts against renal ischemia/reperfusion injury in rats.

PURPOSE: This work investigates the possible renoprotective effects of date palm fruits and seeds extract against renal ischemia and their underlying mechanisms. METHODS: 108-Sprague Dawle male rats were randomly allocated into 6 equal groups differently receiving aqueous or methanolic fruit and seed extracts. Assay of serum creatinine, BUN and TNF-α, morphological examination of the left kidney, markers of the redox state (MDA, CAT, and GSH), the expression of TNFα and Nrf2 genes at the level of mRNA, the expression of caspase-3 and TGF-ß proteins by immunohistochemistry were performed. RESULTS: 45-min renal I/R caused significant deterioration of kidney functions (increase in serum creatinine and BUN) and morphology (P < 0.001) and significant reduction in CAT activity and GSH levels with significant increase in serum TNF-α and MDA concentration and the expression of Nrf2, caspase-3, TNF-α, and TGF-ß in kidney tissues. Pre-treatment with either date palm fruit or seed extracts significantly improved kidney functions and morphology (P ≤ 0.001) with a significant increase in the expression of Nrf2 and CAT activity, and GSH concentration and a reduction in serum TNF-α and expression of caspase-3, TNF-α, and TGF-ß (P < 0.001). CONCLUSIONS: Administration of date palm extracts exhibited a renoprotective effect against renal I/R injury.This renoprotective action might be due to their antioxidants, anti-apoptotic and anti-inflammatory actions. Moreover, aqueous fruit extracts offered powerful renoprotective effect than aqueous seed extracts, and aqueous fruit and seed extracts were generally more effective than methanolic extracts.

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.

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.

Protective effects of antioxidants on linoleic acid-treated bovine oocytes during maturation and subsequent embryo development.

Linoleic acid (LA; n-6, 18:2) is the most abundant polyunsaturated fatty acid in the ovarian follicular fluid and is known to inhibit oocyte maturation and its subsequent development. In the present study, we investigated how its effects on cumulus cell expansion, oocyte nuclear maturation, and blastocyst development are altered by supplementation of the media with vitamin E (VE; 100 µM) and glutathione peroxidase (GPx; 1 µM) either alone or in combination, and whether it has any effect on the mRNA expression of GPx1, GPx4, or superoxide dismutase (SOD2) in the bovine cumulus oocyte complexes (COCs). LA supplementation of the culture media significantly (P ≤ 0.05) reduced the percentage of COCs exhibiting full cumulus cell expansion and the percentage of oocytes reaching metaphase II stage, and lowered the blastocyst rate compared with controls. And these inhibitory effects were associated with a reduction in the relative mRNA expression of GPx1 and SOD2 but not of GPx4 compared with controls. However, VE and GPx, both alone and in combination, completely abrogated the inhibitory effects of LA on nuclear maturation of oocytes and blastocyst rate but failed to do so for cumulus cell expansion. In conclusion, these data suggest that the detrimental effects of LA on oocyte developmental competence are mediated, at least in part, by a reduction in GPx1 and SOD2 mRNA expression. Moreover, VE and GPx may provide protection to most of the inhibitory effects of LA.