Cryopreservation and assessment of genetic fidelity Acorus calamus Linn., an endangered medicinal plant.

BACKGROUND: Acorus calamus Linn. is a medicinally valuable monocot plant belonging to the family Acoraceae. Over-exploitation and unscientific approach towards harvesting to fulfill an ever-increasing demand have placed it in the endangered list of species. OBJECTIVE: To develop vitrification-based cryopreservation protocols for A. calamus shoot tips, using conventional vitrification and V cryo-plate. MATERIALS AND METHODS: Shoot tips (2 mm in size) were cryopreserved with the above techniques by optimizing various parameters such as preculture duration, sucrose concentration in the preculture medium, and PVS2 dehydration time. Regenerated plantlets obtained post-cryopreservation were evaluated by random amplified polymorphic DNA (RAPD) to test their genetic fidelity. RESULTS: The highest regrowth of 88.3% after PVS2 exposure of 60 min was achieved with V cryo-plate as compared to 75% after 90 min of PVS2 exposure using conventional vitrification. After cryopreservation, shoot tips developed into complete plantlets in 28 days on regrowth medium (0.5 mg/L BAP, 0.3 mg/L GA3, and 0.3 mg/L ascorbic acid). RAPD analysis revealed 100% monomorphism in all cryo-storage derived regenerants and in vitro donor (120-days-old) plants. CONCLUSION: Shoot tips of A. calamus that were cryopreserved had 88.3% regrowth using V cryo-plate technique and the regerants retained genetic fidelity. https://doi.org/10.54680/fr24210110412.

The kinetics of goat sperm is negatively affected after freezing in an extender including zinc oxide nanoparticles.

BACKGROUND: Nanotechnology can benefit livestock industries, especially through postharvest semen manipulation. Zinc oxide nanoparticles (Np-ZnO) are potentially an example. OBJECTIVE: To investigate how the addition of zinc oxide nanoparticles (Np-ZnO) affected the characteristics of post-thawed goat semen. MATERIALS AND METHODS: Seminal pools from four Saanen bucks were used. Semen was diluted in Tris-egg yolk extender, supplemented with Np-ZnO (0, 50, 100 or 200 ug/mL), frozen and stored in liquid nitrogen (-196 degree C), and thawed in a water bath (37 degree C / 30 s). Semen samples were evaluated for sperm kinetics by computer-assisted sperm analysis (CASA), and assessed for other functional properties by epifluorescence microscopy, such as plasma membrane integrity (PMi), acrosomal membrane integrity (ACi) and mitochondrial membrane potential (MMP). RESULTS: For total motility (TM), the group treated with 200 ug/mL Np-ZnO was superior to the control. In straight-line velocity (VSL), the control was better than the group containing 200 ug/mL of Np-ZnO. For average path velocity (VAP), the control was higher than with 100 ug/mL Np-ZnO. For linearity (LIN), the control was higher than with 200 µg/mL Np-ZnO. In straightness (STR), the control and 100 µg/mL Np-ZnO were higher than with 200 ug/mL Np-ZnO. In wobble (WOB), the control was better than the 50 µg/mL Np-ZnO treatment. In PMi, ACi and MMP no significant differences were found. CONCLUSION: The addition of Np-ZnO (200 ug/mL) to the goat semen freezing extender improved the total motility of cells, whilst negatively affecting sperm kinetics. https://doi.org/10.54680/fr24210110512.

Effect of melatonin on developmental competence, mitochondrial distribution, and intensity of fresh and vitrified/thawed in vitro matured buffalo oocytes.

BACKGROUND: In livestock breeding, oocyte cryopreservation is crucial for preserving and transferring superior genetic traits. This study was conducted to examine the additional effect of melatonin to maturation and vitrification media on the in vitro developmental capacity, mitochondrial distribution, and intensity of buffalo oocytes. The study involved obtaining ovaries from a slaughterhouse and conducting two phases. In the first phase, high-quality oocytes were incubated in a maturation medium with or without 10-9M melatonin for 22 h (at 38.5°C in 5% CO2). Matured oocytes were fertilized in vitro and cultured in SOF media for seven days. In the second phase, vitrified in vitro matured oocytes were stored in vitrified media (basic media (BM) containing a combination of cryoprotectants (20% Ethyl Glycol and 20% Dimethyl sulfoxide), with or without melatonin, and then stored in liquid nitrogen. Normal vitrified/thawed oocytes were fertilized in vitro and cultured as described. Finally, the matured oocytes from the fresh and vitrified/thawed groups, both with and without melatonin, were stained using DAPI and Mitotracker red to detect their viability (nuclear maturation), mitochondrial intensity, and distribution using a confocal microscope. The study found that adding 10-9M melatonin to the maturation media significantly increased maturation (85.47%), fertilization rate (84.21%)cleavage (89.58%), and transferable embryo (48.83%) rates compared to the group without melatonin (69.85%,79.88%, 75.55%, and 37.25% respectively). Besides that, the addition of melatonin to the vitrification media improved the recovery rate of normal oocytes (83.75%), as well as the cleavage (61.80%) and transferable embryo (27.00%) rates when compared to the vitrified TCM group (67.46%, 51.40%, and 17.00%, respectively). The diffuse mitochondrial distribution was higher in fresh with melatonin (TCM + Mel) (80%) and vitrified with melatonin (VS2 + Mel groups) (76.70%), Furthermore, within the same group, while the mitochondrial intensity was higher in the TCM + Mel group (1698.60) than other group. In conclusion, Melatonin supplementation improves the developmental competence and mitochondrial distribution in buffalo oocytes in both cases(in vitro maturation and vitrification).

Identification of potato varieties suitable for cold storage and reconditioning: A safer alternative to anti-sprouting chemicals for potato sprouting control.

Low temperature storage as an alternative to anti-sprouting chemicals in potato storage may induce reducing sugars (RS) accumulation (i.e. glucose and fructose) in potato tubers. This phenomenon is called "cold induced sweetening" (CIS) and occurs in certain varieties. CIS leads to a decrease in the organoleptic qualities and darkening of processed potato and the accumulation of toxic molecules such as acrylamide. To identify potato varieties suitable for storage at low temperatures, we screened six commercial processing varieties: Lady Claire (LC), Verdi, Kiebitz (KB), Pirol, Agria and Markies for their CIS characteristics and sprout-forming potential after storage at 4 °C and 8 °C. Our findings reveal that 4 °C storage allows for efficient sprout reduction in all six tested varieties for up to 4.5 months of storage. Three CIS-resistant varieties, namely Verdi, Lady Claire and Kiebitz, were identified as able to be stored for up to four months at 4 °C with limited increase in glucose content. Conversely, Pirol, Agria and Markies showed an increase in glucose content with a decrease in storage temperature and can be considered as CIS-susceptible varieties. After processing into crisps, the CIS-susceptible varieties displayed poor crisp color quality (brown to black color crisps) after storage for two months at 4 °C compared to the storage at 8 °C, whereas the CIS-resistant varieties had good crisp color quality (pale yellow color crisps) after storage at both 4 and 8 °C. Interestingly, the trends of total RS and/or glucose content in the CIS-resistant and in the CIS-susceptible varieties were correlated with the trends in Vacuolar Invertase (VInv) gene expression for most varieties, as well as with the trends in acrylamide content after processing. In addition, reconditioning of Markies variety after storage at 4 °C by gradually increasing the temperature to 15 °C resulted in a significant decrease of VInv transcript levels (reduction of 80 %), acrylamide content (reduction of 75 %) and glucose content when compared to a storage at 4 °C without reconditioning. Those results demonstrate that the reconditioning technique is a key factor for a sustainable potato storage and for improving the quality of processed potatoes.

Fatty acid supplementation during warming improves pregnancy outcomes after frozen blastocyst transfers: a propensity score-matched study.

This study aimed to examine the viability of human blastocysts after warming with fatty acids (FAs) using an in vitro outgrowth model and to assess pregnancy outcomes after a single vitrified-warmed blastocyst transfer (SVBT). For the experimental study, we used 446 discarded vitrified human blastocysts donated for research purposes by consenting couples. The blastocysts were warmed using FA-supplemented (FA group) or non-FA-supplemented (control group) solutions. The outgrowth area was significantly larger in the FA group (P = 0.0428), despite comparable blastocyst adhesion rates between the groups. Furthermore, the incidence of outgrowth degeneration was significantly lower in the FA group than in the control group (P = 0.0158). For the clinical study, we retrospectively analyzed the treatment records of women who underwent SVBT in natural cycles between January and August 2022. Multiple covariates that affected the outcomes were used for propensity score matching as follows: 1342 patients in the FA group were matched to 2316 patients in the control group. Pregnancy outcomes were compared between the groups. The rates of implantation, clinical pregnancy, and ongoing pregnancy significantly increased in the FA group after SVBTs (P = 0.0091-0.0266). These results indicate that warming solutions supplemented with FAs improve blastocyst outgrowth and pregnancy outcomes after SVBTs.

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

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

Microplate-Based Cryopreservation of Adherent-Cultured Human Cell Lines Using Amino Acids and Proteins.

With the progression of regenerative medicine and cell therapy, the importance of cryopreservation techniques for cultured cells continues to rise. Traditional cryoprotectants, such as dimethyl sulfoxide and glycerol, are effective in cryopreserving suspended cells, but they do not demonstrate sufficient efficacy for two-dimensional (2D)-cultured cells. In the past decade, small molecules and polymers have been studied as cryoprotectants. Some L-amino acids have been reported to be natural and biocompatible cryoprotectants. However, the cryoprotective effects of D-amino acids have not been investigated for such organized cells. In the present study, the cryoprotective effects of D- and L-amino acids and previously reported cryoprotectants were assessed using HepG2 cells cultured on a microplate without suspending the cells. d-Proline had the highest cryoprotective effect on 2D-cultured cells. The composition of the cell-freezing solution and freezing conditions were then optimized. The d-proline-containing cell-freezing solution also effectively worked for other cell lines. To minimize the amount of animal-derived components, fetal bovine serum in the cell freezing solution was substituted with bovine serum albumin and StemFit (a commercial supplement for stem cell induction). Further investigations on the mechanism of cryopreservation suggested that d-proline protected enzymes essential for cell survival from freeze-induced damage. In conclusion, an effective and xeno-free cell-freezing solution was produced using d-proline combined with dimethyl sulfoxide and StemFit for 2D-cultured cells.

Supplementation of extender with chamuangone-enriched Garcinia cowa leaf extract improves quality parameters and longevity of cold-stored cat semen.

BACKGROUND: Semen preservation by cooling is less expensive, simpler and results in less sperm damage than freezing does. However, spermatozoa can only be preserved for a short period due to the excessive formation of reactive oxygen species (ROS). Although several antioxidants can protect sperms from ROS damage during storage at low temperatures, the use of natural antioxidants derived from plants would be a better alternative. OBJECTIVE: To assess the effects of chamuangone, which can reduce oxidation reactions in cells, on cat semen quality after preservation at 4 degree C for 15 days. MATERIALS AND METHODS: Epididymal sperm samples were collected before being diluted with tris-citric-fructose-egg yolk (TCFE) extender containing different concentrations of chamuangone (0, 50, 100, 150 and 200 ug/mL) and preserved at 4 degree C. Semen samples were evaluated before chilling and then every 3 days after chilling for up to 15 days. Each sample was assessed for sperm motility, viability, DNA integrity, plasma membrane integrity and percentage of spermatozoa with intact acrosomes. RESULTS: A significantly higher sperm motility was observed in the group supplemented with 100 ug/mL chamuangone compared to the control after 6 days of storage. However, the chamuangone concentration at 200 ug/mL did not significantly increase the sperm motility when compared to the control for the entire storage period. CONCLUSION: 100 µg/mL chamuangone can improve sperm characteristics during 15 days of preservation at 4 degree C, keeping sperm alive (49.3 ± 5.2%) and moving (7.1 ± 2.4%). These results can be used for the development of breeding programs using technologically advanced reproductive procedures in domestic and wild cats. https://doi.org/10.54680/fr24110110212.

Evaluation of Kappa-carrageenan supplementation in extender for post-thaw Kajli ram sperm quality.

Cryopreservation is one of the reliable techniques for long-term storage of sperm. The success of this technique depends on the choice of cryoprotectant; therefore, a plethora of literature has reported the effects of different cryoprotective agents so far. Kappa-carrageenan (κ-carrageenan) is a hydrocolloid polysaccharide extracted from red marine seaweed. Its unique property makes it a promising option as a non-colligative cryoprotectant. The current study aims to evaluate the cryoprotective effect of k-carrageenan along with glycerol on ram sperm quality both after equilibration and freezing. Nine Kajli rams were utilized in this experiment for semen collection through an artificial vagina maintained at 42°C. Qualified samples were diluted in tris egg yolk glycerol (TEYG) extender containing different concentrations of k-carrageenan as 0 mg/mL (control), 0.2, 0.5, 0.8 and 1 mg/mL. Post-thaw assessment was done at 37°C after 24 h of storage, which showed a significant improvement (p < .05) in sperm viability, motility, membrane and acrosome integrity in an extender containing k-carrageenan at a concentration of 0.5 mg/mL compared to control. It is concluded from the current study that the combination of glycerol and 0.5 mg/mL concentration of k-carrageenan improved the sperm post-thaw quality.

Meat quality of pigs fed grape pomace in different production systems.

The main objective of this study was to evaluate the effects of supplementation the diet of pigs with grape pomace preserved in silage form (GPS) and its interaction with indoor and outdoor production systems, with and without access to vegetation, on the attributes of meat quality produced. Analyzes of proximal composition, cholesterol content, fatty acid profile, shear force, texture profile and sensory analysis were performed. During cold storage, oxidative stability and objective color were analyzed. Statistical analysis was performed in a 3x2 factorial design (production systems (S) x GPS-feed (F)) and the interaction between them (S*F). The results showed that there was no interaction between the production system and GPS feeding for the attributes evaluated. The proximate composition and fatty acid profile of the muscle remained unchanged. Additionally, it provides higher subjective and objective tenderness, higher red color intensity, and reduces lipid oxidation under refrigeration. The supplementation of pig feed with GPS improve the quality of the meat and constitute a sustainable alternative for the winemaking residue.

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 taurine, cysteine and melatonin as antioxidant supplements to the freezing medium of Prochilodus brevis sperm.

Cryopreservation consist of a set of methods to preserve cells and tissues by drastically reducing the temperature. Among some undesired effects, cryopreservation might generate reactive oxygen species that lead to an increase of oxidative stress, causing damage to cells. This study aimed to test taurine, cysteine, and melatonin on the freezing of Prochilodus brevis sperm and assess its effects on post-thawed sperm quality. Sperm was collected and seven pools were formed (n = 7). They were diluted (1:9) in standard medium (5% glucose, 10% dimethyl sulfoxide and 5% egg yolk) supplemented or not (control) with taurine (0.3, 1.0, 3.16 or 10.0 mM), cysteine (0.3, 1.0, 3.16 or 10.0 mM) or melatonin (0.6, 1.12, 2.0 or 3.56 mM). Post-thawed sperm was evaluated for kinetic (total motility, velocities, and percentage of rapid cells), morphology and membrane and DNA integrity. Differences were found when melatonin was used as an antioxidant. For the variables rapid sperm and sperm velocities, 3.56 mM melatonin presented higher results than the control (melatonin 0 mM). Melatonin 2 mM was similar to 3.56 mM on rapid sperm, average path velocity (VAP) and curvilinear velocity (VCL). No difference was found between concentration 0 mM (control) and taurine treatments. As for cysteine, 0.3 mM presented the best results for rapid sperm than 10 mM, and higher VCL and VAP than 1 mM. Melatonin 3.56 mM presented higher results on kinetic parameters (rapid motility, VCL, VSL and VAP) than other tested antioxidants. Therefore, melatonin 3.56 mM is recommended to be added to the sperm freezing medium of P. brevis.

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

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

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

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

Oocyte vitrification for fertility preservation is an evolving practice requiring a new mindset: societal, technical, clinical, and basic science-driven evolutions.

Infertility is a condition with profound social implications. Indeed, it is not surprising that evolutions in both medicine and society affect the way in vitro fertilization is practiced. The keywords in modern medicine are the four principles, which implicitly involve a constant update of our knowledge and our technologies to fulfill the "prediction" and "personalization" tasks, and a continuous reshaping of our mindset in view of all relevant societal changes to fulfill the "prevention" and "participation" tasks. A worldwide aging population whose life priorities are changing requires that we invest in fertility education, spreading actionable information to allow women and men to make meaningful reproductive choices. Fertility preservation for both medical and nonmedical reasons is still very much overlooked in many countries worldwide, demanding a comprehensive update of our approach, starting from academia and in vitro fertilization laboratories, passing through medical offices, and reaching out to social media. Reproduction medicine should evolve from being a clinical practice to treat a condition to being a holistic approach to guarantee patients' reproductive health and well-being. Oocyte vitrification for fertility preservation is the perfect use case for this transition. This tool is acquiring a new identity to comply with novel indications and social needs, persisting technical challenges, brand-new clinical technologies, and novel revolutions coming from academia. This "views and reviews" piece aims at outlining the advancement of oocyte vitrification from all these tightly connected perspectives.

Views and reviews: fertility preservation from different perspectives.

Fertility preservation for different conditions provides women the chance to buy time that can be invested in improving their well-being, by curing their condition from a holistic perspective, in line with the precepts of modern medicine.

Effects of alpha lipoic acid supplementation on post-thaw quality of drone semen.

This study aimed to determine the effect of alpha-lipoic acid (ALA) on post-thaw quality of bee semen. In the study, semen from sexually mature drone were collected. A series of experiments were carried out in which the retrieved semen was diluted with diluents containing different ALA concentrations or without ALA supplement (control). Cryopreserved sperm were thawed, and evaluated for motility (phase-contrast microscope), plasma and acrosomal membrane integrity, mitochondrial membrane potential, and DNA fregmantation. The results obtained showed that the highest motility after thawing was observed in the groups containing ALA 0.25 mmol (P < 0.05). Likewise, plasma membrane integrity was found to be better preserved in the ALA 0.25 mmol-added group than in other groups. Acrosomal integrity were also higher in the ALA-containing groups than in the control group (P < 0.05). The results of this study show that ALA supplementation especially at 0.25 mmol improved post-thawed sperm motility, plasma membrane functionality, and mitochondrial membrane potantial quality of honeybee semen.

Cysteine supplementation pre-freeze and post-thaw improves integrity and reduces oxidative stress in cryopreserved ram spermatozoa.

Cryopreserved ram sperm is highly sensitive to oxidative stress by reactive oxygen species (ROS) which impair sperm function and integrity. Antioxidants such as cysteine can mitigate the effect of ROS, although the optimal concentration or timing of supplementation is unknown. This study aimed to determine the effect of concentration and timing of cysteine supplementation on the integrity and function of cryopreserved ram spermatozoa. Nine ejaculates were collected from three Texel rams then cryopreserved and supplemented with cysteine (0, 0.5, or 1.0 mg/mL) added pre-freeze (PF), post-thaw (PT) or pre-freeze and post-thaw (PF + PT) generating seven treatments: 1) control 0 mg/mL, 2) PF 0.5 mg/mL, 3) PF 1 mg/mL, 4) PT 0.5 mg/mL, 5), PT 1.0 mg/mL, 6) PF + PT 0.5 mg/mL and 7) PF + PT 1.0 mg/mL. Sperm motility, viability, acrosome integrity, ROS production and penetrability through artificial cervical mucus were assessed post-thaw. Cysteine supplementation reduced ROS production which thereby improved spermatozoa motility, viability, acrosome integrity and penetrability (p < 0.001) Sperm integrity for all parameters was greatest in spermatozoa treated PF + PT with 1.0 mg/mL cysteine, although treatment pre-freeze or post-thaw also improved integrity beyond the control. This study has identified that 1.0 mg/mL cysteine is most beneficial and has highlighted the importance of preventing oxidative stress in spermatozoa post-thaw. These finding can help to mitigate the detrimental effect of cryopreservation on spermatozoa and aid the development of cryopreservation protocols in sheep.

Effects of sodium selenite, cysteamine, bacterially synthesized Se-NPs, and cysteamine loaded on Se-NPs on ram sperm cryopreservation.

During the cryopreservation of sperm, the production of highly reactive oxygen species (ROS) can reduce their viability and fertility. However, the addition of antioxidants can help reduce the harmful effects of ROS. One such antioxidant is selenium, which is a co-factor of the glutathione peroxidase enzyme that is effective in scavenging ROS. Cysteamine can also take part in the structure of this enzyme. The use of nanoparticles can be less toxic to cells than their salt form. To this end, researchers synthesized Se-NPs using the streptococcus bacteria and loaded cysteamine onto the synthesized Se-NPs. The biosynthesis of Se-NPs and cysteamine loaded on Se-NPs was confirmed by UV-visible spectroscopy, X-ray diffraction (EDX), Fourier transforms infrared (FTIR) spectroscopy, and Field Emission Scanning Electron Microscope (FE-SEM). For cryopreservation, ram semen samples were diluted, and different concentrations (0, 1, 5, 25, and 125 µg/mL) of cysteamine, Se-NPs, cysteamine loaded on Se-NPs, and sodium selenite were added. An extender containing no supplement was considered as control group. After cooling the semen samples, they were frozen and stored in liquid nitrogen for evaluation. The samples were thawed and analyzed for mobility, viability, membrane and DNA integrity, and sperm abnormalities, as well as malondialdehyde level (MDA) and superoxide dismutase (SOD). The data was processed using SPSS, and a significance level of p < 0.05 was considered. The results of this experiment showed that adding 1 µg/mL of cysteamine loaded on Se-NPs to the diluent significantly increased the motility, viability, and membrane integrity and SOD of spermatozoa compared to the other treatment groups and control group, and reduced the abnormality, apoptosis, and MDA level of spermatozoa in comparison with the other treatment groups and control group (p < 0.05). In conclusion, the addition of cysteamine loaded on Se-NPs was found to improve the quality of ram sperm after cryopreservation.

Improving chilled and frozen buck sperm characteristics by adding melatonin and L-carnitine to the preservation medium.

This study evaluated the effects of melatonin (MLT) and L-carnitine supplementation on sperm quality and antioxidant capacity during chilled and cryopreservation. Twenty-four ejaculates were collected from six Damascus bucks, 4 ejaculates each, from mid-September to mid-October 2022. The pooled semen from each collecting session was divided into 5 equal aliquots after being diluted (1:10) with Tris-citric acid egg yolk extender. The first aliquot served as a control (treatment-free). MLT was added to the second and third aliquots at low and high doses (LD: 4 and HD: 8 µL/mL) (v/v), respectively, while L-carnitine (LC) was added to the fourth and fifth aliquots at the same aforementioned doses. The aliquots were stored at 4°C for 48 h to assess sperm physical and morphological characteristics, alongside lipids peroxidase (LP) production and glutathione peroxidase (GPX) activity. The optimum doses of MLT and LC that showed potential for maintaining sperm characteristics throughout the chilled storage period were further investigated for protecting the spermatozoa after exposure to cryopreservation stress compared to the control. The results showed higher sperm motility (%) in the MLT-HD group, higher (p ≤ .05) sperm viability (%) in the MLT-LD, and both aliquots of LC at T24 hours of chilled preservation. Normal sperm (%) was higher (p ≤ .05) in both LC-LD and MLT-LD groups than other groups, while sperm acrosome integrity (%) was higher (p ≤ .05) in the LC-LD group. Morphological abnormalities (%) were improved (p ≤ .05) in all treated aliquots compared with control. The mean value of GPX activity was higher (p ≤ .05) in both MLT groups, while the concentration of LP increased (p ≤ .05) in the LC-HD or control groups. Furthermore, supplementing buck sperm medium with 4 µL/mL of MLT or LC improved (p < .05) the sperm characteristics and decreased (p < .05) DNA fragmentation index after thawing.