Ultrastructural examination of cryodamage in Paracentrotus lividus eggs during cryopreservation.

This study examinates the challenges of cryopreserving sea urchin (Paracentrotus lividus) eggs, a task hindered by factors like low membrane permeability and high sensitivity to cryoprotective agents (CPAs). While successful cryopreservation has been achieved for some marine invertebrates, eggs remain problematic due to their unique characteristics. The study explores the impact of various CPAs and cryopreservation techniques on sea urchin eggs, employing scanning and transmission electron microscopy to analyze cellular damage. The findings reveal that exposure to low CPA concentrations (0.5 M) did not induce significant damage to eggs. However, high concentrations (3 M) proved highly detrimental. Every cryopreservation approach investigated in this study resulted in irreversible damage to the sea urchin eggs, rendering them nonviable for future use. The research sheds light on the importance of understanding the structural alterations induced by CPAs and cryopreservation methods. This knowledge is essential for refining cryopreservation methods, potentially paving the way for successful preservation of these challenging cells.

Testing of arctic insect hemolymph as a secondary agent in applied cryopreservation.

BACKGROUND: Cold hardiness of insects from extremely cold regions is based on a principle of natural cryoprotection, which is associated with physiological mechanisms provided by cryoprotectants. OBJECTIVE: Since arctic cold-hardy insects are producers of highly effective cryoprotectants, in this study, the hemolymph of Aporia crataegi L. and Upis ceramboides L. from an extremely cold area (Yakutia) was tested as a secondary component of cryoprotective agents (CPA) for cryopreservation (-80 degree C) of human peripheral blood lymphocytes and skin fibroblasts. MATERIALS AND METHODS: Lymphocytes and skin fibroblasts were treated with various combinations of DMSO and hemolymph extract and step-wise cooled to -80 degree C. Post-cryopreservation cell viability was assessed by vital staining and morphological appearance. RESULTS: Viability was higher when cells were frozen with a mixture containing DMSO and Upis ceramboides hemolymph compared to the cells frozen in DMSO, while cells frozen with DMSO and Aporia crataegi hemolymph did not survive. The fact that hemolymph of not every cold-resistant insect can be used as a secondary agent along with DMSO indicates that only a unique combination of hemolymph components and its compatibility with cells might result in a positive effect. CONCLUSION: Although the use of insect hemolymph as a complementary agent in applied cryopreservation is a problem in terms of practical application, such studies could initiate new trends in the search for the most successful hemolymph-like cryoprotectant systems. https://doi.org/10.54680/fr24210110712.

A novel vitrified cryopreservation approach with stem cell-laden hydrogel microcapsules.

BACKGROUND: Stem cell-laden hydrogel microcapsules construction is important for a wide application in tissue engineering and cell-based medicine, such as building an ideal immune barrier. Challenges are emerging for effectively storing such microcapsules by cryopreservation, and a large proportion of research has been on the cryopreservation of single cells encapsulated into microcapsules without a core-shell structure. OBJECTIVE: To achieve the effective cryopreservation of stem cell-laden hydrogel microcapsules with a core-shell structure. MATERIALS AND METHODS: A novel core-shell alginate hydrogel encapsulation method was used to produce mesenchymal stem cell-laden microcapsules by microfluidic technique. RESULTS: This microcapsule could inhibit ice formation to achieve vitreous cryopreservation with a low concentration (2 M) of penetrating cryoprotectants. CONCLUSION: Cell laden hydrogel microcapsules may have the potential to be the basis of a new strategy of cell cryopreservation and applications. https://doi.org/10.54680/fr24210110212.

Targeted development and optimization of small-molecule ice recrystallization inhibitors (IRIs) for the cryopreservation of biological systems.

Despite the routine use of cryopreservation for the storage of biological materials, its outcomes are often sub-optimal (including reduced post-thaw viability, recovery, and functionality) due to the damage caused by uncontrolled ice growth. Traditional cryoprotective agents (CPAs), including dimethyl sulfoxide (DMSO), fail to prevent damage caused by ice growth and concerns over CPA cytotoxicity have fostered an increased interest in developing improved CPAs and cryoprotection strategies. The inhibition of ice recrystallization by natural antifreeze (glyco)proteins [AF(G)Ps] to improve cryopreservation outcomes has been examined; however, the ice binding properties of these substances and their challenging large-scale production make them poor CPA candidates. Therefore, the development and deployment of biocompatible, small-molecule ice recrystallization inhibitors (IRIs) for use as CPAs is a worthwhile objective. Extensive structure-activity relationship studies on AF(G)Ps revealed that simple carbohydrate derivatives could inhibit ice recrystallization. It was later discovered that this activity could be fine-tuned by delicately balancing the molecule's hydrophobicity and hydrophilicity. Current generation small-molecule IRIs have been meticulously designed to avoid binding to the surface of ice and subsequent biological testing (for both cytotoxicity and cryopreservation efficacy) has demonstrated significant improvements to the cryopreservation outcomes of several cell types. However, an individualized cell-specific approach for the simultaneous assessment of multiple cryopreservation outcomes is necessary to realize the full potential of IRIs as CPAs. This article provides a detailed overview of the development of small-molecule carbohydrate-based IRIs and highlights the crucial cell-specific biological considerations that must be taken into account when assessing cryopreservation outcomes. https://doi.org/10.54680/fr24210110112.

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.

Vitrification of immature oocytes in pigs.

Cryopreservation of oocytes is an important technology for the in vitro gene banking of female germplasm. Although slow freezing is not feasible, porcine oocytes survive vitrification at high rates. Cryopreservation at the germinal vesicle stage appears to be more advantageous than that at the metaphase-II stage. Several factors are considered to affect the success of vitrification and subsequent utilization of immature porcine oocytes such as the device, the protocols for cryoprotectant application, warming, and the post-warming culture. Although live piglets could be obtained from vitrified immature oocytes, their competence to develop to the blastocyst stage is still reduced compared to their non-vitrified counterparts, indicating that there is room for further improvement. Vitrified oocytes suffer various types of damage and alteration which may reduce their developmental ability. Some of these can recover to some extent during subsequent culture, such as the damage of the cytoskeleton and mitochondria. Others such as premature nuclear progression, DNA damage and epigenetic alterations will require further research to be clarified and addressed. To date, the practical application of oocyte vitrification in pigs has been confined to the gene banking of a few native breeds.

Cryopreservation of cerebrospinal fluid cells preserves the transcriptional landscape for single-cell analysis.

Cerebrospinal fluid (CSF) matrix biomarkers have become increasingly valuable surrogate markers of neuropsychiatric diseases in research and clinical practice. In contrast, CSF cells have been rarely investigated due to their relative scarcity and fragility, and lack of common collection and cryopreservation protocols, with limited exceptions for neurooncology and primary immune-based diseases like multiple sclerosis. the advent of a microfluidics-based multi-omics approach to studying individual cells has allowed for the study of cellular phenotyping, intracellular dynamics, and intercellular relationships that provide multidimensionality unable to be obtained through acellular fluid-phase analyses. challenges to cell-based research include site-to-site differences in handling, storage, and thawing methods, which can lead to inaccuracy and inter-assay variability. In the present study, we performed single-cell RNA sequencing (10x Genomics) on fresh or previously cryopreserved human CSF samples from three alternative cryopreservation methods: Fetal Bovine Serum with Dimethyl sulfoxide (FBS/DMSO), FBS/DMSO after a DNase step (a step often included in epigenetic studies), and cryopreservation using commercially available Recovery© media. In comparing relative differences between fresh and cryopreserved samples, we found little effect of the cryopreservation method on being able to resolve donor-linked cell type proportions, markers of cellular stress, and overall gene expression at the single-cell level, whereas donor-specific differences were readily discernable. We further demonstrate the compatibility of fresh and cryopreserved CSF immune cell sequencing using biologically relevant sexually dimorphic gene expression differences by donor. Our findings support the utility and interchangeability of FBS/DMSO and Recovery cryopreservation with fresh sample analysis, providing a methodological grounding that will enable researchers to further expand our understanding of the CSF immune cell contributions to neurological and psychiatric disease.

Post-thaw quality of boar spermatozoa is affected by ejaculate fractions and extenders.

The aim of this study was to investigate the effect of different extenders on the post-thaw (PT) quality of sperm originating from the sperm-rich fraction (SRF) and post-sperm-rich fraction (PSRF) of boar ejaculate. Motility and velocity parameters, analyzed using a computer-assisted semen analysis (CASA) system, and membrane integrity parameters were markedly higher in frozen-thawed (FT) spermatozoa of the SRF in both the Belstville Thawing Solution (BTS) and Androhep Plus (AHP) extenders, irrespective of the post-thaw (PT) storage time. Furthermore, reduced cryo-survival was more marked in FT spermatozoa of the PSRF in both extenders following storage for 60 min. It was found that the SRF-stored samples in the AHP extender for 60 min exhibited significantly higher percentages of spermatozoa with total motility, mitochondrial function and acrosome integrity than those stored in the BTS extender. The findings of this study confirm that components of the ejaculate fractions and extender have varying effects on the cryo-survival of boar spermatozoa.

Adult Stem Cells Freezing Processes and Cryopreservation Protocols.

The development of simple but effective storage protocols for adult stem cells will greatly enhance their use and utility in tissue-engineering applications. Cryopreservation has shown the most promise but is a fairly complex process, necessitating the use of chemicals called cryoprotective agents (CPAs), freezing equipment, and obviously, storage in liquid nitrogen. The purpose of this chapter is to present a general overview of cryopreservation storage techniques and the optimal protocols/results obtained in our laboratory for long-term storage of adult stem cells using freezing storage.

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.

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.

Dog sperm cryopreservation using cryovials and different dilution steps.

BACKGROUND: The conventional sperm freezing method for dog sperm is with straws and includes two-step dilution and a long equilibration time. OBJECTIVE: To develop a more efficient freezing method using cryovials. MATERIALS AND METHODS: Three freezing protocols using cryovials (0.5 mL) were conducted with dog spermatozoa at 1 x 108 sperm/mL: Group 1 spermatozoa were cooled in cryovials and extender 1 (E1) and extender 2 (E1 +1 M glycerol) at 4 degree C for 50 min and then frozen over LN2 for 20 min; Group 2 sperm was cooled and frozen in cryovials with a mixture of E1 and E2 (1:1) in a deep freezer (-80 degree C) for 30 min; Group 3 sperm in cryovials and E1 were cooled at 4 degree C for 20 min, cooled for an additional 20 min after addition of E2 (E1:E2, 1:1), and then frozen using LN2/ vapour for 20 min. The control (Group 4) consisted of spermatozoa in straws being frozen using the conventional freezing method using two-step dilution. All groups were plunged and stored in LN2 after freezing and their functional performance and gene expression determined. RESULTS: Progressive motility and acrosome integrity were highest (P < 0.05) in Groups 2, 3 and 4 (only acrosome integrity). Viability in Group 3 was significantly better that in the other Groups, and the reactive oxygen species (ROS) level and phosphatidylserine (PS) translocation index were significantly lower in Group 2 than the other Groups. The expression of sperm mitochondria-associated cysteine-rich protein (SMCP) and anti-apoptotic B-cell lymphoma 2 (BCL2) genes was highest (P < 0.05) in Group 2 and the expression of pro-apoptotic Bcl2-associated X protein (BAX) was lowest (P < 0.05) in Group 4. CONCLUSION: The sperm frozen using cryovials, one step dilution and the deep freezer (Group 2) proved to be a simple and suitable cryopreservation method for dog sperm. https://doi.org/10.54680/fr24110110312.

The effect of different concentrations of laminarin on the quality of cryopreserved ram semen.

BACKGROUND: Increasingly, sheep breeders are using artificial insemination to produce lambs, so finding methods that preserve ram sperm can be useful. OBJECTIVE: To determine the protective effects of different concentrations of laminarin on ram sperm motility, viability, abnormalities, membrane, and DNA integrity, superoxide dismutase enzyme (SOD) activity, and malondialdehyde (MDA) production after freeze-thawing. MATERIALS AND METHODS: The ejaculates of four rams were collected and stored at 35 degree C. Semen samples were diluted with a tris-base extender containing 100, 200, 400, and 800 ug/mL of laminarin and a control extender containing no laminarin, then frozen in liquid nitrogen after 4 h in the refrigerator. RESULTS: In the treatment of frozen-thawed spermatozoa with 800 ug/mL laminarin, motility, viability, membrane integrity, and DNA integrity were significantly higher than in the control. In spermatozoa that were exposed to 800 ug/mL laminarin after thawing, MDA production was significantly lower than in the control group. The percentage of abnormal spermatozoa in 800 ug/mL laminarin was significantly lower than that in the control. CONCLUSION: The addition of 800 ug/mL laminarin to the freezing extender increases motility, viability, SOD activity, and plasma membrane integrity, while reducing abnormality and MDA production in freeze-thawed ram semen. https://doi.org/10.54680/fr24110110812.

The comet assay as a method for assessing dna damage in cryopreserved samples.

The preservation of the nuclear genome's integrity is paramount for the viability and overall health of cells, tissues, and organisms. DNA, being susceptible to damage under physiological conditions and vulnerable to both endogenous and environmental factors, faces constant threats. To assess DNA damage and repair within individual eukaryotic cells, the comet assay presents itself as a versatile, gel electrophoresis-based, relatively simple, and highly sensitive method. Originally designed to monitor DNA damage and repair within populations of mammalian cells, the comet assay has now found applications across diverse domains, including yeast, protozoa, plants, and invertebrates. This technique has proven invaluable in cryopreservation studies, serving as a valuable adjunct for determining suitable cryopreservation protocols. These protocols encompass choices related to cryoprotectants, sample preparation, as well as storage conditions in terms of time and temperature. In the realm of animal cryopreservation research, the comet assay stands as a gold-standard method for assessing DNA integrity. Nevertheless, when applied in plant-oriented investigations, additional efforts are essential due to the distinct nature of plant cells and associated technical challenges. This review elucidates the fundamental principles underlying the comet assay, discusses its current iterations, and delineates its applications in the cryopreservation of both animal and plant specimens. Moreover, we delve into the primary challenges confronting the comet assay's utility as a monitoring tool in the context of plant sample cryopreservation. https://doi.org/10.54680/fr24110110112.

Mesenchymal Stromal Cells Derived from Canine Adipose Tissue: Evaluation of the Effect of Different Shipping Vehicles Used for Clinical Administration.

Mesenchymal Stromal Cells (MSCs)-based therapies are rapidly gaining interest in veterinary medicine. Cellular therapy represents a new challenge for practitioners and requires precise coordination between the cell processing laboratory and the veterinary clinic. Cryopreservation is the best method to provide fast, in-time, and long-distance delivery of cells for therapeutic applications. However, potentially toxic cryoprotectants and xenobiotic products make the direct administration of cells impracticable for patients. Alternatively, the cells may be resuspended in a ready-to-use vehicle and shipped to the veterinary clinic. In this study, two nutrient-poor vehicles (physiologic saline and ringer lactate solutions) and two nutrient-rich vehicles (the releasate derived from autologous Platelet Poor Plasma and Platelet Rich Plasma) were tested on adipose tissue-derived canine MSCs (AD-MSCs). AD-MSCs stored for 2, 4, or 24 h in the different media were compared regarding mortality, metabolic activity, and replicative capacity. Furthermore, antioxidant activity and the pattern of expression of genes related to AD-MSCs function were performed following 24 h of storage. The results showed that all the different vehicles preserve cell vitality and replication following short-term storage. In long-term storage, the vehicle and cell density affect cell vitality, proliferation, and gene expression (CCL-2, CXCR-4, and TSG-6). Nutrient-rich vehicles seem better suited to preserve cell functionalities in this contest.

Metabolites assay offers potential solution to improve the rooster semen cryopreservation.

Semen cryopreservation represents a promising technology utilized for preserving high-quality chicken varieties in husbandry practices. However, the efficacy of this methodology is significantly impeded by the diminished quality of sperm. Metabolites, as the end products of metabolic reactions, serve as indicators of biological processes and offer insights into physiological conditions. In this study, we investigaged the sperm quality and alteration in metabolic profiles during the cryopreservation of Longyou Partridge Chicken semen. Following artificial semen collection, four groups of semen samples were established based on four points of the cryopreservation process (Ⅰ, fresh semen; Ⅱ, semen added extender and chilled at 4 °C for 30 min; Ⅲ, semen added cryoprotectants; Ⅳ, semen gradient freezed and stored in liquid nitrogen). Semen cryopreservation has a negative effect on the percentage of sperm in a straight-line trajectory (LIN), has no significant effect on total motile sperms (TM) or the proportion of sperm with typical morphology (NM). Metabolites were identified using LC-MS technique and analyses including Principal Component Analysis (PCA), Orthogonal Partial Least Squares Discriminant Analysis (OPLS-DA), Univariate statistical analysis, and the Kyoto Encyclopedia of Genes and Genomes (KEGG) database were employed to identify metabolites. A total of 2471 metabolites had been identified, with the majority of the list being made up of amino acids and their metabolites as well as benzene and substituted derivatives. Group II exhibits 882 metabolites with significantly elevated abundance relative to Group I, alongside 37 metabolites displaying decreased abundance. In Group III, 836 metabolites demonstrate notably augmented abundance compared to Group II, while 87 metabolites exhibit reduced abundance. Furthermore, Group IV showcases 513 metabolites with markedly heightened abundance in comparison to Group III, and 396 metabolites with decreased abundance. Specific metabolites such as 5-Hydroxylysine, Phosphocholine, and alpha-d-glucose-6-phosphate exhibited a progressive decline during the cryopreservation process, correlating with either dilution and chilling, cryoprotectant addition, or freezing. In conclusion, our investigation systematically examined the changes of seminal metabolome and sperm quality throughout the cryopreservation process of rooster semen.

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.

Enhancement potential of Moringa oleifera leaves extract on buffalo bull cryopreserved semen quality and fertilization capacity.

The aim of the present study is to evaluate the effect of Moringa oleifera leave extract (MOLE) on buffalo bull cryopreserved semen quality and fertility. Sixty ejaculates were collected from ten fertile buffalo bulls on a weekly basis for 6 weeks (n = 10 bulls & n = 60) then semen samples were pooled and divided into five groups. The semen of the control group was without additives. The semen of other groups was supplemented with MOLE at doses of 200, 400, 600 and 800 µg/ mL, respectively. One hundred thirty multiparous buffaloes were artificially inseminated with semen supplemented without or with MOLE at dose of 600 µg/ mL. Inclusion of MOLE in semen extender at dose 600 µg/ mL significantly elevated the total motility, progressive motility, membrane integrity and fertilization capacity of the post-thawed spermatozoa, as well as the total antioxidant capacity. However, it significantly decreased acrosomal defects of spermatozoa, and the concentration of malondialdehyde. This study indicated that inclusion of MOLE to semen extender improved the quality and fertility of the post-thawed buffalo bulls' semen through enhancing the activities of the antioxidant enzyme system and decreasing cryodamage of the buffalo bull spermatozoa.

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.

Polyvinyl alcohol addition to freezing extender can improve the post-thaw quality, longevity and in vitro fertility of ram epididymal spermatozoa.

Recovering and cryopreserving epididymal spermatozoa are suitable methods for preserving the genetic potential of livestock and endangered species. Regarding encouraging reports on the use of polyvinyl alcohol (PVA) in cryopreserving various cell types, we conducted this study to examine the impact of PVA on the post-thaw quality, longevity, and in vitro fertility of ram epididymal sperm. In the first experiment, ram epididymal spermatozoa were frozen in extenders containing 6 % glycerol and 0, 0.5, 1, 2, 5, 10, or 15 mg/ml of PVA. Polyvinyl alcohol at concentrations of 0.5, 1, and 2 mg/ml improved the motility and functional membrane integrity (FMI) of the sperm compared with the control group (P < 0.05). In the second experiment, we investigated whether PVA could partially substitute glycerol in the freezing extender. PVA was added at 0, 0.5, 1, and 2 mg/ml to the extenders containing 1 % or 2 % glycerol. After thawing, the sperm motility parameters of the group containing 1 mg/ml PVA and 2 % glycerol were significantly higher than those of the un-supplemented groups (P < 0.05). In the third experiment, the effect of PVA on the post-thaw sperm longevity were examined. Sperm were frozen in 3 extenders: one containing 6 % glycerol and 1 mg/ml PVA (Gly6P1), another containing 2 % glycerol and 1 mg/ml PVA (Gly2P1), and a control extender with 6 % glycerol. After thawing, the quality of the sperm was evaluated. Sperm were then diluted in human tubal fluid (HTF) and incubated at 37 °C for 3 h. Afterwards, the quality of the sperm was evaluated once more. The presence of PVA in the freezing extender improved motility parameters and FMI. Additionally, PVA-containing groups had lower proportions of capacitated and acrosome reacted sperm compared with the control group (P < 0.05). The Gly6P1 group performed better than the other two groups (P < 0.05). In the fourth experiment, sperm from the Gly6P1 and Control groups were used in the IVF process immediately after thawing (T0) and after a 3-h incubation at 37 °C in HTF (T3). Cleavage, blastocyst and hatching rates in both groups were similar at T0, but they were lower in the Control group at T3 (P < 0.05). In conclusion, PVA as an additive to the freezing extender significantly improves post-thaw motility, viability, acrosome integrity, longevity, and fertile lifespan of ram epididymal spermatozoa.