Cryoprotective Medium Research Articles

O-307 Decrease of human sperm hypercondensed chromatin volume after slow freezing revealed by three-dimensional (3D) nuclear morphometry

Abstract Study question Are the 3D morphometry parameters of the human sperm nucleus affected by slow freezing? Summary answer The slow freezing-thawing cycle induces significant alterations on hypercondensed nucleus zone by reducing its volume and splits it into several small zones. What is known already Sperm cryopreservation is an essential technique for infertility care, but it seems to highly affect the genetic and epigenetic integrity of sperm nuclei with poorly identified mechanisms. Moreover, it is known that sperm nuclear alterations may be involved in pregnancy failures. Therefore, sperm nuclear biomarkers are an essential issue to assess the outcome of ART procedures when frozen sperm is used. Thereby, 3D visualization of chromatin is a good way to understand modifications in the organization and composition of sperm chromatin after a slow freezing procedure. Study design, size, duration Ten semen of patients were analyzed (Germetheque biobank) after informed consent. Standard semen parameters, DNA fragmentation, chromatin decondensation, sperm telomere length (STL) and 3D nuclear morphometry were performed before and after slow freezing. Morphometric measurements were performed from a digital representation of the sperm nucleus in 3 dimensions that allow us to measure the volume, flatness and elongation of each sperm nucleus and characterize hypercondensed zones, with live/dead spermatozoa distinction. Participants/materials, setting, methods After addition of cryoprotective medium v/v (Cryosperm®), sperm was frozen with a programmable freezer (NanoDigitcool®). Standards sperm parameters were measured in accordance with WHO guidelines 2021, DNA fragmentation by flow cytometry TUNEL assay, chromatin decondensation by epifluorescent microscopy with Chromomycin A3 and STL using qPCR. 3D nuclear representation was performed by a z-axis image acquisition series of DAPI/PI stained slides with optigrid epifluorescent microscopy. Analyses were carried out using the NucleusJ2.0 software with NODeJ plugin. Main results and the role of chance Freezing induces an alteration of sperm vitality (p < 0.0001) and progressive and total motility (p < 0.001 and p < 0.0001 respectively) and an increase in the percentage of sperm with DNA fragmentation (18.30% fresh vs 31.6% frozen, p < 0.05). We did not observe a modification of the median volume of the sperm nucleus before and after freezing (37.15 [IC = 36.5;38.14] and 37.10 [33.27;37.78] µm3 respectively), as the same for flatness and elongation (2.48 vs. 2.50 a.u. and 2.04 vs. 2.04 a.u. respectively). Nevertheless, five samples showed significant alterations of nucleus volume (p < 0.0001), due to diminution of flatness and more or less elongated. A significant decrease of the total volume of the hypercondensed chromatin zone was observed after freezing (0.94 [0.84;0.99] before vs. 0.61 [0.53;0.69] µm3 after freezing-thawing, p < 0.0001), with an effect size of -1.48 [-2.10; -0.86]. Decrease of total volume of hypercondensed region was measured homogeny in all the samples tested, particularly on live (1.00 vs. 0.68 µm3 respectively, p < 0.0001) compared with dead spermatozoa (0.72 vs. 0.57 µm3, p < 0.05). We showed also a significant increase of the number of hypercondensed zones in each nucleus (p < 0.001), reflecting a split into several parts of the hypercondensed chromatin. Limitations, reasons for caution A 3D-FISH analysis would allow to characterize the hypercondensed chromatin portion, probably as heterochromatin and which we suspect to be the nucleus ring. Moreover, a chromosomes specific marking would allow for the identification of whether whole chromosomes or specific parts of chromosomes (centromeres or telomeres) are affected by this decompaction. Wider implications of the findings This study is the first to make exacts measurements of human sperm nucleus because of a non-decondensing method. We demonstrate that slow freezing affects hypercondensed chromatin probably through a decondensation and modification of the nucleus-specific organization. This new assessment will allow a better proficiency of men fertility preservation techniques. Trial registration number NCT04715828

Monarch butterfly conservation through male germplasm cryopreservation

Monarch butterfly (Danaus plexippus L.) populations have declined in North America. The International Union for Conservation of Nature (IUCN) recently classified the species as endangered, sparking public concern and conservation efforts. Our approach to conservation is through cryopreservation of germinal cells and tissue. The goal of this study was to develop a cryopreservation protocol for monarch spermatozoa to ensure successful long-term storage. Cryopreserved sperm cells would provide a reserve of monarch germplasm, which could be utilized in the event of population loss. In this study, sperm cell bundles collected from male monarch butterflies were cryopreserved in a cryoprotective medium and stored in liquid nitrogen. To determine the post-cryopreservation sperm cell viability, a subsample of preserved sperm bundles were thawed rapidly, and their viability was qualified using a sperm live/dead stain. We are presenting a protocol to preserve and store genetic material and viable sperm bundles of the monarch butterfly. To date, this is the first report of successful cryopreservation of monarch germplasm which sets the foundation for cryostorage and could be extensible to other vulnerable lepidopterans.

FBS-based cryoprotective compositions for effective cryopreservation of gut microbiota and key intestinal microorganisms

ObjectiveThe need for innovative techniques to preserve microbiota for extended periods, while maintaining the species composition and quantitative balance of the bacterial community, is becoming increasingly important. To address this need, we propose an efficient approach to cryopreserve human gut microbiota using a two-component cryoprotective composition comprising fetal bovine serum (FBS) and 5% dimethyl sulfoxide (DMSO). Fetal serum is a commonly utilized component in the freezing media for eukaryotic cells, however, its effects on prokaryotic cells have not been extensively researched.ResultsIn our study, we demonstrated the high efficiency of using a two-component cryoprotective medium, FBS + 5% DMSO, for cryopreservation of human gut microbiota using three different methods. According to the obtained results, the intact donor microbiota was preserved at a level of 85 ± 4% of the initial composition based on fluorescent analysis using the LIVE/DEAD test. No differences in survival were observed when comparing with pure DMSO and FBS media. The photometric measurement method for growth of aerobic bacteria (A. johnsoni), facultative anaerobes (E. coli, E. faecalis), microaerophilic (L. plantarum), and obligate anaerobic bacterial cultures (E. barkeri, B. breve) also demonstrated high viability rates of 94–98% in the two-component protective medium, reaching intact control levels. However, for anaerobic microflora representatives, serum proved to be a more suitable cryoprotectant. Also, we demonstrated that using cryoprotective media with 50–75% FBS content is enough to preserve a significant level of bacterial cell viability, from an economic standpoint.

Optimization of cryoprotectant formulation to enhance the viability of Lactiplantibacillus plantarum NBC99 isolated from human origin

Freeze drying has been well applied in the preparation of high-efficiency viability probiotic powders. However, the process is generally accompanied by probiotic viability deficiency, which poses a problem for further application. In this study, various kinds of cryoprotectant formulations (skim milk, maltodextrin, and sucrose) were tested to enhance the survival of Lactiplantibacillus plantarum NBC99 after freezing and freeze-drying. An I-optimal experimental design-oriented optimization model was presented to optimize the cryoprotective medium, and the highest cell survival was observed with 25% skim milk, 8.71% maltodextrin, and 1.13% sucrose cryoprotectant as the optimum condition. L. plantarum NBC99 has been a good potential strain for the manufacture of an industrial probiotic, and this research has aimed to investigate the long-term protective effects of optimum cryoprotectant formulations on the viability of bacteria. The results showed the potential value of freeze-dried probiotic L. plantarum NBC99 culture for commercialization.

Novel Apoplastic Antifreeze Proteins of Deschampsia antarctica as Enhancer of Common Cell Freezing Media for Cryobanking of Genetic Resources, a Preliminary Study.

Antifreeze proteins (AFPs) are natural biomolecules found in cold-adapted organisms that lower the freezing point of water, allowing survival in icy conditions. These proteins have the potential to improve cryopreservation techniques by enhancing the quality of genetic material postthaw. Deschampsia antarctica, a freezing-tolerant plant, possesses AFPs and is a promising candidate for cryopreservation applications. In this study, we investigated the cryoprotective properties of AFPs from D. antarctica extracts on Atlantic salmon spermatozoa. Apoplastic extracts were used to determine ice recrystallization inhibition (IRI), thermal hysteresis (TH) activities and ice crystal morphology. Spermatozoa were cryopreserved using a standard cryoprotectant medium (C+) and three alternative media supplemented with apoplastic extracts. Flow cytometry was employed to measure plasma membrane integrity (PMI) and mitochondrial membrane potential (MMP) postthaw. Results showed that a low concentration of AFPs (0.05 mg/mL) provided significant IRI activity. Apoplastic extracts from D. antarctica demonstrated a cryoprotective effect on salmon spermatozoa, with PMI comparable to the standard medium. Moreover, samples treated with apoplastic extracts exhibited a higher percentage of cells with high MMP. These findings represent the first and preliminary report that suggests that AFPs derived from apoplastic extracts of D. antarctica have the potential to serve as cryoprotectants and could allow the development of novel freezing media.

Optimizing aquaculture-scale common carp artificial reproduction: a novel approach to sperm cryopreservation using large-volume containers and elevated thawing temperatures

IntroductionThe successful cryopreservation of common carp sperm is crucial for its application in aquaculture and selective breeding programs. This study investigates the efficacy of cryopreserving sperm in large containers (5 mL) with a low dilution rate (1:1) in three different cryoprotective media and thawing in different conditions.MethodsThe developed method utilizes a low-ionic (hypotonic) cryoprotective medium, freezing with a controlled cooling rate, and high-temperature sperm thawing (60°C). The investigation employs a detailed spermatozoon motility assessment.ResultsPost-thaw motility of 32.3% ± 14% and initial curvilinear velocity of 89 ± 20 μm/s across 30 males were observed. Principal component analysis of sperm kinematic characteristics revealed distinct populations of sperm cells exhibiting varying responses to cryopreservation. The developed method achieved successful fertilization comparable to that of the non-frozen control group using sperm from a single cryotube (2.5 mL, approximately 50 * 109 spermatozoa) to fertilize 200 g of eggs (1:120,000 egg:spz).DiscussionThis novel approach demonstrates an effective cryopreservation protocol for common carp sperm in large-volume cryo-containers in combination with low-ionic cryomedia and high thawing temperature, providing methods well-suited for fisheries practices and selective breeding programs. Future studies of the biological properties of different sperm subpopulations in post-thaw sperm samples can contribute to a deeper understanding of sperm biology, improve cryopreservation techniques, and enhance the success rates of assisted reproductive technologies.

Modernization of the cryoprotective medium compoud for intraovarian vitrification of female gametes of Sus scrofa domesticus

Aim. Comprehensive analysis of the morphofunctional state of somatic (cumulus) and germ cells (oocytes) of Sus scrofa domesticus subjected to intraovarian vitrification using silicon dimethylglycerolate (SDMG) are presented.Materials and methods. Fragments of porcine ovaries (FsPO) 15×20 mm in size were gradually kept in cryoprotective agents (CPA) prepared in phosphate-buffered saline (PBS) with 20% fetal bovine serum (FBS): 25 min. in CPA-1 [7.5 % EG (ethylene glycol) with 7.5 % DMSO (dimethyl sulfoxide)] and 15 min. in CPA-2 (15 % EG with 15 % DMSO and 0.5 M sucrose). The composition of the CPA-2 in experimental groups was modified by addition of SDMG (at concentrations of 2 %, 6 %, or 10 %). FsPO were stored in liquid nitrogen. FsPO were devitrified by exposure 1 minute in solution 1 (80 % PBS, 20 % FBS, 0.5 mol/l sucrose) and 5 minutes in solution 2 (80 % PBS, 0.25 mol/l sucrose). The following indicators of cryoresistance of devitrified cumulus-oocyte complexes (COCs) were analyzed: degree of cumulus cells expansion; oocyte morphology and the functional status of lipidome in female gametes (fluorescence intensity of Nile red /lipid droplets complex - FILDs).Results. The addition of SDMG into cryoprotective media reduced the level of denuded oocytes after vitrification. The level of gamete with different degree of cumulus cells expansion (low, medium, high) in the experimental group with 10 % SDMG tended to indicators in the group of native cells. The level of native oocytes with the signs of morphological degeneration (7.7%) had no significant differences with the level of intraovarian vitrified gametes with 10 % SDMG (11 %). The proportion of native oocytes with low FILDs (38.9 %) exceeded the level of oocytes with the above indicator in vitrified oocytes of the control (16.5 %) group and in experimental groups of cells with the addition of 6 % SDMG (4.8 %) and 10 % SDMG (11.8 %, P<0.001).Conclusion. In general, comprehensive monitoring of indicators cryoresistance of COCs in Sus scrofa domesticus subjected to intraovarian vitrification revealed the cryoprotective properties of SDMG. The effects were dose-dependent and were expressed in the stabilization of oocyte-cumulus communication, a decrease in the level of oocytes with the signs of morphological degeneration, and features of the lipidome functioning in intraovarian vitrified female gametes using SDMG at various concentrations.

Open-source hardware- and software-based cryomicroscopy system for investigation of phase transitions in cryobiological research.

The development of inexpensive equipment adapted for the study of a specific biological object is very important for cryobiology. In the presented work, we have proposed a simple system for microscopy utilising open-source platform Arduino. Testing this system showed that it had sufficient sensitivity to determine the physical processes occurring in a cryopreserved sample such as intra- and extracellular water crystallisation and salt eutectic. Utilising this system, we investigated the mechanisms of cryoprotection and cryodamage of testis interstitial cells (ICs) in cryoprotective media, which included cryoprotective agents such as dimethyl sulphoxide (Me2 SO), as well as foetal bovine serum or polymers (dextran, hydroxyethyl starch and polyethylene glycol). It was shown that a serum-/xeno-free medium that included 0.7M Me2 SO and 100mg/mL dextran was able to reduce intracellular water crystallisation in cells, change the structure of extracellular ice, and reduce salt eutectic and recrystallisation. All these effects correlated with better IC survival after cryopreservation in the medium. This medium is potentially less toxic as it has lower concentrations of Me2 SO compared to serum-containing media developed for cryopreservation of testicular cells. This would pave a way for the creation of nontoxic serum-free compositions that does not require removal before use of cryopreserved living cells for laboratory practice or in clinics.

Lipid composition of plasma membranes of rooster sperm (Gallus gallus domesticus) and its dynamics during cryopreservation

The structural features of the plasma membranes of avian sperm make them more sensitive, compared to those of mammals, to low-temperature stress. The qualitative and quantitative composition of membrane lipids can become a determining factor in the development of new effective compositions of cryoprotective media. The purpose of the study was to determine the lipid composition of the plasma membranes of native rooster sperm, the content of carbohydrates and polyols in their cytosol, as well as dynamic changes in the membrane lipidome and cytosol composition under the influence of the cryopreservation protocol, depending on the composition of the cryoprotective medium. The studies were carried out on Rhode Island roosters (n=10), the total and progressive sperm motility and membrane damage were determined. Semen freezing and thawing was carried out using fast protocols. To determine the lipid composition of the plasma membranes of sperm and the composition of their cytosol, a chromatographic analysis method was used. The following were identified in the membranes of native spermatozoa: phospholipids, glycolyllipids and neutral lipids, represented by phosphatidylethalamine, phosphatidylserine, phosphatidylcholine, sphingomyelin and sterol. A change in the ratio between membrane lipids of the inner and outer layers of the plasma membrane of rooster spermatozoa under the influence of the cryopreservation protocol was shown. In native spermftozoa this ratio was 41.2% and 39.4%, respectively, in thawed sperm when using the LCM-control medium – 38.3% and 47.2%, respectively, when using the LCM-T20 medium - 40.7% and 44.5%, respectively. There was a significant decrease, more than 3 times, in the total amount of carbohydrates (fructose, glucose, trehalose) and polyols (glycerol, mannitol, inositol) in the cytosol of frozen/thawed spermatozoa when using the cryoprotective medium LCM-control compared with the values of the native spermatozoa - 0 .1145 mg/ml and 0.0360 mg/ml, respectively. When using the LCM-T20 medium, the change was insignificant and the delta was 5.2%. The effectiveness of using cryoprotective medium LCM-T20 containing trehalose has been proven to maintain the lipid membrane architecture of rooster spermatozoa, the carbohydratepolyol composition of their cytosol and, as a consequence, the morphofunctional usefulness of gametes during the freezing/thawing process.

Changes in kinetic parameters and cytological characteristics of rooster spermatozoa under the influence of technological factors

The problems of fertility reducing of rooster semen in the cycle «native sperm-equilibration-short-term and long-term storage (cryopreservation)» are urgent. The purpose of this study was to determine the effect of different methods of preparation (centrifugation or filtration) of rooster semen on its quality characteristics, depending on the method of removing possible pollutions; to evaluate the change in the composition of the cytosol of spermatozoa of native sperm under the influence of dilution and during short-term storage. Materials and methods. Semen of roosters (n=22) of the Russian white breed was used. Experiment 1: semen was divided into 3 aliquots: I - diluted with synthetic cryoprotective medium LCM in a ratio of 1:1, II - filtered semen diluted with medium (membrane pore diameter 0.2 μm), III - centrifuged (at 3000 rpm in for 10 minutes). Native and frozen/thawed sperm were evaluated in terms of damage to spermatozoa membranes, chromatin, and acrosomes. The composition of carbohydrates and polyols of native spermatozoa was assessed under the influence of dilution and after storage (3 h). The advantage of filtration as a method of technological preparation of semen compared to centrifugation in terms of progressive motility (with rectilinear-translational movement) of sperm (41.0 % versus 27.0 %) and chromatin damage (43.4 % versus 66.4 %) has been shown. The same advantage was observed in frozen/thawed sperm filtered before freezing in terms of progressive motility (25.5 % vs. 5.5 %) and chromatin damage - 16.5 % vs. 33.6 %, respectively. Semen filtration, as a method of technological processing of rooster semen, can be an effective additional step in the preparation of semen for artificial insemination and/or short-term storage. The main component in the composition of the cytosol of native spermatozoa, according to the content of carbohydrates and polyols, was inositol - 73.7 % of ∑ carbohydrates and polyols. The level of inositol decreased during storage by 6.5 times (from 0.030 mg/ml to 0.007 mg/ml). The data obtained let us suppose the role of inositol as the main antioxidant in the cytosol of spermatozoa. Technological factors of storing rooster semen in various modes (short-term at a temperature of 5ºC and long-term at a temperature of -196ºC) have a significant impact on the ratio of sperm cytosol components (carbohydrates and polyols). A significant, 2.5-fold decrease in the relative content of inositol in the cytosol of frozen/thawed spermatozoa, compared with the indicators of native semen, allows us to recommend the introduction of the antioxidant inositol into the composition of cryoprotective media for rooster semen.

Effectiveness of glycerol, DMSO and trehalose in the process of cryopreservation of drone semen from the species apis mellifera

Cryopreservation of economically important or endangered animal species is becoming an increasingly popular practice in the field of reproductive biotechnologies. The mass extinction of honey bee (Apis Mellifera) colonies in the last decade requires the use of new chemical components in cryopreservation media for the storage of male gametes. A number of studies demonstrate conflicting data regarding the success rate and cytotoxicity of glycerol and dimethyl sulfoxide and to a lesser extent Trehalose. Dimethyl sulfoxide is an organosulfur compound with low cryotoxicity which transforms into solids below room temperature and this property makes it suitable for cryoprotectant. The kosmotropic properties of glycerol allow it to create hydrogen bonds with water molecules, making it difficult to form intracellular ice crystals in the cryoprotective media. Due to its high-water retaining properties Trehalose can be used as cryoprotectant, but its effectiveness has not yet been thoroughly researched in relation to the cryopreservation of bee drone spermatozoa. The research aims to compare the effectiveness of each of the specified cryoprotectants regarding the preservation of spermatozoa from Apiss Mellifera drones.

Comparative effectiveness of cryoprotective diluents for rooster seed

The preservation and use of frozen/ thawed male sperm is important for maintaining the biodiversity of poultry and will eventually play an important role in breeding, allowing greater flexibility in breeding programs. This use of thawed semen is advisable only at a high level of its fertilizing ability. Trehalose is a promising natural component in cryoprotective media for rooster semen due to its exceptional ability to neutralize cold stress. The purpose of the study was to determine the effectiveness of using trehalose as part of a medium for cryopreservation of rooster semen based on the Russian development - Leningrad cryoprotective medium (LCM-control). The composition of the LCM-T20 experimental medium with the addition of trehalose at a concentration of 9.5 mM has been developed. The mobility indicators of thawed rooster semen (Rhode Island Red breed, ♂n=10) did not differ significantly depending on the composition of the medium. Significant differences (p <0.05) were obtained in terms of egg fertilization: 86.0% when using the LCM-T20 medium and 79.0% using LCMcontrol and in assessing the state of the vitelline membrane of the egg yolk (p <0.001). The fertilization rate of eggs at a level acceptable for thawed semen of 50% was maintained until the 10th day (20th day of egg collection) from the last insemination when using the LKS-T20 medium. The functional usefulness of thawed sperm was maintained for 15 days (the 25th day of egg collection) from the last insemination when using the LCM-T20 media - egg fertilization was 15.0%, the average number of points of interaction of sperm with the vitelline membrane of the egg yolk was 345.2 pcs/cm2. When using the LCM-control diluent, the functional ability of thawed spermatozoa was absent during the same control periods. The achieved high level of egg fertilization when trehalose was included in the cryoprotective diluent in combination with the method of freezing rooster semen in pellets confirms the feasibility of introducing the LCMT20 diluent into the technology for preserving the genetic diversity of local and indigenous breeds of chickens using in vitro methods.

Bat-derived cells use glucose as a cryoprotectant

Evolution of heterothermy in environments with variable temperatures has allowed bats to survive food scarcity during seasonal climatic extremes by using torpor as a hibernation strategy. The controlled reduction of body temperature and metabolism through complex behavioural and physiological adaptations at organismal, organ, cellular and molecular levels includes the ability of tissues and cells to adapt to temperature alterations. Based on the prediction that cells of different tissues cultured in vitro would differ in their ability to withstand freezing and thawing of the medium, we determined the survival rate of bat-derived cells following exposure to −20 °C for 24 h in media with no cryoprotective agents or medium supplemented by glucose in concentration range 0–3333 mM. Cell survival rates were determined in relation to availability of glucose in the medium, organ origin, cell concentration and bat species. In general, increased glucose helped cells survive at sub-zero temperatures, though concentrations up to 80-fold higher than those found in chiropterans were needed. However, cells in glucose-free phosphate buffered saline also survived, suggesting that other mechanisms may be contributing to cell survival at low temperatures. Highest in vitro viability was observed in nervus olfactorius-derived cell cultures, with high survival rates and rapid re-growth under optimal conditions after exposure to −20 °C. Kidney cells from different bat species showed comparable overall survival rate patterns, though smaller chiropteran species appeared to utilise lower glucose levels as a cryoprotectant than larger species. Our in vitro data provide evidence that cells of heterothermic bats can survive sub-zero temperatures and that higher glucose levels in important tissues significantly improve hibernation survival at extremely low temperatures.

A model for preservation of thymocyte-depleted thymus.

DiGeorge syndrome is a disorder caused by a microdeletion on the long arm of chromosome 22. Approximately 1% of patients diagnosed with DiGeorge syndrome may have an absence of a functional thymus, which characterizes the complete form of the syndrome. These patients require urgent treatment to reconstitute T cell immunity. Thymus transplantation is a promising investigational procedure for reconstitution of thymic function in infants with congenital athymia. Here, we demonstrate a possible optimization of the preparation of thymus slices for transplantation through prior depletion of thymocytes and leukocyte cell lineages followed by cryopreservation with cryoprotective media (5% dextran FP 40, 5% Me2SO, and 5% FBS) while preserving tissue architecture. Thymus fragments were stored in liquid nitrogen at -196°C for 30 days or one year. The tissue architecture of the fragments was preserved, including the distinction between medullary thymic epithelial cells (TECs), cortical TECs, and Hassall bodies. Moreover, depleted thymus fragments cryopreserved for one year were recolonized by intrathymic injections of 3×106 thymocytes per mL, demonstrating the capability of these fragments to support T cell development. Thus, this technique opens up the possibility of freezing and storing large volumes of thymus tissue for immediate transplantation into patients with DiGeorge syndrome or atypical (Omenn-like) phenotype.

Machine learning and hypothesis driven optimization of bull semen cryopreservation media

Cryopreservation provides a critical tool for dairy herd genetics management. Due to widely varying inter- and within-bull post thaw fertility, recent research on cryoprotectant extender medium has not dramatically improved suboptimal post-thaw recovery in industry. This progress is stymied by the interactions between samples and the many components of extender media and is often compounded by industry irrelevant sample sizes. To address these challenges, here we demonstrate blank-slate optimization of bull sperm cryopreservation media by supervised machine learning. We considered two supervised learning models: artificial neural networks and Gaussian process regression (GPR). Eleven media components and initial concentrations were identified from publications in bull semen cryopreservation, and an initial 200 extender-post-thaw motility pairs were used to train and 32 extender-post-thaw motility pairs to test the machine learning algorithms. The median post-thaw motility after coupling differential evolution with GPR the increased from 52.6 ± 6.9% to 68.3 ± 6.0% at generations 7 and 17 respectively, with several media performing dramatically better than control media counterparts. This is the first study in which machine learning was used to determine the best combination of constituents to optimize bull sperm cryopreservation media, and provides a template for optimization in other cell types.

Influence of Technological Stages of Preparation of Rooster Semen for Short-Term and Long-Term Storage on Its Quality Characteristics

There is a problem of declining quality of rooster semen in the "native semen-equilibrium-short-term and long-term storage (cryopreservation)" cycle. The aim of this study was to determine the effects of various methods of preparing rooster semen on its qualitative characteristics, taking into account the method of removing possible contaminants (centrifugation or filtration), and to evaluate the change in the composition of the cytosol of the spermatozoon of the native semen, during equilibration of the diluted semen and during short-term storage. In this study, semen from roosters (n = 22) of the Russian White breed was used. Experiment 1: semen was divided into 3 aliquots: I-was diluted with synthetic cryoprotective medium (1:1 with LCM control, II-was filtered (membrane pore Ø 0.2 μm), and III-was centrifugated (at 3000 rpm for 10 min). Native and frozen/thawed semen was evaluated. Experiment 2: the composition of carbohydrates and polyols of the spermatozoa of native semen was evaluated during equilibration and after storage (3 h). The results of Experiment 1 showed an advantage in the quality of filtered semen compared to centrifuged in terms of progressive motility (41.0% vs. 27.0%) and chromatin integrity (56.6% vs. 33.6%). Results from frozen/thawed samples of filtered semen compared to centrifuged in terms of progressive motility were 25.5% vs. 5.5%, respectively, and in terms of chromatin integrity-83.5% vs. 64.4%, respectively. The results of Experiment 2 showed the main component in the composition of the native spermatozoa cytosol in assessing the content of carbohydrates and polyols was inositol-75.6%. The content of inositol decreased during storage by 6.5 times (from 0.030 mg/mL to 0.007 mg/mL), proposing the role of inositol as the main antioxidant in the cytosol of spermatozoa, which makes it biologically justified to introduce inositol into the composition of synthetic diluents, including cryoprotective ones.

Melatonin Supplementation for the Cryopreservation of Canine Sperm.

Antioxidants can be used in sperm cryopreservation protocols to reduce oxidative stress that occurs due to the cryopreservation process. The aim of this study was to evaluate the effects of melatonin supplementation on quality and oxidative stress parameters in cryopreserved canine sperm. Eighteen sperm ejaculates were collected from 6 Frenchie Bulldog males (3 collections per male). Sperm motility parameters, membrane integrity, and sperm morphology were analyzed before the cryopreservation process. The extender used in cryopreservation was composed of Tris-egg yolk and ethylene glycol 5% was added as a cryoprotectant. The cryoprotective medium was supplemented with 1.0, 1.5, 2.0, 2.5, and 3.0 mM melatonin, and the control group (without melatonin). Post-thaw sperm was evaluated as described for fresh sperm and oxidative stress parameters (lipid peroxidation, catalase, and superoxide dismutase). Post-thaw sperm motility parameters, membrane integrity, sperm morphology, and oxidative stress parameters did not differ (p > 0.05) among the control group and samples supplemented with melatonin. The results of this study showed that melatonin supplementation had no positive or negative effect on the parameters evaluated. Thus, it is suggested that different concentrations of melatonin be tested to assess its effectiveness as an antioxidant in the cryopreservation process in canine sperm.

Validation of a SARS-CoV-2 RT-PCR assay: a requirement to evaluate viral contamination in human semen

Research questionIs it possible to validate an accurate and reliable method for direct detection of SARS-CoV-2 by reverse transcription polymerase chain reaction (RT-PCR) in human semen fractions? DesignThis qualitative improvement study aimed to provide a prospective validation of SARS-CoV-2 detection in male semen. The SARS-CoV-2 genome was detected by multiplex real-time RT-PCR on patient samples that underwent routine semen analyses for infertility at the Center for Reproductive Medicine at the University Hospital of Clermont-Ferrand. Samples comprised surplus semen collected for treatment with assisted reproductive technology. Seminal fluid and spermatozoa fractions were isolated with density gradient centrifugation and cryopreserved. Positive samples were prepared with a standard of inactivated SARS-CoV-2 particles. ResultsThe analytical method was validated in both seminal fluid and spermatozoa fractions. In both semen fractions, the assay was repeatable, reproducible and showed high sensitivity with a limit of detection of 0.33 SARS-CoV-2 genome copies/µl. The limit of quantification was 1 copy of the SARS-CoV-2 genome/µl. The method was effective regardless of semen quality (normal and altered sperm parameters), number of spermatozoa or the cryoprotectant media used to freeze spermatozoa. ConclusionThis validated RT-PCR assay provided accurate and reliable screening of SARS-CoV-2 in seminal fluid and spermatozoa fractions. This method is essential to ensure protection against viral contamination in the cryobanking process.

CRYOPRESERVATION OF REPRODUCTIVE CELLS OF MALE RUSSIAN STURGEON

Currently, cryopreservation of sperm is recognized as one of the promising ways to preserve the genetic diversity of fish, not only rare and endangered species, but also aquaculture objects. Scientific research was carried out in the scientific research center "Fisheries" of the S. Seifullin Kazakh Agro Technical University and the JSC "Republican Center for livestock breeding "Asyl Tulik" in 2022. The purpose of the research was to study methods of cryopreservation of Russian sturgeon sperm using cryoprotectors using dimethyl sulfide oxide with a concentration of 5% and 10% and methanol with a concentration of 3% and 8%. In the course of the research, generally accepted methods for assessing and freezing the sperm of the studied object were used. During the experimental work, the method of freezing the reproductive cells of the Russian sturgeon with a 15-minute exposure to -21°C in a box with subsequent immersion in a Dewar vessel was studied. The survival and lifetime of spermatozoa after defrosting were also studied. According to the results of the assessment of the quality of defrosted sperm of the Russian sturgeon, it was found that the most effective cryoprotective medium for cryopreservation is based on methanol with a concentration of 8%. The number of spermatozoa with translational movements in the three groups ranged from 23.8% to 31.2%. At the same time, the lifetime of spermatozoa in this cryoprotective medium was 118s. Methanol with a concentration of 8% provides the best resistance to oxidative stress experienced by cells during freezing. The results of the research make it possible to create a cryobank of the Russian sturgeon gene pool at fish hatcheries to preserve genetic diversity.

High sperm concentration during cryopreservation decreases post-thaw motility percentage without compromising in vitro fertilization outcomes in common carp

To avoid mechanical compression of spermatozoa during the cryopreservation, a high dilution ratio with the cryoprotective medium is applied. Due to this, the sample volume increases, which entails increased cryobank capacity. Moreover, the high volume of cryomedias does not allow to fertilize of large volumes of eggs in practical artificial reproduction of common carp aquaculture. The current study demonstrated that reasonable lowering of dilution rates might still be effective for carp sperm cryobanking —elevation of spermatozoa concentration up to 13 × 109 spz mL−1 resulted in a significant decrease in post-thaw sperm motility percentage to 20% compared with 39% in 0.5 × 109 spz mL−1. Nevertheless, no significant differences in sperm kinetic parameters (VCL and LIN) were found in this case. The fertilization outcome (embryo development and hatching rates) was similar after applying thawed sperm samples with “optimal” (2.2–2.4 × 109 spz mL−1) and “sub-optimal” (11.0–13.0 × 109 spz mL−1) concentrations (sperm/egg ratio at fertilization was in the range of 0.3–4.5 × 105 spz/egg). Thus, applying a low dilution rate such as one part of sperm to one of the cryoprotective mediums is favorable for decreasing cryo-storage space and the sperm volume needed to fertilize big egg numbers. The experiment also shows that the 4.5 × 105 spz/egg ratio is not sufficient for good fertilization, and it is necessary to use higher sperm concentrations per egg or improve the method of fertilization.