Liquid Nitrogen Vapor Research Articles

Sperm Cryopreservation in Canaries to Protect Endangered Songbird Species: Comparison of Different Cryoprotectants.

Sperm cryopreservation is a rather complex process that needs to be adapted to wild and domestic bird species to ensure adequate efficiency. This study aimed to determine the usability of different cryoprotectants in the cryopreservation of Gloster canary sperm. For this purpose, sperm samples were collected from 12 2-year-old male Gloster canaries three times a week using cloacal massage for 4 weeks. After individual evaluation, sperm samples from the canaries were combined. Mixed sperm were divided into two groups in the study. Overall, 8% dimethyl sulfoxide (DMSO) and ethylene glycol (EG) were used as cryoprotectants. Sperm samples were drawn into straws after adding Dulbecco's Modified Eagle Medium (DMEM) extender with high glucose ratio and two different cryoprotectants in a 1:1 ratio and frozen to -80°C with liquid nitrogen vapour and then stored in liquid nitrogen at -196°C. Frozen-thawed semen samples were evaluated regarding motility, vitality, plasma membrane integrity (hypoosmotic swelling test [HOST]), density and abnormal spermatozoa rate. The highest motility value after freezing and thawing was determined in the EG group with 31.667%±4.773%. In addition, vitality, plasma membrane integrity and normal sperm morphology were statistically significantly higher in the EG-frozen group, whereas head and tail abnormality was low (p<0.05). This study determined that a DMEM extender containing 8% EG was more advantageous than a DMEM containing DMSO regarding spermatological parameters and could be used for long-term storage of canary sperm.

Investigation of the efficacy of different cryoprotectants in the freezing of testicular tissue and epididymal sperm: Spermatological parameters, tissue viability and PARP-1 gene expression

The presented study covers testicular tissue and epididymal spermatozoa cryopreservation processes in bulls and aims to investigate the effects of these applications on spermatological parameters, cell viability in testicular tissue, and the expression of the PARP-1 gene, a DNA repair enzyme. Testes of 20 bulls over 2 years old, slaughtered in a slaughterhouse, were used in the study. After spermatological evaluations, the semen obtained from the cauda epididymis was frozen in liquid nitrogen vapor according to the straw method and stored in liquid nitrogen (−196 °C). Testicular tissue pieces obtained from the testicles were frozen by the slow freezing method in cryotubes in diluents containing Dimethylsulfoxide (DMSO) and Ethylene Glycol (EG) cryoprotectants and stored in liquid nitrogen (−196 °C). The total motility (TM) (85.89 ± 12.83 %), progressive motility (PM) (54.02 ± 15.77 %), and kinematic parameter values of fresh sperm were significantly higher compared to the TM (57.62 ± 13.13 %), PM (29.60 ± 10.76 %), and kinematic parameter values after thawing (P < 0.05). Significant decreases in plasma membrane integrity (PMI) and viability and an increase in chromatin condensation and morphological disorders in the head, middle part, and tail regions were observed in post-thaw semen samples (P < 0.05). When the effects of DMSO and EG on cell viability after thaw in frozen testicular tissue were evaluated, it was observed that the cell viability values of testicular tissues frozen with EG (45.70 ± 10.00) were statistically significantly lower than those frozen with DMSO (51.20 ± 7.70) (P < 0.05). When the effects of both cryoprotectants on gene expression in tissue and semen samples were examined, it was determined that gene expression increased on average 0.19 ± 0.27 times in the tissue samples in the DMSO group compared to fresh tissue samples and 0.17 ± 0.19 times in the tissue samples in the EG group. It was determined that gene expression levels increased by an average of 1.20 ± 1.08 times in post-thaw epididymal spermatozoa samples compared to fresh semen samples. The results show that cryopreservation can activate cellular repair mechanisms by stimulating PARP-1 gene expression and affect gene expression by activating specific pathways in tissues and cells.

Effects of astaxanthin supplementation during vitrification and liquid nitrogen vapor freezing on motility, morphology, survival, reactive oxygen species (ROS), and DNA fragmentation of post-cryopreserved human sperm.

To investigate the effect of astaxanthin supplementation in cryopreservation media on post-thawed sperm motility, viability, morphology, reactive oxygen species (ROS), and DNA fragmentation in two cryopreservation techniques using vitrification and liquid nitrogen vapor freezing. Thirty normozoospermic semen samples were used in the study. Post-prepared semen samples were divided into 1) non-cryopreserved control, 2) and 3) vitrified without (V) and with astaxanthin 0.5 µM (V+ATX), 4) and 5) frozen in liquid nitrogen vapor without (L) and with astaxanthin 0.5 µM (L+ATX). Cryopreservation using vitrification and liquid nitrogen vapor freezing significantly decreased sperm motility and viability and increased ROS levels. However, no changes were seen in sperm morphology or DNA fragmentation. The addition of astaxanthin in cryopreservation media significantly increased post-thawed motility in both vitrification (77.6±8.9% vs. 69.0±9.5% in V+ATX and V) and vapor freezing (57.0±13.3% vs. 47.7±14.6% in L+ATX and L); it significantly increased sperm viability in vitrification (75.0±11.9% vs. 65.9±11.1% in V+ATX and V), and significantly decreased ROS level in both vitrification (4.7 (2.6-8.3) RLU/sec/106 vs. 10.6 (9.4-16.0) RLU/sec/106 in V+ATX and V) and vapor freezing (4.6 (3.3-10.5) RLU/sec/106 vs. 10.3 (7.9-18.6) RLU/ sec/106 in L+ATX and L). Astaxanthin supplementation in cryopreservation media did not affect sperm morphology or DNA fragmentation. Astaxanthin supplementation improved post-cryopreserved sperm motility, decreased ROS levels in both vitrification and liquid nitrogen vapor freezing and improved sperm viability only in the vitrification technique.

An Endogenic Origin for Titan's Rampart Craters: Assessment of Explosion Mechanisms

AbstractRampart craters are a class of lakes or depressions in Titan's north polar region that have morphological attributes suggestive of an explosive origin. Two previous studies have proposed that rampart craters form via nitrogen or methane vapor explosions analogous to terrestrial maar explosions. We propose a new terrestrial analog for rampart craters: gas emission craters (GECs) found in permafrost zones. We evaluate the explosive origin of Titan's rampart craters by modeling the dispersal of material from an explosive vent. The dimensions of nine rampart craters with radar‐bright ramparts were used to model the explosion process. The model yields a range of explosion conditions (e.g., gas mass and reservoir depth) producing ejecta dispersal patterns matching the observed features. We find that gas masses of 1011–1014 kg are required to produce a rampart crater. We examine two explosion scenarios: (a) rapid, maar‐like vaporization and explosion of liquid nitrogen or methane, and (b) more gradual gas accumulation and explosion akin to a GEC driven by methane released from destabilizing clathrates. If Titan's crust is composed of pure water ice, the calculated gas pressures are consistent with a rapid, maar‐like explosion mechanism. If the subsurface is predominantly composed of organic materials or clathrate, either scenario may be plausible. Further research on the composition and tensile strength of Titan's subsurface are required to discriminate between hypotheses. Nevertheless, we conclude that explosive dispersal of ejecta from a vent can account for the morphologies of Titan's rampart craters and may contribute to atmospheric methane replenishment.

Immunofluorescence and biochemical investigation of the protective effects of naringin and diosmin on the freezability of Merino ram semen

BACKGROUND: Various antioxidant substances are added to sperm extenders to protect spermatozoa against oxidative stress and cryodamage. OBJECTIVE: To investigate the effects of the flavonoid diosmin (DIO) and a flavanone glycoside naringin (NAR) on the freezability of ram semen. MATERIALS AND METHODS: In this study, six Merino rams were used during the breeding season. The ejaculates were pooled after collection from the rams. Pooled ejaculates were divided into six groups: control, NAR 1 mM, NAR 2 mM, NAR 4 mM, DIO 2 mM, and DIO 4 mM, and then diluted with a TRIS-based diluent. The pooled semen was equilibrated, placed in 0.25 mL pipettes with 10 × 107 sperm cells in each pipette, and frozen in liquid nitrogen vapor. After 24 h, the pipettes were thawed at 37°C for 25 s and analyzed in terms of spermatological parameters. RESULTS: The highest plasma membrane integrity ratio was found in the DIO 4 mM group, whereas a statistically significant difference was found between the NAR 1 mM and NAR 2 mM groups (p&lt;0.05). While the DIO 4 mM group had the highest acrosome integrity rate, a statistically significant difference was found between the other groups (p&lt;0.05). Mitochondrial activity was the highest in the NAR 4 mM, DIO 4 mM and DIO 2 mM groups (p&lt;0.05). In the analysis of the sperm membrane lipid profile, it was observed that the DIO group had the highest lipid-phospholipid ratio. In sperm membrane protein profile analysis, it was found that both additives exerted protective effects at different levels. The highest total protein content was seen in the DIO 4 mM and NAR 4 mM groups. 8-hydroxydeoxyguanosine (8-OhDG) positivity was more common in the control group than in the DIO and NAR groups. Cu-Zn superoxide dismutase (SOD) expression was lower in the control group and more intense in all other groups. Positive results were especially observed in the acrosome of the sperm cells. CONCLUSION: The addition of NAR and DIO to the ram semen extender increased the quality of sperm parameters after the freeze-thaw process.

Effect of different concentrations of inulin on ram sperm quality during cryopreservation

BACKGROUND: In reproductive biotechnology, sperm cryopreservation has a vital role to play. Cryopreservation of sperm produces reactive oxygen species (ROS), which disrupt sperm function and structural competence. Numerous protective chemicals, including fructans, have been used during sperm cryopreservation. OBJECTIVES: To evaluate the effect of different concentrations of the fructosan inulin on ram sperm quality parameters, superoxide dismutase (SOD) activity, and malondialdehyde (MDA) production after freezing and thawing. MATERIALS AND METHODS: The pooled samples from four healthy rams were divided into seven equal aliquots and diluted in a Tris-base extender supplemented with 1, 2, 4, 8, 16, and 28 mM of inulin or without inulin supplementation (control). By using liquid nitrogen vapor, the semen was frozen and stored at 196°C. RESULTS: The total motility, viability, and DNA integrity were significantly improved after freezet-hawing with 28 mM inulin, compared to other treatment groups (P&lt;0.05). A Tris-based extender containing 16 and 28 mM of inulin displayed the highest levels of ram sperm membrane integrity when compared with the control (p&lt;0.05). The abnormality of ram sperm was increased during freeze-thawing at control and 1 mM of inulin, compared to 16 and 28 mM of inulin (P&lt;0.05). Additionally, 28 mM of inulin decreased MDA and increased SOD activity in ram sperm in comparison with the other treatments (P&lt;0.05). CONCLUSION: As a result, 28 mM of inulin could be beneficial for the cryopreservation industry and reduce the harmful effects of freeze-thawing on ram sperm.

L-carnitine supplementation in conventional slow and ultra-rapid freezing media improves motility, membrane integrity, and fertilizing ability of dog epididymal sperm

This study aimed to assess the impact of L-carnitine (LC) supplementation in conventional-slow (CS) and ultra-rapid (UR) freezing media on post-thaw quality and fertilizing ability of dog epididymal spermatozoa. Sperm samples were collected from 60 epididymides obtained from 30 adult orchiectomized dogs via retrograde flushing. Twenty pooled sperm samples were then created (3 epididymal samples/pool). Four treatments were established according to the freezing method (CS and UR) and LC supplementation (5 and 0 mM [control, Co]): CS-LC5, CS-Co, UR-LC5, and UR-Co. The CS freezing involved exposing 0.25 mL straw to liquid nitrogen vapors (LN2), while UR freezing submerged 30-µL drops of sperm samples directly into LN2. Sperm kinematics, membrane integrity, and fertilizing ability (by heterologous in vitro fertilization using bovine oocytes) were evaluated for all treatments. Post-thaw results revealed that the CS freezing treatments resulted in significantly higher values (P < 0.05) of curvilinear and average-path velocities, and beat-cross frequency compared to the UR freezing treatments, regardless of LC supplementation. The CS-LC5 and UR-LC5 treatments cryoprotected the sperm by increasing (P < 0.05) the percentage of ‘live-sperm/intact-acrosome’ compared to their controls treatments CS-Co and UR-Co. Regarding fertilizing ability, the CS-LC5 treatment yielded a higher percentage (P < 0.05) of pronuclei formation compared to both UR treatments. The UR-LC5 treatment, however, obtained greater percentage (P < 0.05) than their control UR-Co. In conclusion, supplementation with L-carnitine in conventional-slow and ultra-rapid freezing improved sperm motility, plasma, and acrosome membranes integrity and fertilizing ability of dog epididymal spermatozoa.

The characteristics of frozen-thawed rooster sperm using various intracellular cryoprotectants

Cryopreservation of rooster semen is essential for conserving genetic resources, genetic improvement, and increasing productivity. However, the nature of avian sperm presents a global issue in ensuring superior frozen semen for artificial insemination. Thus, the present study aimed to evaluate the impact of using dimethylacetamide (DMA), dimethyl sulfoxide (DMSO), and ethylene glycol (EG) as cryoprotectants on post-thawed sperm motility, quality, antioxidant indicators, and fertilizing capacity. Twice a week, fresh semen ejaculates were collected from 15 adult roosters and immediately evaluated to constitute a pool from clean and qualified samples. The pooled semen was further diluted at a ratio of 1:2 (v/v) with an extender and then subjected to a freezing protocol in a liquid nitrogen vapor after adding a cryoprotectant solution containing 6% of either DMA, DMSO, or EG, respectively. After thawing, characteristics of sperm motion, quality, antioxidants, and fertilizing ability were evaluated and compared to fresh and cooled semen as controls. The results demonstrated that semen cooling negatively affected some parameters of sperm motility, quality, antioxidant biomarkers, and fertility. In comparison to the DMSO and EG groups, employing DMA considerably (P < 0.05) raised the percentages of sperm progressive motility, viability, plasma membrane intactness, and DNA integrity. The DMA group showed a significant increase in the catalase and glutathione reduced antioxidant enzyme activity and a reduction in nitric oxide and lipid peroxidation. After artificial insemination, the DMA and DMSO groups exhibited considerably (P < 0.05) better rates of hatchability and fertility than the EG group. It is concluded that freezing extenders containing 6% DMA is better than DMSO or EG to improve the post thaw semen quality and fertility in chickens.

Poor Sperm Chromatin Condensation Is Associated with Cryopreservation-Induced DNA Fragmentation and Cell Death in Human Spermatozoa.

Background/Objectives: Semen cryopreservation is routinely performed in fertility clinics for a variety of reasons, including fertility preservation and storage of donor sperm, yet the freeze-thaw process leads to cellular damage via ice crystal formation, osmotic shock, and supraphysiological levels of oxidative stress. Sperm resistance to damage during the freeze-thaw process varies widely, yet the intrinsic factors associated with sperm cryotolerance are largely unknown. The study aimed to investigate whether poor chromatin condensation renders sperm vulnerable to DNA fragmentation and cell death induced by the freeze-thaw process. Methods: Participants (n = 51) from the general community who met the inclusion criteria collected a semen sample after 3-8 days of abstinence. Neat semen samples underwent traditional semen analysis, aniline blue (AB)-eosin staining for chromatin condensation, the terminal deoxynucleotidyl transferase dUTP nick end labelling (TUNEL) assay for DNA fragmentation, and the Annexin V assay for apoptosis/necrosis, prior to being cryopreserved using the liquid nitrogen vapour method and stored at -196 °C. Stored samples were later thawed at room temperature and processed using density gradient centrifugation. Motile sperm concentration, DNA fragmentation and apoptosis/necrosis were analysed in post-thaw samples. Results: As indicated by a significant interaction effect in linear mixed models, an increased proportion of AB-positive sperm in the pre-freeze sample exacerbated the adverse effect of freezing on sperm DNA fragmentation (p = 0.004), late apoptosis (p = 0.007), and necrosis (p = 0.007). AB-staining was positively correlated with all three parameters in the post-thaw sample (all rs ≥ 0.424, all p < 0.01) and remained significant after adjusting for neat sperm concentration (all partial rs ≥ 0.493, all p < 0.01). Similarly, AB-staining was significantly correlated with the percentage point change in sperm DNA fragmentation (rs = 0.366, p = 0.014) and necrosis (rs = 0.403, p = 0.009), both of which remained significant after adjusting for neat sperm concentration (both partial rs ≥ 0.404, both p < 0.01), and borderline significantly correlated with percentage point change in late apoptosis (rs = 0.307, p = 0.051). Conclusions: Sperm with poorly condensed chromatin may be more susceptible to cellular damage during the freeze-thaw process, independent of pre-freeze sperm concentration. These findings may help to explain the intrinsic variation in sperm resistance to cryodamage within and between individuals that is poorly understood.

The contribution of different sperm parameters to better explain ram semen cryoresistance

In order to determine an effective procedure for explaining ram sperm cryoresistance and develop a new model for breeders classification, a retrospective study was conducted using sperm analysis data obtained over two consecutive years from a total of 82 sessions of ram semen cryopreservation. In each session, fresh ejaculates from eight males were collected via artificial vagina, pooled and frozen in liquid nitrogen vapors. After thawing, a total of 19,084 sperm tracks and 11,319 morphometric measurements were analysed. Clustering analyses were applied to establish motile and morphometric sperm subpopulations. Additionally, plasma and acrosome membrane integrity, as well mitochondrial activity using flow cytometry immediately after sperm thawing and following hypoosmotic shock test (HOST) was assessed. To develop a Ram Sperm Cryoresistance Index, Principal Component Analyses (PCA) using 22 variables were conducted. In the first PCA, the parameters that best explain cryoresistance include total motility (TM), motile subpopulation 2 (motSP2, which groups slow, very linear spermatozoa with low lateral head displacement), morphometric subpopulation 1 (morphSP1, grouping spermatozoa with the smallest head size and lowest shape values), sperm plasma membrane integrity immediately after thawing and following hypoosmotic shock test. These parameters collectively account for 77.34 % of the accumulated variance. To emphasize their importance, a second PCA was performed, revealing significant higher weighting coefficients for the quantity (TM) and quality (motSP2) of sperm movement after thawing, compared to the head size and shape of the thawed sperm (morphSP1). Furthermore, HOST Viability played a more decisive role than what was observed under isotonic conditions.

EFFECT OF CRYO-STORAGE ON STORABILITY OF PAPAYA SEEDS

A fruit crop of significant economic importance, papayas are often propagated from seeds. Based on their freezing injury even at low moisture content, papaya seeds are classified as "intermediate seeds"; and rapid loss of viability in storage is assumed to be the primary cause of poor germination; thus, the development of appropriate technology is necessary to prolong the storability of papaya seeds. One such effective technology for prolonged seed storability is cryo-storage. The purpose of the current study was to investigate how cryostorage affects papaya seed storability. For this investigation, TNAU Papaya variety CO 8 seeds were utilised. Five distinct treatments were applied to the seeds, which were then kept for approximately six months at liquid nitrogen's vapour phase (-140°C). The biochemical parameters such as protein content, free amino acid, total carbohydrates, free sugars, total lipids, and dehydrogenase activity were measured on a monthly basis along with seed germination and seedling vigour in the cryopreserved papaya seeds. Results indicated that papaya seeds that had been vitrified (seeds with a 10% moisture content that had been soaked in loading solution for 20 minutes, then immersed in Plant Vitrification Solution 2 (PVS2) for another 20 minutes, packed in cryovials and kept in cryo-storage at -140°C) compared to papaya seeds that were packed in cloth bags and stored in ambient conditions had higher seed germination, dry matter production and vigour index at six months after storage. In comparison to fresh seeds, the results also showed that, after six months of cryo-storage, biochemical proximate, protein content, total carbohydrate content, total lipid content, and dehydrogenase activity were maintained in the aforementioned treatment with minimum reduction, while electrical conductivity, free amino acid content, and free sugar content gradually increased with the storage period at minimum rate. In crux, vitrified papaya seeds packaged in cryovials and kept in cryo-storage at -140°C preserved their quality for six months, meeting Indian Minimum Seed Certification Standards. This could be useful for storing papaya seeds for longer periods of time. Keywords: Papaya, vitrification, differential scanning calorimetry, liquid nitrogen, cryo-storage

Dynamics of Condensation and Evaporation of Liquid Nitrogen in a Closed Vessel Pressurized with Helium, Nitrogen, and Their Mixture

Dynamics of Condensation and Evaporation of Liquid Nitrogen in a Closed Vessel Pressurized with Helium, Nitrogen, and Their Mixture

Investigation of the protective effects of different forms of selenium in freezing dog semen: Comparison of nanoparticle selenium and sodium selenite.

This study aimed to investigate the protective effects of nanoparticle selenium (SeNP) and sodium selenite (SS) on preventing oxidative stress during the freezing process of dog semen. A total of six dogs were used in the study. The ejaculate was collected from dogs three times at different times by massage method. A total of 18 ejaculates were used and each ejaculate was divided in five experimental groups. The experimental groups were designed to tris extender containing no antioxidants control, 1 μg/mL SeNP1, 2 μg/mL SeNP2, and 1 μg/mL SS1 and 2 μg/mL SS2. Extended semen were equilibrated for 1 h at 4°C, then frozen in liquid nitrogen vapour and stored in liquid nitrogen (~-196°C). After thawing, semen samples were evaluated in terms of CASA motility and kinematic parameters, spermatozoa plasma membrane integrity and viability (HE Test), spermatozoa morphology (SpermBlue) and DNA fragmentation (GoldCyto). Antioxidant enzyme activity (glutathione peroxidase; GPX, superoxide dismutase; SOD, catalase; CAT) and lipid peroxidation (malondialdehyde; MDA) were evaluated in frozen-thawed dog sperm. When the results were evaluated statistically, the progressive motility, VCL, and VAP kinematic parameters in the SeNP1 group were significantly higher than the control group after thawing (p < .05). The highest ratio of plasma membrane integrity and viable spermatozoa was observed in the SeNP1 group, but there was no statistical difference found between the groups (p > .05). Although the ratio of total morphological abnormality was observed to be lower in all groups to which different selenium forms were added, compared to the control group, no statistical difference was found. Spermatozoa tail abnormality was significantly lower in the SeNP1 group than in the control and SS2 group (p < .05). The lowest ratio of fragmented DNA was observed in the SeNP1 group, but there was no statistical difference was found between the groups (p > .05). Although there was no statistical difference between the groups in the evaluation of sperm antioxidant profile, the highest GPX, SOD and CAT values and the lowest lipid peroxidation values were obtained in the SeNP1 group. As a result, it was determined that 1 μg/mL dose of SeNP added to the tris-based extender in dog semen was beneficial on spermatological parameters, especially sperm kinematic properties and sperm morphology, and therefore nanoparticle selenium, a nanotechnology product, made a significant contribution to the freezing of dog semen.

Good Manufacturing Practice-Compliant Cryopreserved and Thawed Native Adipose Tissue Ready for Fat Grafting.

Background: As adipose tissue-derived mesenchymal stem cells are becoming the tool of choice for many clinical applications; standardized cryopreservation protocols are necessary to deliver high-quality samples. For this purpose, the cryopreservation and thawing of native adipose tissue under GMP conditions could represent an extremely useful and powerful tool for the direct reinfusion of the tissue, and consequently, of its stromal vascular fraction. Methods: In this study, 19 samples of adipose tissue were cryopreserved and characterized before and after storage in liquid nitrogen vapors. Of these 19 samples, 14 were processed in research and 5 in a GMP-compliant environment. Storage with and without cryopreservation medium was also evaluated. After one week to three months of storage, samples were thawed, washed, enzymatically digested, and characterized with flow cytometry. Results: The results show that there is a loss of nearly 50% of total nucleated cells during the cryopreservation/thawing process. Non-GMP and GMP samples are comparable for all parameters analyzed. This study also allowed us to exclude the cryopreservation of adipose tissue without any cryopreservation medium. Conclusions: The data shown in this work are consistent with the idea that native adipose tissue, if properly processed and controlled, could be a useful source of cells for regenerative medicine, keeping in mind that there is a clear difference in the quality between fresh and thawed samples.

Effect of resveratrol supplementation in conventional slow and ultra-rapid freezing media on the quality and fertility of bull sperm

The study investigated the impact of resveratrol (RES) on bull sperm cryopreservation employing conventional slow (CS) and ultra-rapid (UR) freezing methods on sperm quality and in vitro fertility. Twenty-four ejaculates from four bulls were divided into four groups based on the cryopreservation method and RES addition: CS-RES (n = 80), CS-Co (n = 80), UR-RES (n = 24), and UR-Co (n = 24). The CS freezing involved exposing sperm straws with 5% glycerol to liquid nitrogen (LN2) vapors, while UR freezing submerged sperm drops with 100 mM sucrose directly into LN2. Overall, sperm kinematic parameters and integrity of plasma and acrosome membranes significantly decreased (P < 0.001) after cryopreservation. Post-thaw values of motilities (total [TM] and progressive [PSM]), velocities (curvilinear and straight-line), beat cross frequency (BCF), and sperm with intact plasma membrane/intact acrosome (PI-/PNA-) were higher (P < 0.05) with CS-RES and CS-Co treatments compared to UR-RES and UR-Co treatments. CS-RES treatment resulted in greater percentages (P < 0.05) of TM, PSM, PI-/PNA-, and fertility (blastocyst rate) than their control, CS-Co; while UR-RES showed higher BCF values (P < 0.05) than its control, UR-Co. Additionally, UR-RES treatment exhibited lower oxidative stress percentages than UR-Co (P < 0.05). This study presents the following conclusions: (1) the CS freezing resulted in better cryosurvival of bull sperm than UR freezing; (2) the RES supplementation to CS freezing medium improved sperm motility, membrane integrity, and fertility; and (3) despite low cryosurvival sperm and fertility, the RES addition to ultra-rapid freezing medium reduced oxidative stress.

Fractional thermal load in cryogenically cooled Yb:YLF and Yb:YAG lasers

We present a method for the direct measurement of the fractional thermal load (FTL) in cryogenically cooled laser crystals. The experimental methodology involves characterizing the liquid nitrogen evaporation rate in a dewar containing the laser crystals, allowing for the accurate determination of FTL. The FTL is measured to be 1.7 × quantum defect (QD) for Yb:YLF and 1.5 × QD for Yb:YAG under continuous wave lasing conditions. The measured FTL values are then used to calculate the temperature distribution inside the crystals as a function of pump power, and the simulation results are found to be in very good agreement with the in-situ temperature measurements using contactless optical luminescence thermometry. The method and findings presented in this work hold great potential to benefit laser engineers and scientists working with cryogenic lasers to address and overcome temperature-dependent handicaps.

In vivo study of porous NiTi cryotweezers for bone tissue cryotherapy

This study examined the effects of liquid nitrogen vapor on osteogenesis in the rabbit femur. Cryotweezers made of porous nickel titanium alloy (nitinol or NiTi) obtained by self-propagating high temperature synthesis were used in this experiment. The porous structure of the cryotweezers allows them to hold up to 10 g of liquid nitrogen after being immersed for 2 min, which completely evaporates after 160 s. To study the effects of liquid nitrogen evaporation on osteogenesis, a rabbit femur was perforated. The formed holes were subjected to cryotherapy with varying exposure times. It was found that a 3 s exposure time stimulates osteogenesis, which was manifested in a greater number of osteoblasts in the regenerate compared to the control sample without liquid nitrogen. It was observed that increasing the exposure to 6, 9 or 12 s had a destructive effect, to varying degrees. The most severe damage was exerted by a 12 s exposure, which resulted in the formation of osteonecrosis areas. In the samples exposed to 6 and 9 s of cryotherapy, destruction of the cytoplasm of osteocytes and osteoclasts was observed.

Cryopreservation of ram semen: baicalein efficiency on oxidative stress, chromatin integrity, viability and motility post thaw.

Baicalein (B) has potential antioxidant properties, but it has not been tested as a ram semen extender. This study aimed to assess the impact of B on various sperm parameters and determine its potential influence on semen quality after the freeze-thawing process. During the breeding season, ejaculates were obtained from four rams with the aid of an artificial vagina. The collected mixed semen samples were divided into four groups: control (C; 0), B0.5 (0.5 mM), B1 (1 mM), and B2 (2 mM). After semen extension, the samples were loaded into 0.25 mL straws and stored for 2 h at 4°C prior to freezing in liquid nitrogen vapor and thawed in a water bath at 37°C. Among the groups, B0.5 demonstrated the highest progressive motility results, while B1 and B2 exhibited reduced motility (p < 0.05). In terms of high mitochondrial membrane potential, plasma membrane and acrosome integrity, and viability, B0.5 showed significantly superior outcomes to the other B groups (p < 0.05), although it was not significantly better than C. B1 displayed the highest plasma membrane integrity levels (p < 0.05). Notably, B2 displayed the lowest total antioxidant status levels among the groups (p < 0.05). The findings of this study suggested that the in vitro spermatological characteristics of ram spermatozoa such as progressive motility and chromatin integrity can be protected from the freeze-thawing process by using the 0.5 mM dose of baicalein as a semen extender. The treatment of sperm freezing might benefit from further in-depth research on the role of B in the improvement of cryoinjury and its underlying processes.

Modeling of a liquid nitrogen droplet evaporating inside an immiscible liquid pool

Evaporation of liquid nitrogen in another immiscible liquid occurs in many industrial applications. Existing oversimplified one-dimensional (1D) quasi-steady models, although can quantitatively predict the evaporation rate by introducing an empirical fitting parameter, rely on configurations inconsistent with experimental observation so more rigorous models are required to get in-depth physical insights and improve modeling capability. This study proposes a 2D quasi-steady-state theoretical model, free of fitting parameters, that predicts the bubble growth rate and estimates the heat transfer rate for a liquid nitrogen droplet evaporating inside a liquid pool within the spherical bubble regime. The droplet's shape and position within a spherical bubble are determined by the equilibrium between the gravitational force and the upward pressure force resulting from the vapor flow between the droplet and the pool. The vapor layer thickness is calculated to be on the order of 10 microns. Notably, the primary contribution to heat transfer arises from the lower portion of the droplet, leading to local heat flux values up to approximately six times higher at the bottom compared to the top. The predicted bubble growth is quantitatively consistent with experimental data within the capillary spherical bubble regime. Furthermore, the overall heat transfer rate Q exhibits a distinct scaling relationship with the volume ratio between the bubble and droplet, yielding Q∼(Vb/Vd)−0.1.

Effects of shilajit addition to honey bee diet on semen freezing

This study aimed to investigate the effects of adding shilajit to the diet of honey bees on semen freezing. A total of 5 groups were formed in the research, one of which was a control group (SH-0) and the other four were an experimental group (SH-1, SH-2, SH-3, SH-4). A total of 25 study colonies were used, 5 in each group. While the SH-0 group was formed without using any additives, the experimental groups were fed with the addition of shilajit (1/1 sugar/water) in different doses (5, 10, 15, 20 mg/L) to the honey bee diet. The collected semen samples were frozen in liquid nitrogen vapor and then stored in liquid nitrogen at -196°C. Then, semen samples were thawed at 37°C and evaluated to determine spermatological parameters (motility, acrosome integrity, plasma membrane integrity, spermatozoa concentration). It was also examined in terms of total oxidant and total antioxidant. Compared to the control group, it was determined that all shilajit doses significantly increased spermatological parameters such as motility, hypoosmotic swelling test (HOST), acrosome integrity (p&amp;lt;0.001), but did not significantly affect the spermatozoa concentration value (p &amp;gt;0.05). Although there is no statistical difference between the groups in terms of semen TAS (Total Antioxidant Status) and TOS (Total Oxidant Status) values, which are oxidative stress parameters, the numerical increase in TAS values in SH-2 and SH-3 groups; striking. As a result, it was determined that adding shijajit to the honey bee diet positively affected the post-thawing spermatological parameters of frozen bee semen.