Airflow drying of mammalian cells has not been successful yet, with one obstacle being the improper drying time of cells-laden trehalose droplets. To evaluate the major factors affecting the drying kinetics of aqueous trehalose droplets. A numerical analysis was applied to the evaporation behavior of aqueous trehalose droplets for the preservation of biomaterials. Factors such as convection caused by droplet contraction, vapor diffusion, and Stefan flow around droplets were taken into account in the analysis. Reducing the size and initial concentration of the droplets helps to achieve rapid drying of droplets. Forced convection can effectively enhance the initial drying rate of droplets, which may mitigate cell damage caused by hypertonic solutions. Upon drying, however, the rapid formation of an outer glassy shell inhibits the further drying of trehalose droplets. Simple enhancement of convection does not facilitate complete droplet vitrification due to the formation of an outer glassy shell that prevents water movement. https://doi.org/10.54680/fr25210110712.

Antioxidant is important to prevent ROS attack in boar sperm during the freezing-thawing process. To study whether hydrogen (H2) could improve the survival of boar sperm upon the freeze-thaw process. The effects of hydrogen in pre-diluent, freezing extender and thawing solution were investigated. Sperm viability, progressive motility, acrosomal integrity, mitochondrial activity, as well as the levels of malonaldehyde (MDA) and total antioxidant capacity (T-AOC) were measured. Hydrogen in pre-diluent, freezing extender and thawing solution significantly improved sperm quality parameters (including sperm viability, progressive motility, acrosomal integrity and mitochondrial activity), increased total antioxidant capacity (T-AOC) and decreased malondialdehyde (MDA) of post-thawed boar sperm. The use of hydrogen in all three solutions improved the quality of post-thawed boar sperm. Hydrogen protects boar sperm against ROS-induced damages during cooling and the freezing-thawing process. Doi.org/10.54680/fr25310110512.

Aquaporins (AQPs) are essential proteins that facilitate the rapid movement of water and cryoprotective agents (CPAs) during the cryopreservation process, and ensure the cryo-tolerance of sperm cells. This study evaluated the preservation of aquaporin levels in human sperm after undergoing freezing using natural deep eutectic solvents (NADES) as CPAs for cryoprotection. From June 2021 to October 2022, 35 semen samples with normal sperm parameters were acquired from the Mehr Infertility Treatment Institute in Rasht, Iran. The samples were divided into several groups for analysis: control group (not frozen), group frozen with SpermFreeze Solution, and groups frozen with different NADESs, including ChS, ChX, ChU, ChG, GlyP, and EtP. After thawing, various aspects for each group were assessed, including the integrity and condensation of sperm chromatin, viability, motility, integrity of acrosome, and the expression of AQP1, AQP3, AQP7, AQP8, and AQP9 genes. The analysis of gene expression revealed that freezing with ChS and GlyP preserved the expression of the AQP1 and AQP3 genes compared to the control group. Regarding AQP7 and AQP8, significant differences were not observed in expression levels between certain NADES groups (e.g., ChS, ChU, and GlyP) and the control group. Additionally, samples frozen with specific NADESs, such as ChS, ChG, EtP, and GlyP, exhibited preserved levels of AQP9 expression when compared to the control group. These findings emphasize the importance of NADES in preserving the expression of aquaporins in cryopreserved human sperm and their important fertility parameters. Doi.org/10.54680/fr24310110512.

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. 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. 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. 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. 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.

Cryopreservation of spermatozoa is a biotechnology used for fertilization purposes and preservation of genetic material in various domestic species. To determine the efficacy of two different commercial semen diluents in the cryopreservation of epididymal semen of domestic cats. Twenty male cats aged between 1- 3 years and weighing 2.5- 4.5 kg were used in the study. The testicular tissues removed from the cats were immediately brought to the laboratory in physiological saline and the epididymal parts were trimmed in commercial semen extenders (INRA 96, Group I; OPTIXCELL, Group II). Diluted semen samples were cooled to 4°C and filled into 0.25 mL straws. Semen samples were frozen in a programmable semen freezing device and then placed in a liquid nitrogen container at -196 C. Semen samples were thawed at 38 degree C for 25 s. Thawed semen samples were evaluated in terms of motility and kinematic parameters using CASA. No statistical difference was found between the groups in terms of total motility, progressive motility, and velocity parameters at 4 degree C. The rate of spermatozoa at slow speeds was found to be lower in group II. In addition, after freezing and thawing process, no statistical difference was observed between the groups in terms of motility, kinematics, and velocity parameters. Both commercial semen extenders can be used for cryopreservation of cat epididymal semen. https://doi.org/10.54680/fr24610110712.

'Dingjiaba' is an important Prunus persica cultivar (cv) mainly grown in the Hexi corridor in northwest China, which has an inherited strong cold tolerance. To compare the transcriptome and physiology data of leaves of cvs 'Dingjiaba' (D) and 'Kanoiwa' (K) following cold treatment at different time periods, in order to gain new insights into the mechanisms of cold adaptation in 'Dingjiaba'. We analyzed the transcriptomic and physiological data of leaves of D and K cvs exposed to 0 h (D0/K0), 2 h (D2/K2), 6 h (D6/K6) and 12 h (D12/K12) cold stress. Low temperature stress caused membrane damage and led to increased rate of electrolyte leakage and increased MDA content. Cold stress induced the accumulation of soluble sugars, soluble proteins and proline in leaves of both cvs, with a lower increase in K compared to D. Transcriptome analysis identified 4,631, 5,069, 5,662 and 3,886 differentially expressed genes (DEGs) between D0 and K0, D2 and K2, D6 and K6 and D12 and K12, respectively. The differentially expressed genes significantly enriched in metabolic pathways and biosynthesis of secondary metabolites. We further validated the reliability of sequencing data of the RNA-Seq with Real-Time Quantitative PCR, which suggested that the expression trend of the RNA-Seq were same as RT-PCR. These results provide novel insights into a series of molecular mechanisms underlying physiological metabolism and defense. https://doi.org/10.54680/fr24210110312.

Semen cryopreservation is a complex process during which there is alteration in the expression of sperm and seminal plasma proteins, molecular weight of protein or loss of membrane proteins during the process. In order to compensate for these changes, different membrane stabilizers are used in freezing semen extenders. However, there is scarcity of such studies during cryopreservation of goat semen. To investigate the effect of membrane stabilizers on sperm membrane protein expression during cryopreservation of goat semen. A total of 36 semen ejaculates from nine Assam Hill Goat bucks aged 2 to 2.5 years was collected by artificial vagina method. Three membrane stabilizers, each at two different concentrations viz. 50 and 80 mM sucrose, 50 and 100 mM trehalose, and 100 and 150 ng per mL IGF-1 (insulin-like growth factor 1 protein) were added to Tris-citric acid fructose egg yolk glycerol (TCFEYG) extender and semen samples were cryopreserved. The sperm membrane protein profile was studied in fresh and cryopreserved semen by SDS-PAGE. SDS- PAGE of sperm membrane extract of fresh semen revealed the presence of 24 protein bands with molecular weights ranging from 10 kDa to 240 kDa. Samples supplemented with 50 mM sucrose and 80 mM sucrose revealed 21 protein bands with molecular weights ranging from 10 kDa to 240 kDa. All the 21 protein bands were same as those observed in the sperm membrane of fresh spermatozoa, except that the 23 kDa, 29 kDa and 42 kDa bands were absent in frozen semen. Similarly, frozen semen extended with 50 mM trehalose and 100 mM trehalose revealed 22 protein bands with molecular weights ranging from 10 kDa to 240 kDa, but lacking the 29 kDa and 42 kDa bands. Proteins with molecular weights of 29 kDa, 130 kDa and 240 kDa were absent in frozen semen supplemented with 100 ng per mL IGF-1 and 150 ng per mL IGF-1. The present study revealed that supplementation of tris basic extender with trehalose at 100 mM and or IGF-1 at 100 ng/mL or 150 ng per mL improves the post-thaw semen characteristics and protects certain fertility related sperm membrane proteins. Doi.org/10.54680/fr23510110612.

Longan honey (LH) has the potential as a natural extracellular cryoprotectant to maintain the integrity of intact preantral follicles against the cryoinjury during ovarian vitrification. This research determined the cryoprotective effects of logan honey on preantral follicles integrity of rat ovary post-vitrification. After vitrification, the follicle index was determined by observing the ovarian histological sections made using the paraffin method with hematoxylin-eosin staining and analyzed using Optilab 3.0 and Image Raster software. The results showed that the combination of ethylene glycol (EG) with LH and a dose variation of 7.5 %-15 % (KP1-KP4) increased the density of follicles, the number of intact follicles in G2 and G3, with a decrease in the atretic follicles in G1 better compared to the use of EG only (KKP1-KKP2). The differences in the histological structur e of preantral follicles affected the doses of LH needed by each type of follicle to maintain the integrity of the follicular structure from cryoinjury effects. The comparison of the G2 total follicle index values were KKP1 (90.7 ± 18.3), KKP2 (115.6 ± 9.9) < KP1 (193.6 ± 10.7), KP2 (189.3 ± 10.5), KP3 (182.2 ± 27.1) and KP4 (169.4±8.8). Among the variations in the dose of LH 7.5 % - 15%, the highest increase in the G3 index value was found in primary (51.7 ± 9.8), tertiary (43.1 ± 8.8), secondary (33.9 ± 4.7), and primordial (28.7 ± 2.5) follicles of KP3 (7.5 % of LH). The primary and tertiary follicular stages maintain the best integrity and can be used after the vitrification of rat ovaries. doi.org/10.54680/fr23110110712.

Goldfish Carassius auratus is a popular ornamental fish extensively cultured worldwide. Sperm cryopreservation is a common fish breeding method that ensures sperm availability around the year. Studies on cryopreservation of goldfish sperm, especially on the suitability of cryoprotectant types and pre-freezing time, are scarcely available. To determine the most suitable type of cryoprotectant and pre-freezing for the successful cryopreservation of goldfish sperm. A completely randomized design with two factors was utilized in this study. The first factor is the type of cryoprotectants, which included methanol, ethanol, ethylene glycol, glycerol, and DMSO. The second is pre-freezing times of 10, 20, 30, and 40 min at each of the pre-freezing temperatures of 4 degree C, -10 degree C, and -79 degree C, meaning that the total times for the ramping down of temperature were 30, 60, 90 and 120 min, respectively. The Ringer solution and 10% egg yolk were used as extender and extracellular cryoprotectant. The sperm was stored at -179 degree C for 7 days. The ANOVA test showed that cryoprotectants and pre-freezing significantly affected the motility, viability, and fertility of goldfish sperm after freezing in liquid nitrogen for 7 days (P<0.05). Furthermore, 10% DMSO combined with 15% egg yolk with an pre-freezing time of 20 min can maintain sperm motility, viability, and fertility higher than other treatments, by 79%, 80%, and 33%, respectively. The agarose gel electrophoresis showed no DNA fragmentation in all samples, including fresh sperm. We conclude that 10% DMSO combined with 15% egg yolk and 20 min pre-freezing is the best treatment for goldfish sperm cryopreservation. DOI: 10.54680/fr23310110412.

The cryopreservation of oocytes through vitrification is quite successful but oocyte vitrification is still being standardized because of their structural and molecular sensitivity to the cooling and freezing processes. To evaluate the effect of different cryoprotectant concentrations on post-thaw morphology and viability of immature oocytes in sheep. Vitrification was achieved in three vitrification solutions comprised of different concentrations of the cryoprotectants ethylene glycol + DMSO, viz., (G1) 20%, (G2) 30%, (G3) 40% ethylene glycol + DMSO in equal ratio. Cryopreservation was in open pulled straws. Post vitrification evaluation was done after 1 week's storage in liquid nitrogen based on morphological evaluation and viability using trypan blue dye. The present study revealed non-significantly higher morphologically normal oocytes in G3 (74.7%) followed by G2 (70.3%), and the lowest in G1 (66.6%). Morphological defects were observed in 33.3 %, 29.6% and 25.2% of oocytes after cryopreservation in 20% (G1), 30% (G2) and 40% (G3) vitrification solutions, respectively. The results were non-significantly different between vitrification solution groups. However, the viability of post thaw immature oocytes was 95.6%%, 84.4% and 81.1% after vitrification in G1 (20%), G2 (30%) and G3 (40%), with viability being significantly highest (P<0.05) in G1 (20%) and lowest in G3 (40%). Cryoprotectant concentrations enable the maintenance of normal morphology and minimize cryoinjury during vitrification of immature oocytes.