Freezing Protocol Research Articles

Improvement of Workflows and Assay Reproducibility by The Introduction of "Assay-Ready" Culturing of MDCK Cells for Transport Studies.

In previous studies, we established and validated three Madin Darby Canine Kidney MDCKII cell lines, recombinantly modified with zinc finger nuclease (ZFN) technology. Here, we investigated the applicability of seeding these three canine P-gp deficient MDCK_ZFN cell lines, directly from frozen cryopreserved stocks without previous cultivation for efflux transporter and permeability studies. This technique is referred to as "assay-ready" and allows for highly standardized conduction of cell-based assays and shorter cultivation cycles. To obtain a rapid fitness of the cells for that purpose, a very gentle freezing and thawing protocol was applied. Assay-ready MDCK_ZFN cells were tested in bi-directional transport studies and compared to their traditionally cultured counterparts. Long-term performance robustness, human effective intestinal permeability (Peff) predictability and batch to batch variability were assessed. Efflux ratios (ER) and apparent permeability (Papp) results were highly comparable between assay-ready and standard cultured cell lines with R2 values of 0.96 or higher. Papp to Peff correlations obtained from passive permeability with non-transfected cells were comparable independent of the cultivation regime. Long-term evaluation revealed robust performance of assay-ready cells and reduced data variability of reference compounds in 75% of cases compared to standard cultured MDCK_ZFN cells. Assay-ready methodology for handling MDCK_ZFN cells allows more flexibility in assay planning and reduces performance fluctuations in assays caused by cell aging. Therefore, the assay-ready principle has proven superior over conventional cultivation for MDCK_ZFN cells and is considered as a key technology to optimize processes with other cellular systems.

Cryopreservation of sperm in the spotted scat, Scatophagus argus (Linnaeus, 1766) - Conservation and aquaculture perspectives

This study emphasizes the need for sperm quality assessment and cryopreservation strategy in the spotted scat Scatophagus argus as the occurrence of male population of this species is limited. Analyses of sperm quality were made including ionic content and osmolality of seminal plasma and blood serum. Sodium chloride (0.9%) was optimized as the extender with a dilution ratio of 1:3 (milt:cryomedium) for all experiments. Cryoprotectants including dimethyl sulfoxide (DMSO), dimethyl acetamide (DMA), glycerol (GLY), propylene glycol (PG), ethylene glycol (EG) and methanol (MeOH) were screened at various concentrations and equilibration times. Cryopreservation was performed using the cryoprotectants DMSO, GLY, EG, and MeOH individually as well as in combinations. Experiment 1 was done with DMSO, GLY, EG and MeOH (5, 10 and 15%); experiment 2 was done with DMSO (5%) + GLY (5%), DMSO (5%) + EG (5%), DMSO (5%) + MeOH (5%). Simultaneously, experiment 3 was done with both cryoprotectants and co-cryoprotectants viz. DMSO (5%) + egg yolk (5%), DMSO (5%) + coconut water (5%), DMSO (5%) + tomato water (5%) and DMSO (5%) + honey (5%). The cooling sample was optimized and performed with a one-step freezing protocol of −5 °C/min from 25 °C to −150 °C. The results indicated that samples subjected to experiment 2 using DMSO (5%) + EG (5%) for 60 days of storage registered maximum motility (82.33 ± 2.51%), duration time of sperm motility (10.45± 1.01 min) and viability (84.14 ± 5.29%) of post-thaw cryopreserved sperm. Morphology and ultrastructure of post-thaw sperm showed smooth surface topography and normal architecture of cellular organelles. Biochemical analysis indicated a reduction in the protein content of post-thaw cryopreserved sperm. DNA integrity (Comet assay) showed 6.30 ± 1.00% of tail DNA in post-thaw sperm, whereas fresh sperm registered 2.41 ± 1.85%. Functional integrity using the HOST of post-thaw sperm showed a maximum of 82.66 ± 1.15% with DMSO (5%) + EG (5%). Finally, fertility of post-thaw sperm using mature eggs of wild-caught females indicated the entry of sperm by breaking the vitelline membrane. Overall it was demonstrated that the DMSO (5%) + EG (5%) was amenable to the long-term preservation of sperm. Therefore, this protocol can be used as an alternative for male broodstock development under captive breeding program.

COMPARISON BETWEEN RAPID AND SLOW CRYOPRESERVATION PROTOCOLS FOR RAM SEMEN

Information regarding adequate freezing protocols of ram semen considering freezing distance and time during cryopreservation has not been adequately reported. Therefore, this study aimed to compare two freezing protocols for Najdi ram's semen. In the rapid freezing protocol, the straws were frozen at 5 cm over the surface of liquid nitrogen for 15 minutes. While in the slow freezing protocol, the straws were frozen at 8 cm over the surface of liquid nitrogen for 20 minutes. The semen was collected from five rams and extended with tris egg yolk glycerol cryodiluent. The extended semen was chilled slowly to 50C within two hours and equilibrated for two hours before being frozen on the liquid nitrogen vapor and cryopreserved at -1960C. There was no significant (P >0.005) effect of the freezing protocol on the sperm’s total motility, plasma membrane integrity, DNA integrity, and abnormalities. However, the vitality, fast progressive motility, straight-line velocity, average pathway velocity, linearity, and wobble were significantly higher in rapid freezing than in slow freezing protocol. In conclusion, cryopreservation of ram semen using rapid freezing (5 cm for 15 mins) protocol was better than slow freezing (8 cm for 20 mins) protocol regarding post-thawing semen quality. Key words: freezing protocol; cryopreservation; egg yolk; ram; semen

Cryoprotection in Human Mesenchymal Stromal/Stem Cells: Synergistic Impact of Urea and Glucose

Standard freezing protocols of clinically relevant cell lines commonly employ agents such as fetal bovine serum and dimethyl sulfoxide, which are a potential concern from both a regulatory and a patient safety perspective. The aim of this work was to develop formulations with safe and well tolerated excipients for the (cryo-) preservation of cell therapy products. We evaluated the cryoprotective capabilities of urea and glucose through measurements of cell metabolic activity. Freezing of clinically relevant human mesenchymal stromal/stem cells and human dermal fibroblasts at ≤ - 65°C at equimolar ratios of urea and glucose resulted in comparable viabilities to established dimethyl sulfoxide. Pre-incubation of human mesenchymal stromal/stem cells in trehalose and addition of mannitol and sucrose to the formulation further enhanced cell viability after freeze-thaw stress. Other cell types assessed (A549 and SK-N-AS) could not satisfactorily be preserved with urea and glucose, highlighting the need for tailored formulations to sustain acceptable cryopreservation.

A Dynamic Membrane-Active Glycopeptide for Enhanced Protection of Human Red Blood Cells against Freeze-Stress.

Intracellular delivery of freezing-tolerant trehalose is crucial for cryopreservation of red blood cells (RBCs) and previous strategies based on membrane-disruptive activity usually generate severe hemolysis. Herein, a dynamic membrane-active glycopeptide is developed by grafting with 25% maltotriose and 50% p-benzyl alcohol for the first time to effectively facilitate entry of membrane-impermeable trehalose in human RBCs with low hemolysis. Results of the mechanism acting on cell membranes suggest that reversible adsorption of such benzyl alcohol-grafted glycopeptide on cell surfaces upon weak perturbation with phospholipids and dynamic transition toward membrane stabilization are essential for keeping cellular biofunctions. Furthermore, the functionalized glycopeptide is indicative of typical α-helical/β-sheet structure-driven regulations of ice crystals during freeze-thaw, thereby strongly promoting efficient cryopreservation. Such all-in-one glycopeptide enables achieving both high cell recovery post-thaw >85% and exceptional cryosurvival >95% in direct freezing protocols. The rationally designed benzyl alcohol-modified glycopeptide permits the development of a competent platform with high generality for protection of blood cells against freeze-stress.

Sperm cryopreservation does not affect live birth rate in normozoospermic men: analysis of 7969 oocyte donation cycles.

Does sperm cryopreservation influence the reproductive outcomes of normozoospermic patients in oocyte donation cycles? After controlling for confounders, the use of cryopreserved semen from normozoospermic patients does not affect pregnancy and live birth rates after elective ICSI. Sperm cryopreservation by slow freezing is a common practice in ART. While frozen-thawed semen typically presents reduced motility and vitality, its use for ICSI is generally considered adequate in terms of reproductive outcomes. Nevertheless, most studies comparing reproductive outcomes between fresh and cryopreserved sperm include patients with severe male factor (testicular sperm, oligo-, and/or asthenozoospermia) or women of advanced maternal age, where the altered quality of the gametes can partially mask the full effect of freezing/thawing. The study included a retrospective cohort of 7969 couples undergoing their first oocyte donation cycle between January 2013 and December 2019 in one large clinic, using normozoospermic semen from the male partner. All cycles involved elective ICSI, fresh oocytes, and a fresh embryo transfer, either at cleavage or blastocyst stage. Two study groups were established based on the sperm status: fresh (n = 2865) and cryopreserved (n = 5104). A slow freezing protocol was used for all sperm cryopreservation. Sperm washing, capacitation, and selection prior to ICSI were performed identically for fresh and frozen-thawed samples, using pellet swim-up. Fertilization rate (FR), pregnancy (biochemical and ongoing), and live birth rates were compared between study groups using univariate and multivariate regression analyses. Male and female age, sperm concentration and motility after ejaculation, and number of oocytes inseminated were similar between cycles using fresh or cryopreserved sperm. Analysis by Student's t-test did not indicate a significant difference in FR between fresh and cryopreserved sperm (P = 0.0591); however, after adjusting for confounders, this difference reached statistical significance: 74.65% FR for fresh (CI 95%: 73.92-75.38) versus 73.66% for cryopreserved sperm (CI 95%: 73.11-74.20), P = 0.0334. The adjusted regression analysis revealed higher odds of biochemical pregnancy when using fresh sperm (odds ratio (OR): 1.143, P = 0.0175), but no significant effects of sperm cryopreservation were observed for ongoing pregnancy (OR: 1.101, P = 0.0983) and live birth (OR: 1.082, P = 0.1805). Caution should be exerted when extrapolating these results to different protocols for sperm cryopreservation and selection, or to IVM, advanced maternal age and classical IVF cycles, which were excluded from analysis. Owing to the retrospective nature of the study, some uncontrolled for variables may affect the results. Sperm cryopreservation does not affect pregnancy and live birth rates in normozoospermic patients, and although it may lower FR s slightly, this would not be clinically relevant. In line with previous studies that included patients with an apparent male or female factor, sperm cryopreservation is a safe and convenient technique. The study received no external funding and all authors have no conflicts of interest to declare. N/A.

Germplasm cryopreservation in bulls: Effects of gonadal tissue type, cryoprotectant agent, and freezing-thawing rates on sperm quality parameters

Germplasm preservation is crucial for reproductive programs involving farm and endangered species. This study describes the effects of slow-uncontrolled cryopreservation protocols on bovine sperm associated with testicular or epididymal tissues. Samples from the testis or epididymis (cauda) were cut into ∼0.5 or 1 cm3 fragments and cryopreserved using Me2SO (Dimethyl Sulfoxide) or glycerol-based cryoprotectants. Sperm were collected from testicular or epididymal tissue before and after freezing-thawing (38 °C or 40 °C) and kept at room temperature (RT) or 4 °C during handling. The parameters studied were viability, membrane integrity (HOS), motility, acrosome integrity, chromatin, and morphology. Pre-freezing parameters were lower in testicular sperm than epididymal: HOS+ and DNA integrity (P < 0.05). Normal-% pre-freezing testicular sperm morphology was lower than epididymal (43.3 ± 1.8% vs. 65.3 ± 14.8%). All testicular RT-kept sperm parameters decreased post-freezing, except for acrosome integrity, which remained constant (P > 0.05). There were no differences in Me2SO-frozen tissue sizes (P > 0.05). All epididymal RT-kept sperm parameters dropped post-freezing except for the constant DNA integrity (P > 0.05). 4oC-kept sperm were fitter than those at RT (P < 0.05). 4oC-kept testicular sperm viability, DNA, and membrane integrities declined after 38 °C or 40 °C thawing (P < 0.05). Acrosome integrity and motility remained unchanged after freezing (P > 0.05). 4oC-kept epididymal sperm acrosome integrity, motility, and HOS+% severely dropped post-thawing (P < 0.05). Viability and DNA integrity were unchanged (38 °C vs. 40 °C; P > 0.05). Overall, post-freezing sperm morphology was unaffected (P > 0.05), but Dag defect was significantly lower in testicular samples (P < 0.05). Whole-epididymis parameters were maintained up to 24h at 4 °C (P > 0.05). In conclusion, testis-epididymis freezing protocols should use small tissue pieces, Me2SO-based cryoprotectants, and 4°C-kept samples to reduce sperm damage.

Semen cryopreservation in Bornean Sun Bear (Helarctos malayanus euryspilus)

Semen cryopreservation is one common tool for extended genome storage and serves as insurance for declining biodiversity. In the endangered Bornean sun bear, semen was collected with electroejaculation and this is the first attempt at semen cryopreservation for this subspecies. Chilled semen with motility of more than 50 % was extended with Caniplus freezing medium, and cryopreserved with slow freezing protocol. A total of 22 cryopreserved semen straws were evaluated with computer-assisted sperm analysis and conventional semen evaluation tests as well as additional functional tests including evaluation of the acrosome (Rose Bengal Fast Green stain), plasma membrane (hypoosmotic swelling test), and chromatin (toluidine blue stain). Post-thaw semen quality was compromised with poor viability (27.6 ± 11.2 %), motility (8.3 ± 7.1 %), and progressive movement (1.3 ± 2.0 %) but maintained high integrity for the acrosome (71.1 ± 10.8 %), plasma membrane (54.7 ± 9.6 %), and chromatin (89.7 ± 7.6 %). The total post-thaw sperm abnormality was 51.7 ± 10.7 %, predominantly bent tail (28.0 ± 7.6 %) and proximal droplets (20.0 ± 11.8 %). Although this current cryopreservation was not a success, the compromised frozen-thawed semen may be a valuable resource in assisted reproductive technologies such as intracytoplasmic sperm injection. The three additional functional tests demonstrated were simple and affordable and hence are recommended to be part of the routine semen evaluation. Further research is required to develop species-specific cryopreservation protocols and to explore other assisted reproductive technologies in the Bornean sun bear.

Development of effective hematopoietic stem cell freezing protocols

Development of effective hematopoietic stem cell freezing protocols

Improvement of slow freezing protocol for adult Caenorhabditis elegans

Improvement of slow freezing protocol for adult Caenorhabditis elegans

Rapid Freezing of Skeletal and Cardiac Muscles Using Isopentane Cooled with Liquid Nitrogen and Tragacanth Gum for Histological, Genetic, and Protein Expression Studies.

Histological and molecular genetic evaluation of skeletal and cardiac muscles is an indispensable part of understanding muscle biology and the pathology of muscle disorders. Proper processing of the muscle tissue is a prerequisite for optimal evaluation. However, the processing of skeletal muscle samples often comes with many challenges. One of the commonly used methods of frozen tissue preparation involves optimal cutting temperature compound (OCT compound) embedding. This method is considered optimal for the processing of most of the routinely studied tissue samples. However, the processing of skeletal muscle samples using this method is often unsuitable as it causes artifacts and low DNA, RNA, and protein yield and quality due to the slow freezing of skeletal muscle tissues that allows ice crystals to form. One of the most suitable methods for skeletal muscle tissue processing for histological, genetic, and molecular studies is rapid freezing of freshly collected tissue samples using isopentane cooled with liquid nitrogen and tragacanth gum, which provides distinct advantages in consuming less time, preserving the cell morphology, and helping higher nucleic acids and protein yields. This chapter describes a protocol for rapid freezing of freshly collected skeletal muscle tissues using isopentane pre-chilled with liquid nitrogen and tragacanth gum. Skeletal muscle tissue samples processed using this protocol can be used for histological and immunological staining investigations and studies requiring DNA, RNA, and proteins from these tissues.

Example for process validation in biobanking: Fit for purpose testing of a cryopreservation method without isopentane.

Background: The freezing process of tissue samples is crucial for the preservation of morphological and molecular features. Several biobanking guidelines describe freezing techniques for optimal outcomes. As the Vetbiobank standard freezing protocol does not comply with those recommendations in detail, a process validation was performed to demonstrate that samples are suitable for downstream applications. Here we give a formal example of a process validation in the biobanking setting, as required by the biobanking guideline ISO 20387 (2018). Methods: Three different freezing protocols, freezing in liquid nitrogen, freezing via isopentane precooled on dry ice and freezing via liquid nitrogen vapor, were assessed based on morphological integrity of mouse liver and muscle tissue samples. Samples were either frozen in cryotubes (without Optimal Cutting Temperature compound, OCT) or in cryomolds (with OCT). The protocol providing the best results was validated for reproducibility and robustness in terms of defined acceptance criteria for morphological evaluability, A260/A280 ratio, and RNA integrity number values (RIN). In addition, performance tests were run by gene expression analyzes of selected, tissue specific biomarkers to confirm that processed samples are fit for purpose. Results: From the three applied freezing protocols, freezing in liquid nitrogen generated best results. Reproducibility acceptance criteria were met for both, morphological integrity and RNA quality. The freezing method was robust for the tested tissue types and the application of OCT, with exception of liver tissue, where it led to a significant decrease of the RIN value. Gene expression analyzes showed good comparability of results regardless of the applied freezing method. Conclusion: Freezing of tissue samples in liquid nitrogen provides samples of adequate quality for subsequent RNA investigations. A negative impact of OCT on the RIN value of liver samples was observed, which was independent from the applied freezing protocol and showed no impact on subsequent gene expression analysis.

Cryosurvival of buffalo semen extended in tris-egg yolk supplemented with a combination of different concentrations of butylated hydroxytoluene and taurine

Cryopreservation is accompanied with much detrimental premature sperm capacitation, decreased livability and fertility consequences. Taurine is considered as an antioxidant ameliorating the semen value post freeze-thawing. The purpose of this investigation was to elucidate the impact of Tris-diluent enriched with a mixture of taurine and different concentrations of butylated hydroxytoluene on buffalo bull semen preservability. Semen samples were extended in a Tris extender. Variable concentrations of butylated hydroxytoluene (BHT) (0.5, 1.0 and 2.0 mM) were set in ethyl alcohol in prewarmed (37oC) test tubes. Then taurine (60 mM) was added into each tube. Extended semen samples were added into the test tubes and put at 37oC for 5 minutes to permit the permeation of BHT into the spermatozoa. The control test tubes were Tris extender with (zero BHT and zero taurine). The test tubes were exposed to the freezing protocol. Semen was assessed and conception rate was carried out. The sperm membrane integrity (HOST) was eminently higher in TTB1 and TTB2 if compared to the control post cooling (Table 1). The post thawing results (Table 2) revealed significant improvement in sperm motility in TTB2 if compared to the control and other concentrations. Alive sperm % was kept in all concentrations as the control. Sperm abnormalities were kept in TTB2 and TTB3 as the control. Sperm membrane integrity (HOST) was significantly improved in all significantly as compared to the control. The acrosomal integrity was kept in TTB1 and TTB2 concentrations the same as the control The conception percent (Table 3) was higher in TTB2. It could be concluded that, the post cooling results exhibited the best semen quality in TTB1and TTB2. The post -thawing results revealed that, most improved sperm parameters and conception rate were in TTB2.

Laparoscopic ovarian tissue harvesting for cryopreservation from a child with galactosemia

To describe an approach to fertility preservation by a multidisciplinary team of reproductive endocrinology and infertility, pediatric gynecology and surgery, and genetics experts via ovarian tissue harvesting and cryopreservation for a toddler with galactosemia. Galactosemia is associated with progressive primary ovarian insufficiency (POI) and early intervention with ovarian tissue cryopreservation may help preserve fertility. Video description of a tissue harvesting and cryopreservation technique. Academic institution. 16-month-old female with classic galactosemia. At 6 months of age, despite good metabolic control, the infant's antimüllerian hormone (AMH) level was <0.015 ng/ml; luteinizing hormone level was 3.1 mIU/ml; and follicle stimulating hormone level was 30.2 mIU/ml. She was referred by her geneticist to the reproductive endocrinology and infertility specialist for fertility preservation. The AMH levels and pelvic magnetic resonance imaging findings of the patient were monitored over the next 9 months. Although the magnetic resonance imaging exam showed the presence of a dominant follicle in the right ovary and multiple small antral follicles in both ovaries at the age of 8 months, her laboratory assessment at the age of 14 months suggested impending POI (estradiol level <11.80 pg/mL; LH, 3.3 mIU/ml; follicle stimulating hormone, 35.97 mIU/ml; AMH, 0.03 ng/mL). At 16 months of age, given the low AMH levels, right ovary was laparoscopically harvested, so that a sufficient reserve of primordial follicles may be cryopreserved for fertility preservation. We dissected the mesosalpinx initially to separate the ovary from the tube in a manner that minimized the effects of cauterization on the ovary and preserved the fallopian tube. Successful harvesting and cryopreservation of the ovarian tissue containing primordial follicles. The right ovary, which measured 20 × 3 × 3mm, was bisected under a stereomicroscope along the hilum, trimmed to the cortical thickness of 1 mm and sliced into eight 4 × 4-mm pieces. These were then frozen with an established slow freezing protocol. The child was discharged the same day and had an uneventful postoperative course. A subsequent histological examination showed presence of primordial follicles, albeit at a reduced density for her age. Ovarian tissue cryopreservation is feasible in very young female children with rare genetic disorders associated with POI. We illustrated the unique aspects of performing these procedures in very young children.

Testicular tissue cryopreservation for fertility preservation in prepubertal and adolescent boys: A 6 year experience from a Swiss multi-center network.

Testicular tissue cryopreservation is the only option of fertility preservation in prepubertal boys. While it is considered experimental, since procedures to obtain mature spermatozoa from prepubertal testicular tissue are still under development, testicular tissue cryopreservation programs have emerged worldwide. Our aim was to study the feasibility and safety of a program of testicular tissue cryopreservation in prepubertal and adolescent boys facing gonadotoxic treatment in three University hospitals in Switzerland. Testicular tissue cryopreservation was accepted by 90% of families, with a total of 35 patients included. The average patient age was 8.5 years (range 7 months to 18.5 years). Malignancies were the most common diagnosis (31 patients, 88.6%) with 16 (45.7%) solid tumors and 15 (42.9%) hematological malignancies. Four (11.4%) patients had a benign condition. The main indication for testicular tissue cryopreservation was conditioning for hematologic stem cell transplantation (25 patients, 71.4%). Testicular tissue was cryopreserved according to the freezing protocol of Louvain Catholic University (Belgium), which includes either only immature testicular tissue freezing, or mature and immature testicular tissue freezing depending on the age of the patient and the presence or absence of haploid cells. The median number of spermatogonia per tubule cross-section was 2 (range 0–6) and spermatozoa were found in only one patient. Tumoral cells were found in one testicular biopsy of a leukemic patient. There were two minor adverse events and none of them required medical treatment or surgical revision. Five patients died during follow-up. Our data demonstrate the feasibility and safety of a program of testicular tissue cryopreservation coordinated by a multidisciplinary team of fertility preservation. Despite the experimental aspect of the procedure, the acceptation rate was high, which highlights the willingness of families and patients to participate in testicular tissue cryopreservation.

Validation of double freezing protocol for Beetal buck (Capra hircus) spermatozoa.

The study aimed to validate the double versus single freezing protocol for Beetal buck (Capra hircus) spermatozoa in tris-citric acid (TCA) based extender both in terms of quality and fertilization potential. Computer-assisted sperm motion and kinematic (CASA) variables, i.e. total (%), and progressive motilities (TM and PM, %) and rapid velocity (RV, %), average path (VAP, μm/s), straight line (VSL, μm/s) and curved line velocities (VCL, μm/s), straightness, (VSL/VAP, %) and linearity, (VSL/VCL, %) as well as supra-vital plasma membrane integrity (SV-PMI, %), mitochondrial membrane potential (MMP, %), viable/intact acrosome (V-IACR, %) and DNA integrity (DNA-I, %) had significantly greater values (p < .05) during single freeze-thawing as compared with the double freeze-thawing at 0, 30, 90, 150 and 210 days, respectively. All CASA and other assays alone did not show significant differences (p > .05) between both freeze-thaw cycles at all treatment durations, respectively. No statistical significance (p > .05) was observed for the in vivo fertility between single (n=84/141=59.72%) and double freeze-thawing (n=72/136=52.9%) cycles, respectively. In conclusion, sperm motion, kinematics, plasma membrane, acrosome, mitochondria and DNA integrities and in vivo fertility are acceptable after the double freezing protocol despite being lower than after one freeze cycle in Beetal buck.

Evaluation of Russian sturgeon (Acipenser gueldenstaedtii) Semen Quality and Semen Cryopreservation.

Simple SummaryThe decline in sturgeon populations, including Russian sturgeons, has reached alarming levels, threatening these species with extinction. The requirements for the meat of these fish, caviar and other products used in the textile, medical and food industries are constant or even on the rise. Reproductive biotechnologies seem to be the main solution to these problems, but the many unknowns related to the particularities of the species still place them far from the efficiency reached in some species of domestic animals. The development of fish farms for sturgeons and the intensification of reproductive biotechnologies is the main way in which the natural habitats affected by depopulation can be repopulated and to respond to the consumption and industrial requirements of the population. Our purpose is to contribute with additional novel information on the cryopreservation of sturgeon spermatozoa, as well as the evaluation of semen quality with the aim of developing a cryopreservation technique suitable for commercial breeding facilities.The alarming decline in sturgeon populations doubled by growing demands for sturgeon products, urge us to prevent the loss of these species. Fish stocking in natural habitats and developing fish farms are viable solutions, which can be successfully implemented with the help of reproductive biotechnologies. Despite the fact that semen cryopreservation is admittedly an important step for saving the Russian sturgeon, a reproducible standard method with good results has yet to be identified. Sperm quality assessment is essential for quantifying the impact of cryopreservation on spermatozoa. The purpose of our study was to provide additional information regarding semen cryopreservation and semen quality evaluation for the Russian sturgeon. Our study method is based on the use of two yolk-free extenders (with different cryoprotectants: DMSO and methanol) for freezing semen, using a simple freezing protocol. Parameters such as volume, concentration, motility, morphology and membrane integrity were evaluated. In conclusion, cryopreservation of Russian sturgeon spermatozoa using an extender containing methanol as cryoprotectant led to high egg fertilization percentages (72.67 ± 5.4%) even if the total motility values recorded for thawed semen were quite low (18–25%). Additionally, we identified two optimal stains for morphological studies and morphometry (Spermac stain kit and Trypan Blue Solution).

Sodium dodecyl sulphate, N-octyl β-D glucopyranoside and 4-methoxy phenyl β-D glucopyranoside effect on post-thaw sperm motion and viability traits of Murrah buffalo (Bubalus bubalis) bulls

Sodium Dodecyl Sulphate (SDS), N-Octyl β-D Glucopyranoside (NOG), 4-Methoxy Phenyl β-D Glucopyranoside (4-MPG) as ice recrystallization inhibitors were added to Tris Egg Yolk Glycerol (TEYG) semen extender for cryopreservation of semen of buffalo bulls. Post-thaw sperm motion and viability traits were evaluated. Pilot study involved six semen ejaculates (2 ejaculates/bull, from three bulls); second experiment was conducted using twenty seven semen ejaculates (9 ejaculates/bull, from 3 bulls) and in third experiment three semen ejaculates (one bull) were used. Eight concentrations of SDS (2, 1, 0.5, 0.25, 0.15, 0.125, 0.0625 and 0.0312%), twelve concentrations of NOG (33, 22, 11, 5.5, 2.5, 0.75, 0.5, 0.25, 0.125, 0.0625, 0.03125 and 0.0156 mM), and, eleven concentrations of 4-MPG (220, 165, 110, 55, 50, 25, 12.5, 6.25, 3.125, 1.56 and 0.78 mM) were supplemented in TEYG semen extender to evaluate the post-thaw sperm motility and viability traits. Computer Assisted Sperm Analysis (CASA) was used to measure the kinetic and functional parameters for sperm motion traits, Hypo Osmotic Swelling Test (HOST) for sperm plasma membrane integrity, Eosin Nigrosin staining for viability and Rose Bengal staining for sperm abnormalities for all the experiments except for pilot study where only Total Motility (TM) and Rapid Progressive Motility (RP) were evaluated. Three freezing protocols; i) Normal P24 (freezing rate of −30 °C min−1 from 4 °C to −15 °C; −40 °C min−1 from −15 °C to −60 °C; and −50 °C min−1 from −60 °C to −140 °C; and then plunged in liquid Nitrogen at −196 °C); ii) Moderate P25 (freezing rate of −30 °C min−1 from 4 °C to −15 °C; −50 °C min−1 from −15 °C to −60 °C; and −50 °C min−1 from −60 °C to −140 °C; and then plunged in liquid Nitrogen at −196 °C); and iii) Rapid P26 (freezing rate of −30 °C min−1 from 4 °C to −15 °C; −60 °C min−1 from −15 °C to −60 °C; and −50 °C min−1 from −60 °C to −140 °C; and then plunged in liquid Nitrogen at −196 °C) were evaluated using SDS 0.125% in TEYG semen extender. SDS ≤0.125%, NOG ≤0.0625 mM and 4-MPG ≤ 3.125 mM in TEYG buffalo semen extender improved significantly (p < .05) the kinetic and functional parameters as compared to the other Ice Recrystallization Inhibitors (IRIs) concentrations used for cryopreservation of buffalo bull semen in the pilot study. SDS 0.125% supplementation was the best IRI among all which resulted in improved kinetic and functional parameters of bull semen in second experiment. Conclusion was drawn that buffalo bull semen cryopreservation using sodium dodecyl sulphate, 0.125% as IRI in TEYG semen extender along with freezing protocol P 25 revealed optimum kinetic and functional parameters for post-thaw spermatozoa.

Potential Application of Cryogenic Freezer on Turbot Sperm Cryopreservation

BACKGROUND: Cryopreservation is a common practice to preserve fish sperm for prolonged periods. OBJECTIVE: To examine the effect of different freezing protocols on sperm characteristics, fertilization, and hatching rate of turbot. MATERIALS AND METHODS: Milt was obtained from ten 8-year-old turbot (54.3 ± 1.7 cm in length and 3,106 ±283 g in weight) at the peak of spawning season. Six batches of milts with &gt;90% motility was pooled and diluted to 1:3 by adding dimethyl sulfoxide (DMSO, 10%) as cryoprotectant. Then straws filled with semen were subjected to three freezing protocols (cooling rates). Sperm characteristics were assessed using sperm class analyzer before and after cryopreservation. Cryopreserved and fresh sperm were used for artificial fertilization to assess fertilization and hatching rates. RESULTS: Cryopreservation protocol has significantly deleterious effects on total motility, progressive motility, curvilinear velocity, straight line velocity, average path velocity, linearity index, straightness index, oscillation index, and amplitude of lateral head displacement of sperm. However, the beat frequency of cryopreserved sperm was found to be similar to control sperm. The fertilization rate of sperm subjected to three freezing protocols were similar, varying between 65.3 % and 75.6 %, and the hatching rates varied from 51.2% to 70.7%. CONCLUSION: The results show the potential application of cryopreservation in fish hatcheries.

Fresh Versus Frozen Stool for Fecal Microbiota Transplantation-Assessment by Multimethod Approach Combining Culturing, Flow Cytometry, and Next-Generation Sequencing.

The objective of this work was to compare the quality of FMT preparations made from fresh feces with those made from feces frozen at –30°C without any pre-processing or cryopreservation additives. The research hypothesis was that such preservation protocol (frozen whole stool, then thawed and processed) is equipotent to classical fresh FMT preparation. For that, three complementary methods were applied, including: (i) culturing in aerobic and anaerobic conditions, (ii) measuring viability by flow cytometry, and (iii) next-generation sequencing. Flow cytometry with cell staining showed that the applied freezing protocol causes significant changes in all of the observed bacterial fractions. Alive cell counts dropped four times, from around 70% to 15%, while the other two fractions, dead and unknown cell counts quadrupled and doubled, with the unknown fraction becoming the dominant one, with an average contribution of 57.47% per sample. It will be very interesting to uncover what this unknown fraction is (e.g., bacterial spores), as this may change our conclusions (if these are spores, the viability could be even higher after freezing). Freezing had a huge impact on the structure of cultivable bacterial communities. The biggest drop after freezing in the number of cultivable species was observed for Actinobacteria and Bacilli. In most cases, selected biodiversity indices were slightly lower for frozen samples. PCoA visualization built using weighted UniFrac index showed no donor-wise clusters, but a clear split between fresh and frozen samples. This split can be in part attributed to the changes in the relative abundance of Bacteroidales and Clostridiales orders. Our results clearly show that whole stool freezing without any cryoprotectants has a great impact on the cultivability and biodiversity of the bacterial community, and possibly also on the viability of bacterial cells.