Effects Of Different Cryoprotectants Research Articles

Basic study on cryopreservation of rat calvarial osteoblasts with different cryoprotectants.

Cryopreservation is a method adopted for storage of autologous skulls. Herein, this current research sought to explore the effects of different cryoprotectants on the biological characteristics of rat calvarial osteoblasts after cryopreservation. Neonatal Sprague-Dawley rats were selected and their skull tissues were isolated. The skull tissues were allocated into the refrigerating-3M, refrigerating-6M, M199-3M, M199-6M, povidone iodine-3M, and povidone iodine-6M groups according to the usage of cryoprotectants and treatment time (month) and the fresh group. Osteoblasts were isolated from skull tissues in each group through digestion. The histomorphology of the skull was evaluated by H&E staining and cell morphology was observed by microscopy. The viability, proliferation, apoptosis, and osteogenic activity of osteoblasts were assessed by trypan blue staining, MTT, flow cytometry, and alkaline phosphatase (ALP) staining. The skull histomorphology and osteoblast morphology were similar between the fresh and refrigerating groups. Osteoblast viability was weakened after cryopreservation. The longer the refrigeration time, the lower the number of living cells and the higher the apoptosis rate. However, cryopreservation using different cryoprotectants did not evidently affect osteoblast proliferation and ALP activity. Different cryoprotectants show no apparent effect on the osteogenic activity of rat calvarial osteoblasts after cryopreservation.

Impact of the Use of Cryoprotectants in the Production of Freeze-Dried Soluble Coffee from Cold Brew Arabica Coffee

AbstractCold brew is a method of coffee extraction that uses low temperature, preserving the volatile compounds of coffee. Freeze-drying allows the preservation of coffee features and nutritional value. The aim of this study was to evaluate the effects of different cryoprotectants in cold brew extracts as a basis for freeze-dried coffee production. Thus, the Coffea arabica extracts and the soluble coffee were characterized concerning caffeine content, antioxidant capacity, total phenolic compounds, and antimicrobial activity to verify the potential of this method. The extracts did not show antimicrobial activity with a high soluble solid content. It was observed that the cold extraction methods were efficient regarding the caffeine content, antioxidant capacity, and total phenolic compounds. Freeze-dried coffees also did not show antimicrobial activity, and they maintained the water and humidity activity standards. In general, cryoprotectants displayed an unfavorable influence on the extract and freeze-dried coffee in the analyses performed. The coffee extract without cryoprotectants had a higher antioxidant capacity (88.12%) and content of phenolic compounds (7.74 mg AG/mL of the coffee extract). Only for the analyses of soluble solids, the cryoprotectants mannitol and fructose showed promising results (14.03 °Brix, 14.40 °Brix, 11.33 °Brix, respectively). Thus, for the analyses conducted, the cryoprotectants did not lead to significant advantages for this process.

48 Effect of different cryoprotectants and slow freezing on viability of Saiga (Saiga tatarica) fibroblasts

48 Effect of different cryoprotectants and slow freezing on viability of Saiga (Saiga tatarica) fibroblasts

Effect of different cryoprotectants on the stability and survivability of freeze dried probiotics

Probiotics are of great economic importance mainly because of the various health benefits it offers. It is important for the probiotics to maintain its viability and health-promoting properties during the manufacturing process and storage. Freeze- drying is a common method to preserve the probiotics. The probiotic bacteria were freeze-dried using different cryoprotectants and its effect on the stability and viability of the probiotics were studied. The freeze dried culture was stored at a temperature of -20°C and 4°C to check for the survival rate. The plate count method was used to determine the survival rate of bacterial cells before and after freeze-drying. The inactivation rate constant k, was determined. Production of vitamins - Riboflavin and Cobalamin in probiotic bacteria was analysed by HPLC and the presence was confirmed by FTIR. Yoghurt was prepared using monoculture and co-culture of Lactobacillus sp. and Lactococcus sp. The proximate analysis indicated that the yoghurt was good source of protein, fat and energy.

Correction to: Effects of different cryoprotectants on the viability of microencapsulated Lactobacillus plantarum CJLP133 during long-term storage

Correction to: Effects of different cryoprotectants on the viability of microencapsulated Lactobacillus plantarum CJLP133 during long-term storage

The Effect of Cryoprotectants and Storage Conditions on the Transfection Efficiency, Stability, and Safety of Lipid-Based Nanoparticles for mRNA and DNA Delivery.

Lipid-based nanoparticles have recently shown great promise, establishing themselves as the gold standard in delivering novel RNA therapeutics. However, research on the effects of storage on their efficacy, safety, and stability is still lacking. Herein, the impact of storage temperature on two types of lipid-based nanocarriers, lipid nanoparticles (LNPs) and receptor-targeted nanoparticles (RTNs), loaded with either DNA or messenger RNA (mRNA), is explored and the effects of different cryoprotectants on the stability and efficacy of the formulations are investigated. The medium-term stability of the nanoparticles was evaluated by monitoring their physicochemical characteristics, entrapment and transfection efficiency, every two weeks over one month. It is demonstrated, that the use of cryoprotectants protects nanoparticles against loss of function and degradation in all storage conditions. Moreover, it is shown that the addition of sucrose enables all nanoparticles to remain stable and maintain their efficacy for up to a month when stored at -80°C, regardless of cargo or type of nanoparticle. DNA-loaded nanoparticles also remain stable in a wider variety of storage conditions than mRNA-loaded ones. Importantly, these novel LNPs show increased GFP expression that can signify their future use in gene therapies, beyond the established role of LNPs in RNA therapeutics.

Effects of different cryoprotectants on the viability of microencapsulated Lactobacillus plantarum CJLP133 during long-term storage

Effects of different cryoprotectants on the viability of microencapsulated Lactobacillus plantarum CJLP133 during long-term storage

Development of Solid Lipid Nanoparticles as Dry Powder: Characterization and Formulation Considerations.

Solid lipid nanoparticles (SLNs) are lipid-based colloidal systems used for the delivery of active compounds. Although SLNs have many benefits, they show important issues due to physical and chemical instability phenomena during storage. For these reasons, it is highly desirable to have a dried SLN formulation available. Therefore, the aim of the project was to identify suitable methods to obtain a dry powder formulation from an SLN suspension. The nanoparticle suspension was dried using both freeze- and spray-drying techniques. The suitability of these methods in obtaining SLN dry powders was evaluated from the analyses of nanotechnological parameters, system morphology and thermal behavior using differential scanning calorimetry. Results pointed out that both drying techniques, although at different yields, were able to produce an SLN dry powder suitable for pharmaceutical applications. Noteworthily, the freeze-drying of SLNs under optimized conditions led to a dry powder endowed with good reconstitution properties and technological parameters similar to the starting conditions. Moreover, freeze-thaw cycles were carried out as a pretest to study the protective effect of different cryoprotectants (e.g., glucose and mannitol with a concentration ranging from 1% to 10% w/v). Glucose proved to be the most effective in preventing particle growth during freezing, thawing, and freeze-drying processes; in particular, the optimum concentration of glucose was 1% w/v.

In situ visual and content changes analysis of coumarins in Radix Angelicae dahuricae by LSCM combined with LC-MS technology

In this study, a simple, accurate and green localization method of coumarins in Radix Angelicae dahuricae was established with fresh Radix Angelicae dahuricae as research material to reveal the distribution and accumulation of coumarins, based on frozen section and fluorescence imaging technology. The best frozen section conditions were established by comparing the effects of different cryoprotectants on the quality of Radix Angelicae dahuricae's frozen sections, according to the loss of coumarins and the complexity of the operation process. The coumarin components in Radix Angelicae dahuricae at different stages were located and quantitatively analyzed, and coumarin components distribution positions and content changes were identified using fluorescence imaging combined with LC-MS technology. The results showed that 30 μm slice thickness with 15 % glycerin protectant treatment is the best condition for frozen section. Fluorescence imaging showed that coumarins in Radix Angelicae dahuricae were mainly distributed in secretory tissue, the content over different periods showed an “S” curve of growth and coumarins reached their highest content in early September. The distribution and accumulation of coumarins in roots were revealed, which provided a reference for the synthesis and metabolism mechanism of metabolites in medicinal plants and the quality evaluation of traditional Chinese medicine.

Relationship Between Toxicity of Cryoprotectants, Osmotic and Oxidative Stresses In Awassi Ram Sperm

BACKGROUND:The relationship between the toxicity of cryoprotectants and their osmotic and/or oxidative stresses remains to be further investigated .OBJECTIVE:To investigate the toxic effects of different cryoprotectants and osmotic stress on Awassi ram sperm and to determine the relationship between oxidative and antioxidative status of the sperm.MATERIALS AND METHODS:Pooled sperm samples were exposed to sucrose solutions of different concentrations (75 to 900 mOsm) and isosmotic condition (290-325 mOsm) was re-established by adding HEPES buffered Tyrode's lactate. Sperm samples were mixed with 0.5, 1.0 and 1.5 M of glycerol, methanol, 2-methoxyethanol, dimethylacetamide or 1,2propanediol for 5 min and returned to isosmotic condition. Sperm samples were exposed to cryoprotectants at 4°C for 2 hours and isosmotic conditions were re-established. Motility, viability, acrosome integrity and oxidative or antioxidative parameters were determined.RESULTS:Treatment with hypo- or hyperosmotic sucrose solution reduced motility and viability without affecting acrosome integrity. The addition and removal of glycerol and dimethylacetamide (1.0 or 1.5 M) decreased sperm motility, while cryoprotectants had no effect on viability except for 1.5 M glycerol. Chilling significantly reduced the motility and viability of the sperm, but not the acrosome integrity. Rapid addition or removal of cryoprotectants also did not affect the acrosome integrity. Cryoprotectants changed only the ceruloplasmin level, while there were significant post-chilling differences in lipid hydroperoxide, paraoxonase and ceruloplasmin levels.CONCLUSION:Cryoprotectants without other additives have limited protection and glycerol can be toxic to spermatozoa. The oxidative stress plays a role in cryoprotectant toxicity and chilling stress.

Effect of cryoprotectants on the formation of advanced glycation end products and acrylamide in fried fish cakes

The effects of different cryoprotectants (sorbitol, sucrose, and polyphosphates) were investigated on the formation of advanced glycation end products (AGE) and acrylamide (AA) in fried fish cakes. AGE and AA were mainly formed in the crust. AA was undetectable in the core. Sorbitol could significantly reduce AA and fluorescent AGE contents in fried fish cakes, and hardly influenced formation of Nε-carboxymethyl lysine (CML). The addition of sucrose alone had no significant effect on the formation of AGE and AA in fried fish cakes. Polyphosphates significantly reduced the CML and fluorescent AGE contents by 43.58% and 49.81%, but increased the AA content by 9.48%. The highest CML content was found in the fried fish cake with mixed cryoprotectants. It was due to the combined action of sucrose, polyphosphates and low moisture content which further strengthened MR. The results suggest that sucrose and polyphosphate should not be used in fried surimi products at the same time. Additionally, sorbitol is suitable for adding to surimi for fried food.

Effect of Different Cryoprotectant Agents on Mitochondrial Distribution and Oocyte Developmental Competence in the Buffalo (Bubalus Bubalis )

Oocytes cryopreservation in mammalian species has gained a rapid pace during the past couple of decades emphasizing its importance in various assisted reproductive technologies .Aim of this work was to study effects of different cryoprprotectant agents: Ethylene Glycol (EG), Dimethyl sulfoxide (DMSO) and combination of them (EG+DMSO) on the viability, mitochondrial distribution and intensity of in vitro matured vitrified/ thawed buffalo oocytes by determine: 1) the effect of different cryoprotectants on the viability and developmental competence of vitrified/thawed in vitro matured buffalo oocytes. 2) The effect of different cryoprotectants on the mitochondria distribution and intensity of vitrified/thawed in vitro matured buffalo oocytes. Ovaries were collected from EL-Warak slaughter house, Cairo, Egypt. Excellent and good oocytes were in vitro matured in TCM-199 +IGF for 22 h in incubator at 38.5 ₒC in 5% CO2 and humidified atmosphere. Morphologically normal matured oocytes with first polar were placed in equilibration solution (VS1) for 1 min then oocytes were transferred to (VS2) for 30 sec. VS1 is half concentration of VS2 which contain EG 40% (EG group) or DMSO 40% (DMSO group) or EG 20%+DMSO 20% (Combination group). Oocytes were loaded in sterile 0.25 ml straws and stores in liquid nitrogen for 7-10 days. Morphological changes (normal and abnormal) of vitrified thawed in vitro mature buffalo oocyte were detected under invertd microscope. Mitotracker red stain and confocal microscope (Zeiss 710) used to study the mitochondrial distribution and Hochest dye to study the viability of in vitro vitrified/ thawed matured buffalo oocytes. The obtained results revealed that using a combination of EG+DMSO for vitrification resulted in the best quality of in vitro vitrified thawed buffalo oocytes which was demonstrated by significantly higher percent of morphologically normal recovered oocytes and transferable embryos when compared with EG and DMSO. More mitochondria were diffusely distributed in the fresh oocytes (93.33%) than in all vitrified groups oocytes: DMSO group (63.33%), EG group (79.99%) and DMSO+ EG group (79.99%). The Mean No. of mitochondrial intensity of recovered mature oocytes vitrified in EG+DMSO group (186.22) was significantly higher (P < 0.05) than those vitrified in EG group (153.33) and DMSO group (146.98). In conclusion: Combination of EG+DMSO cryoprotectants improve the viability and developmental competence of in vitro matured vitrified/thawed buffalo oocytes.

Cryopreservation of sheep ovarian tissue: Effect of different cryoprotectants and equilibration regimen on ovarian follicles structure

Cryopreservation of sheep ovarian tissue: Effect of different cryoprotectants and equilibration regimen on ovarian follicles structure

10.4314/sajas.v40i5.65338

The study evaluated the effect of different cryopro tectants on post-thaw survival and motility of Kolbroek sperm. Semen from Kolbroek boars was collected with the gloved hand technique. Ejaculates wer e diluted with Beltsville thawing solution (BTS) at a ratio of 1 : 1 prior to freezing. Semen was dilute d with egg yolk tris; thereafter, one of the three cryopro tectants (14% glycerol, 14% DMSO or 7% glycerol + 7% DMSO) were added. Diluted samples were then loaded into 0.5 mL straws and cooled with a programmable freezer. Thereafter the semen straws were plunged d irectly into liquid nitrogen (-196 °C) and stored f or 48 h. Frozen straws were thawed at 39 °C for a minute and evaluated for sperm motility and survival at 0, 30 , 60 and 90 min post-thaw. The post-thaw sperm survival frozen using glycerol as a cryoprotectant was significantly higher immediately after thawing, com pared to DMSO, however, similar to the combination of glycerol and DMSO. There was no significant difference on motility rate immediately (0 min) post-thaw between the three cryoprotectants. Sperm cryopreser ved with glycerol exhibited a significantly higher percentage motility at 30, 60 and 90 min post-thaw than in the other cryoprotectants. Based on sperm motility, glycerol was a better cryoprotectant for cryopreservation of Kolbroek boar sperm.

Studi Pendahuluan: Pengaruh Krioprotektan yang Berbeda terhadap Persentase Kerusakan dan Penetasan Embrio Ikan Lele Dumbo (Clarias gariepinus)

Tujuan penelitian ini adalah mengetahui pengaruh krioprotektan yang berbeda dalam kriopreservasi embrio terhadap persentase kerusakan dan penetasan embrio ikan lele dumbo (Clarias gariepinus). Embrio ikan lele pada fase gastrula diberi perlakuan larutan dimethyl sulfoxide (DMSO), propylene glycol (PG), madu, dan kombinasinya dengan konsentrasi 5% untuk masing-masing perlakuan. Embrio disimpan pada suhu 4 dan 0ºC, masing-masing selama 30 menit, 1, 2, 3, 4, 5, dan 6 jam. Thawing embrio dilakukan pada suhu 28ºC. Selanjutnya, embrio diinkubasi dalam akuarium pada suhu 28ºC. Hasil penelitian menunjukkan bahwa perbedaan krioprotektan dalam kriopreservasi embrio fase gastrula memberikan perbedaan kerusakan dan penetasan embrio ikan lele pada lama waktu penyimpanan yang berbeda. Persentase kerusakan embrio meningkat seiring dengan lama waktu penyimpanan dan suhu penyimpanan yang berbeda. Kombinasi larutan krioprotektan DMSO dan madu serta PG dan madu menunjukkan persentase kerusakan embrio yang lebih rendah dan persentase penetasan embrio yang lebih tinggi dibandingkan perlakuan krioprotektan lainnya (p&lt;0,05).

EFFECT OF DIFFERENT CRYOPROTECTANTS ON THE POST-THAW SPERM CHARACTERISTICS AND IN VIVO FERTILITY OF BUFFALO (Bubalus bubalus) BULL SEMEN

This study aimed to investigate the effect of different cryoprotectants, glycerol (GLY) or ethylene glycol (EG) or dimethyl sulfoxide (DMSO) on sperm characteristics, and in vivo fertility of frozen-thawed buffalo-bull semen. A total of 85 ejaculates collected by artificial vagina from buffalo-bulls of proven fertility were used in this study. The collected ejaculates were examined for volume, motility, viability, morphology, and sperm cell concentration. The qualifying ejaculates (≥ 3 mass motion, &gt; 70% progressive motility and viability, &lt; 15% abnormal morphology and &gt; 1 x 109 sperm cells/mL) were pooled and diluted with Tris-based diluent containing either 7% GLY or 5% EG or 5% DMSO. After 4 h equilibration time, the diluted semen was loaded in 0.5 mL straws, labeled, sealed and frozen stored until analysis. Frozen straws were thawed and evaluated for progressive motility, viability, hypo-osmotic swelling test (HOST), acrosomal membrane integrity, and acrosome reaction (AR) in response to calcium ionophore A23187. Moreover, in vivo fertility was calculated after artificial insemination (AI) of 75 buffalo-cows (25 female/cryoprotectant) treated with double doses of prostaglandin F2α (PGF2α) 11 days interval. The proportions of progressive motility, viability and intact-acrosome were higher (p &lt; 0.05) in extender containing 7% GLY compared to 5% EG and 5% DMSO. The proportion of intact-plasma membrane was comparable (p ≥ 0.05) between GLY and EG but higher than that of DMSO. A time-dependent increase in the % AR and % relative AR was recorded in the three cryoprotectants with clear significant (p &lt; 0.01) difference among them at 30 and 60 min incubation, respectively. Moreover, GLY yielded higher pregnancy rate (52%) than EG (32%) and DMSO (16%). In conclusion, GLY is recommended for preservation of buffalo-bull semen in order to maintain sperm plasma membrane integrity and improve in vivo fertility of frozen-thawed buffalo-bull semen.Key words: buffalo-bull; frozen-thawed semen; cryoprotectant; estrus synchronization; In vivo fertility

Preparation and Characterization of Freeze-dried Liposomes Loaded with Amphotericin B

Background: Amphotericin B (AmB) is a drug of choice in the therapy of systemic fungal infection because of its board-spectrum antifungal activity. However, its conventional formulation has many side effects such as acute and chronic nephrotoxicity. Liposomes have been developed to reduce the drug’s toxicity. However, they had to meet strict stability criteria. In general, liposomes can be freeze-dried to inhibit liposomes infusion, phospholipids degradation during storage. Liposomal size usually increases after freeze-drying because of being influenced by many factors in freezing, lyophilizing and rehydration processes. Therefore, cryoprotectants are used to stabilize liposomal vesicles during freeze-drying process. &lt;/P&gt;&lt;P&gt; Objective: In the present study, we developed AmB liposomal suspension and lyophilized liposomes loaded with AmB, evaluated the effect of different cryoprotectants on the characterization of freeze-dried AmB liposomes. &lt;/P&gt;&lt;P&gt; Methods: In this study, AmB liposomes were prepared from hydrogenated soy phosphatidylcholine, distearoylphosphatidylglycerol and cholesterol by thin lipid film hydration method using different hydrate mediums likely: Glucose solution, citrate buffer, phosphate buffer. High-pressure homogenization and extrusion methods were used to reducing vesicles size. Dynamic light scattering was used to characterize liposomal size, and size distribution. HPLC method was used to assay drug and determine entrapment efficiency. Liposomal suspension was lyophilized with different cryoprotectants: Sucrose, mannitol, lactose, trehalose and glycerol. Differential scanning calorimetry was used to study lyophilized cake. &lt;/P&gt;&lt;P&gt; Results: We found that liposomal suspension with hydration medium10 mM citrate buffer pH 5.5 had a small average size (&lt;100nm) and narrow distribution (PDI &lt;0.2). Sucrose and trehalose stabilized vesicles size during freezing process, and lyophilized liposomes with sucrose and trehalose had an elegant appearance, yellow, compact cake. DSC study showed that sucrose and trehalose in lyophilized cake were amorphous. The cake was rehydrated within 10 seconds to form liposomal suspension, in which vesicles size was less than 140 nm. &lt;/P&gt;&lt;P&gt; Conclusion: We have developed successfully AmB liposomal suspension and lyophilized liposomes loaded with AmB. Sucrose and trehalose can be used as cryoprotectants in the freeze-drying and reconstitution process.

Effects of different cryoprotectants on microemulsion freeze-drying

Abstract In this study, the effects of different cryoprotectants, i.e., mannitol, d -trehalose, lactose, and glycine, were investigated during the evening primrose oil microemulsion freeze-drying process. The glass transition temperature, particle size and dispersion, and appearance of the microemulsions before and after freeze-drying were compared to identify the best cryoprotectant by using differential scanning calorimetry and Dynamic light scattering. In summary, mannitol was shown to be the best cryoprotectant for the evening primrose oil microemulsion due to its high glass transition temperatures, small rehydrated microemulsion particle sizes, uniform particle size distribution, and ability to produce crystal-like freeze-dried powders. Increasing the ratio of cryoprotectant to evening primrose oil generally produced better freeze-dried powders. The optimal mannitol to evening primrose oil ratio for microemulsion freeze-drying was determined to be 6:1.

Effects of five cryoprotectants on proliferation and differentiation-related gene expression of frozen-thawed bovine calf testicular tissue.

The cryopreservation of testicular tissue is a potential method for preserving male fertility. However, the effect of cryopreservation on bovine calf testicular tissue is scarce. This study investigated the effect of different cryoprotectants on bovine calf testicular tissue at the molecular level. Testicular tissue from ten immature bovine calves (6months) was collected after slaughter and cryopreserved in an extender containing different concentrations of the following five cryopreservation solutions (CP): bovine serum albumin (BSA) with 5% dimethyl sulfoxide (DMSO), trehalose with 5% DMSO, DMSO and glycerol and ethylene glycol (EG). After 7-day cryopreservation, the expression levels of three spermatogonial stem cell (SSC)-related genes, octamer-4 (OCT4), KIT ligand (MGF/SCF) and kit oncogene (C-KIT), were investigated by quantitative PCR (qPCR). The cell viability was highest for the tissues preserved with 30mg/ml BSA (77.82%±1.22) and 40mg/ml trehalose (74.23%±1.16) compared with other groups (p<0.05), and the level of expression of the three genes was highest with 30mg/ml BSA (p<0.05). Compared with other CPs, the 30mg/ml BSA and 40mg/ml trehalose have the better cryopreserve protection. The 30mg/ml BSA is the most viable media for the cryopreservation of testicular tissue from cattle.

Bovine oocyte membrane permeability and cryosurvival: Effects of different cryoprotectants and calcium in the vitrification media

The cryopreservation process must be improved to enhance oocyte cryosurvival and functionality. Two protocols with different cryoprotectants (CPAs), containing either ethylene glycol (EG), dimethyl sulfoxide (DMSO) and sucrose (EGDMSO) or 1,2-propanediol and sucrose (PrOH) were evaluated. In both protocols, calcium (Ca2+) free or -containing base media were tested. Oocytes were subjected to vitrification or only exposed to CPAs without immersion in liquid nitrogen. Oocyte's viability, cortical granules location and competence for development after fertilization were assessed. Finally, fatty acid composition and membrane permeability of oocytes exposed to CPAs were analyzed. Independently of Ca2+ concentration in the vitrification media, the development rates were higher in oocytes vitrified with EGDMSO protocols (p = 0.0005). After warming, higher cleavage rates were obtained in EGDMSO + Ca2+ compared to the PrOH without Ca2+ protocol (p = 0.02). Oocytes exposed to PrOH without Ca2+ presented lower cleavage rates compared to control (p = 0.04). An enhanced premature zona hardening in vitrified oocytes as well as lower concentrations of the fatty acids c11:18:1 and 20:4n-6 in cumulus oocyte complexes exposed to PrOH protocols were identified. The oocytes minimum volume and permeability were affected by the exposure to PrOH and Ca2+ (p ≤ 0.007). In conclusion, the most effective protocol for bovine oocytes cryopreservation combines EG and DMSO, independently of Ca2+ concentration in the media. A higher toxicity and an incomplete depletion of water during PrOH loading may hamper oocyte viability. The type of CPAs and Ca2+ interfered differentially on oocyte pathways to functionality, and this should be considered when choosing a cryopreservation protocol.