Biopreservation and Biobanking

This study assessed the outcomes of nonsurgical embryo recovery (NSER) after superovulation (SOV) in five locally adapted Brazilian breeds of sheep and goats. The objective was to evaluate the feasibility and efficiency of using SOV combined with a less-invasive embryo collection technique for supplying the Brazilian animal gene bank with germplasm from specific genotypes of interest. Morada Nova (n = 20), Santa Inês (n = 20), and Somalis (n = 20) ewes received an intravaginal progesterone (330 mg) device for 9 days, while Canindé (n = 15) and Moxotó (n = 15) goats received an intravaginal medroxyprogesterone acetate (60 mg) device for 6 days. All females received 133 mg of porcine follicle-stimulating hormone (pFSH) administrated in six decreasing doses 12 hours apart, starting 60 hours before device removal, plus 37.5 μg of d-cloprostenol at the fifth and sixth pFSH dose. Donors in estrus were mated with fertile males. The corpora lutea (CL) number was assessed by ultrasonography 1 day before NSER. On day 6.5 or 7 after estrus, NSER was performed following hormonally induced cervical relaxation. A total of 97% of sheep and 90% of goats responded with estrus, and among those, 91% of sheep and 85% of goats presented a CL. In ewes, the numbers of CL were greater (p < 0.05) in the Santa Inês breed, while similar (p > 0.05) CL numbers were found among the goat breeds. All viable embryos were freezable (excellent and good quality) and the number per donor was 7.8 for sheep and 4.9 for goats. All parameters of NSER efficiency, embryo yield, and fertility post-NSER did not differ (p > 0.05) between breeds among each species. The SOV-NSER procedures applied for an embryo biobank supply of locally adapted Brazilian breeds of small ruminants were efficient regarding production of cryopreservable embryos, and preservation of donor fertility. Therefore, SOV followed by NSER is recommended for embryo biobank assembly in sheep and goats.

This study investigated the cryoprotectant effects of dimethylformamide (DMF), ethylene glycol (EG), and dimethyl sulfoxide (DMSO) as substitutes for glycerol (GLY) in a soybean lecithin (SL)-based extender in the cryopreservation of buck sperm. In this study, the semen of three Saanen bucks was individually extended in SL supplemented with 5% GLY (control), DMF, EG, or DMSO. After this, the extended semen was cryopreserved and two straws from each group were thawed (37°C for 30 seconds), pooled, and analyzed for sperm motion parameters, plasma membrane integrity (PMI), acrosomal integrity (ACI), and high mitochondrial membrane potential (HMMP). Samples were analyzed after 15 minutes (T0) and after 2 hours of incubation at 37°C (T2). The results revealed higher values of motility (total and progressive) and sperm motion parameters for DMF than the other cryoprotectants (p < 0.0001). PMI and HMMP did not differ (p > 0.05) between GLY and DMF, but ACI was higher (p < 0.01) for DMF compared with GLY. Based on these results, DMF and GLY samples were used in heterologous in vitro fertilization assays by using bovine oocytes (n = 337) obtained from a slaughterhouse. No differences (p > 0.05) were observed between GLY and DMF for unfertilized (GLY: 38.8%; DMF: 25.33%), pronucleus (GLY: 25.68%; DMF: 27.92%), and cleavage rates (GLY: 35.52%; DMF: 46.75%). Based on these results, it is concluded that DMF preserves sperm motion characteristics and ACI better than GLY, EG, and DMSO, and it is the penetrating cryoprotectant of choice for the cryopreservation of buck sperm in SL extender.

Oxidative stress is a major contributory factor to cellular damage during semen cryopreservation and results in a decreased fertilizing capacity of cryopreserved bull sperm. The inclusion of exogenous antioxidants sometimes exerts deleterious effects on sperm quality. Thus, enhancing the endogenous production of antioxidants is a requirement. This study aimed to investigate the effect of milk type heated at different temperatures on the antioxidant potential of extenders, and the subsequent post-thaw quality parameters and in vivo fertility of buffalo bull semen. Cow (C) and buffalo whole milk (B) were used separately for semen extender preparation, heated at five different temperatures (T₁ = 90°C, T₂ = 100°C, T₃ = 110°C, T₄ = 120°C, T₅ = 130°C) for 10 minutes. Reactive sulfhydryl groups were measured in each subgroup by Ellman's reagents as CT₁ = 143.2 μM, CT₂ = 147.4 μM, CT₃ = 151.5 μM, CT₄ = 157.2 μM, CT₅ = 161.8 μM, BT₁ = 168.3 μM, BT₂ = 172.5 μM, BT₃ = 176.7 μM, BT₄ = 196.3 μM, and BT₅ = 205.7 μM. All semen samples were cryopreserved in milk-based extenders by using standard procedures. Post-thaw quality parameters including total and progressive motility, mitochondrial membrane potential, plasma membrane integrity, and acrosome integrity were found to be higher (p < 0.05) in the group (BT₃) containing buffalo milk heated at 110°C, whereas in the same group, lipid peroxidation was found to be lower (p < 0.05) as compared with other treatment groups and control group. In vivo fertility of cryopreserved buffalo sperm was compared among BT₃, CT₁ (conventionally used milk extender), and a Tris egg yolk extender group. The fertility rates [47% (54/114), 30% (33/108), and 36% (37/103)] were higher (p < 0.05) in BT₃ as compared with other groups. This study suggests that buffalo milk heated at 110°C has high antioxidant potential and improves post-thaw quality and in vivo fertility of cryopreserved buffalo bull semen.

The present study was performed to investigate the effects of supplementing flaxseed oil (FO) or vitamin E (VE) or their combination to an extender for Simmental bull semen cryopreservation. In experiment 1, different concentrations of FO (0, 10, 100, and 1000 ng/mL) and VE (0.05, 0.1, and 0.2 mg/mL) were added to the extenders. In experiment 2, FO, VE, and FO + VE were added and a control group was included. Sperm viability, motility, motion parameters, acrosome integrity and membrane integrity, endogenous antioxidant indices, reactive oxygen species, and malondialdehyde levels were evaluated after semen thawing. A higher percentage of viability, motion parameters, endogenous antioxidant indices, and membrane integrity was observed after supplementation with 10 ng/mL FO or 0.1 mg/mL VE compared with the control group (p < 0.05). Also, combined supplementation of 10 ng/mL FO +0.1 mg/mL VE further improved the quality of frozen–thawed sperm by regulating viability, motion parameters, membrane integrity, and endogenous antioxidant indices compared with the FO or VE alone (p < 0.05). These results indicated that FO (10 ng/mL) + VE (0.1 mg/mL) could further improve the protective effects on bull sperm post-thaw.

Freezing of sperm is known as an important part of assisted reproduction. However, many studies have illustrated that cryopreservation negatively affects the quality and fertility rate of sperm. This study aimed to evaluate the effects of trehalose and pentoxifylline (PTX) in diluents on cooled and frozen-thawed Markhoz goat sperm. Preassessed samples were pooled and diluted with a basic diluent using trehalose and PTX. The cooled sperm showed significant improvement. The motion characteristics of cryopreserved sperm were evaluated based on computer-assisted system analysis. In this study, we investigated the viability, membrane integrity, malondialdehyde concentration, total abnormality, acrosome integrity, and seminal hyaluronidase enzyme. Also, the hypo-osmotic swelling test, mitochondrial activity, apoptotic features, caspase activity, chromatin dispersion test, active mitochondria, and reactive oxygen species (ROS) activity were assessed as complementary parameters. The data illustrate that the total motility, progressive motility, average path velocity (VAP), straight-line velocity (VSL), curvilinear velocity (VCL), and the ratio of sperm chromatin dispersion, viable sperm were improved significantly (p < 0.05) using 3 mM PTX alone or 3 mM PTX plus 50 mM trehalose, while other characteristics indicate significant enhancement by 3 and 6 mM PTX and 50 and 70 mM trehalose alone or in combination, except amplitude of lateral head displacement (ALH), beat/cross frequency (BCF), and intracellular ROS-(O⁻), which demonstrate no significant difference among treatments. In conclusion, this study indicates that addition of 3 and 6 mM PTX alone or with 50 and 70 mM trehalose seems to reduce the damage caused by cooling and cryopreservation processes.

This study investigated the effect of ascorbic acid (vitamin C) and proline amino acid alone or together on the quality and fertility of frozen/thawed honey bee spermatozoa. The experiments were designed to compare a single ascorbic acid, a single proline amino acid, and different combinations of ascorbic acid with proline amino acid on the cryopreservation of honey bee semen based on sperm motility, viability, intact membrane (hypo-osmotic swelling test), and fertility rates. Eight cryopreserved study groups comprised Control II with no supplement, along with groups with ascorbic acid (2 mg), proline 25 mM, proline 50 mM, proline 100 mM, and combination groups of both ascorbic acid (2 mg) and proline 25 mM, proline 50 mM, and lastly proline 100 mM groups, respectively. Using 50 mM proline in the tested groups had the greatest impact on sperm motility, viability, the percentage of spermatozoa with intact membrane, and fertility. The cryopreservation process caused a gradual decrease in motility, viability, intact membrane (p < 0.05), and fertility rates (p < 0.01) in all the tested research groups as against the fresh semen control group. Successful honey bee sperm cryopreservation and fertility are achievable when using an appropriate sperm freezing protocol and antioxidant. Proline amino acid as an antioxidant in semen extender had a more beneficial influence on sperm quality parameters and fertility. The success of cryopreservation with antioxidants is related to the chosen antioxidant in a dose-dependent manner.

Cell lines are valuable tools to safeguard genetic material from species threatened with extinction that is mainly due to human action. In this scenario, the puma constitutes a species whose population is being rapidly reduced in the ecosystems it inhabits. For the first time, we characterized puma skin-derived cell lines and assessed these cells after extended culture (experiment 1) and cryopreservation (experiment 2). Initially, we identified and characterized four dermal fibroblast lines using morphology, ultrastructure, and immunofluorescence assays. Moreover, we evaluated the effects of culture time (1st, 3rd, and 10th passages) and cryopreservation on their morphology, ultrastructure, viability, metabolism, proliferative activity, reactive oxygen species (ROS) levels, mitochondrial membrane potential (ΔΨm), and apoptosis. The cells showed a typical spindle-shaped morphology with centrally located oval nuclei. The cells were identified as fibroblasts by staining for vimentin. In vitro culture after the 1st, 3rd, and 10th passages did not alter most of the evaluated parameters.

Cells in the 3rd and 10th passages showed a reduction in ROS levels (p < 0.05). The ultrastructure revealed morphological damage in the prolongments, and nuclei of cells derived from the 3rd and 10th passages. Moreover, cryopreservation resulted in a reduction in ΔΨm compared with that of noncryopreserved cells, suggesting that the optimization of cryopreservation methods for puma fibroblasts is essential. In conclusion, we found that viable fibroblasts could be obtained from puma skin, with slight changes after the 10th passage in in vitro culture and cryopreservation. This is the first report on the development of cell lines derived from pumas.

Preservation and transportation are essential for the clinical application of chimeric antigen receptor T (CAR-T) cells. This study aimed to optimize a cryopreservation solution for CAR-T cells and evaluate the antitumor efficiency of CAR-T cells using this optimized solution in vitro and in vivo. First, the stability of the cryopreservation solution for CAR-T infusion was detected by the L27 (3⁷) orthogonal experiment. Subsequently, osmolality and pH were analyzed for the preservation reagent. Additionally, apoptosis and CAR expression of CAR-T cells were measured by flow cytometry, and the cytotoxicity was determined by calcein-AM staining. The results showed that cryopreservation solutions used in this study demonstrated high chemical stability, which induced only 2% CAR-T cells apoptosis in optimal solutions, which were slightly lower than other commercial solutions. Moreover, the CAR expression was not significantly affected by preservation with these solutions. There were no significant differences in the cytotoxicity between fresh and thawed CAR-T cells cryopreserved in the cryopreservation solutions in vivo and in vitro. This study developed a new cryopreservation solution for CAR-T cells, and it was safe and also had negligible effects on the CAR-T cells antitumor activity.