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Somatic cells can be used for rescuing wild mammals of ecological and economic importance, such as red-rumped agouti, through their application in advanced technologies. Thus, appropriate cell isolation, culture, and storage through cryopreservation can ensure the future safe use of these cells. We aimed to establish and evaluate the effects of culture time (second, fifth, and eighth passages) and cryopreservation on the morphology, viability, metabolism, proliferative activity, reactive oxygen species (ROS) levels, mitochondrial membrane potential (ΔΨm), and apoptosis on somatic cells derived from red-rumped agouti skin. Initially, we identified six dermal fibroblast lines by morphology, immunophenotyping, and karyotyping assays. In vitro culture after the second, fifth, and eighth passages, as well as the cryopreservation conditions used did not affect the metabolism or level of apoptosis. Nevertheless, cells in the fifth passage featured a reduction in proliferative activity and an increase in ROS levels when compared to second and eighth passage cells. Moreover, cryopreservation resulted in reduced ΔΨm when compared to non-cryopreserved cells. Additionally, cryopreserved cells showed a reduction in viability immediately after thawing; nevertheless, the viability of these cells was re-established after 11 days of in vitro culture and was similar to that of non-cryopreserved cells. In conclusion, we have shown that viable fibroblasts can be obtained from red-rumped agouti skin, featuring minimal changes after eight passages in in vitro culture systems. Additionally, adjustments to the cryopreservation protocol are necessary to reduce cellular oxidative stress caused by low temperatures. • Viable fibroblasts can be obtained from red-rumped agouti skin. • In vitro culture system until the eight passage does not affect the quality of agouti skin fibroblasts. • An optimization of the composition of the cryopreservation solution could increase cellular quality after thawing in agoutis. • Our data provide an important basis for the formation of a cell bank for red-rumped agouti. [ABSTRACT FROM AUTHOR]

Cellular reprogramming mainly involves induction of reactivation of genes responsible for nuclear plasticity, a process that can be performed in vitro through production of cloned embryos by somatic cell nuclear transfer or by induction of cells into the pluripotent state through exogenous transcription factor expression. While these techniques are already well known and utilized in mice and rats, their application in other rodent species would be greatly beneficial, especially for conservation purposes. Within the diverse Rodentia order, wild species stand out as they play an important role in balancing the ecosystem by facilitating seed diversion, soil aeration, and consequently, reforestation. Many of these species are currently approaching extinction, and application of techniques, such as nuclear reprogramming, aimed at species conservation and multiplication and to produce stem cells is of interest. Thus, in this review, we aimed to present the evolution and success of nuclear reprogramming, mainly highlighting its potential application for the conservation of wild rodents. [ABSTRACT FROM AUTHOR]

Currently, it has been observed that a considerable segment of the jaguar population is declining mainly because of hunting, and destruction and fragmentation of habitat. Given this scenario, efforts of the scientific community have been concentrated on the development of conservation strategies, such as the formation and use of somatic sample banks. We aimed to assess the effects of cryopreservation techniques of the ear skin of jaguar [slow freezing (SF) or direct vitrification in cryovials (DVC) or solid-surface vitrification (SSV)] on the morphological analysis and cell ability during the culture. All cryopreserved fragments regardless of the technique used, showed a reduction in the dermis and total thickness of the skin. Although a collagen matrix similar to the control group (fresh) has been observed only for the fragments from SF and SSV groups, all cryopreserved techniques were able to maintain normal patterns of the fibroblasts. Moreover, DVC and SSV methods maintained the proliferative activity of the tissues even after warming. After the culture, SF and SSV techniques were efficient for the recovery of the somatic cells according to most of the evaluated parameters, especially with regard to the duration of culture and cell metabolic activity. In conclusion, SSV was found to be a more efficient technique for cryopreserving jaguar skin when compared to DVC and SF. These results are relevant for the formation of somatic resource banks of this species, directed at cryopreserving adequate samplings of different individuals and generations for future applications in regenerative medicine, and assisted reproductive technologies. • SSV was the most adequate for cryopreservation of jaguar ear skin. • SSV can be applied to skin cryopreservation for the genetic conservation. • The biological reserve for this endangered species opens possibilities for the storage of genes that would be lost. [ABSTRACT FROM AUTHOR]

Considering the importance of efficiently obtaining somatic resource banks as an ex-situ conservation strategy for wild mammals, we evaluated two techniques (slow freezing - SF and solid-surface vitrification - SSV) for the cryopreservation of ear cartilage and skin from six-banded armadillos. Additionally, we analyzed the effects of two combinations of intracellular cryoprotectants (3.0 M or 6.0 M ethylene glycol - EG and dimethyl sulfoxide - Me 2 SO) on SSV. Tissues not subjected to cryopreservation were used as controls. All samples were evaluated for morphological analysis and cell ability during culture. The thickness of the basal layer was similar to the control only for tissues derived from SF, while SSV ensured the preservation of the cartilage thickness. Moreover, fragments derived from SF and SSV, especially in the 3.0 M EG-Me 2 SO group, resulted in dermis thickness and total skin similar to the control. All cryopreserved techniques maintained normal patterns of the fibroblasts, epidermal cells, and melanocytes. While only SF-derived fragments maintained the number of degenerated chondrocytes similar to the control, no difference was observed between groups for normal chondrocytes and lacunae. Moreover, SSV maintained the collagen fibers percentage of the tissues even after warming. After culture, SF and SSV techniques were efficient for the recovery of the somatic cells in all parameters evaluated, except the day of subconfluence, which was greater for the SSV group with 6.0 M EG-Me 2 SO. In summary, SSV, especially with 3 M EG-Me 2 SO, was as efficient as SF in preserving ear skin and cartilage derived from six-banded armadillos. • Solid-surface vitrification with 3 M EG-Me 2 SO was as efficient as slow freezing in preserving skin of six-banded armadillos. • Solid-surface vitrification is suggested as the most convenient technique than slow freezing for six-banded armadillos • The efficiency of the vitrification depends on the use of lower concentrations of permeating cryoprotectants at 3 M • Our data provide an important basis for the formation of tissue bank for six-banded armadillos. [ABSTRACT FROM AUTHOR]

The use of natural antioxidants in culture media can be an alternative to minimize the negative effects of oxidative stress produced by culture conditions. Essential oil from Syzygium aromaticum (EOSA) has therapeutic properties, including antioxidant activity in different cell types, and could be an interesting antioxidant agent during in vitro maturation (IVM) of bovine oocytes. Therefore, we sought to evaluate the antioxidant effect of the EOSA on bovine IVM, levels of reactive oxygen species (ROS), mitochondrial membrane potential (ΔΨm), and subsequent preimplantation embryonic development. Then, viable oocytes were matured in vitro under five sets of conditions: EOSA0 (without antioxidants), EOSA10 (10 μg/mL of EOSA), EOSA15 (15 μg/mL of EOSA), EOSA20 (20 μg/mL of EOSA), and CYS (100 μM of cysteamine). These oocytes were used in three experiments. In the first experiment, oocytes were evaluated for IVM according to the expansion and viability of cumulus cells, the presence of the first polar body, and metaphase II. In the second experiment, denuded oocytes were evaluated for an antioxidant effect by labeling them with H2DCFDA (ROS levels) and MitoTracker Red (ΔΨm). In the third experiment, denuded matured oocytes were artificially activated and embryos were cultured for eight days. In the first experiment, no difference was observed in the IVM rates (P > 0.05). Nevertheless, EOSA15, EOSA20, and CYS improved the viability of cumulus cells after IVM, with EOSA20 viability higher than that of EOSA0 (P < 0.05). In the second experiment, although no difference has been observed for ROS levels (P > 0.05), oocytes derived from the EOSA15, EOSA20, and CYS groups showed significantly lower ΔΨm compared to the EOSA0 group. In the third experiment, although no difference in cleavage rates was observed, EOSA20 improved the blastocyst/total oocyte and blastocyst/cleavage oocyte rates when compared to EOSA0 (P < 0.05). Moreover, the rates of the EOSA20 group were similar to that of the CYS group (P > 0.05). Additionally, embryos derived from EOSA15 and EOSA20 showed a higher number of cells when compared to those derived from EOSA0 (P < 0.05). Therefore, EOSA, at 20 μg/mL, increased the viability of cumulus cells, promoted a reduction of in ΔΨm, and improved embryonic development in bovine oocytes. In conclusion, EOSA, added to the IVM medium, could be an interesting alternative for the reduction of damage caused by the oxidative stress in bovine oocytes. • EOSA at 20 μg/mL during in vitro bovine oocyte maturation improves parthenogenetic embryonic development. • EOSA at 15 μg/mL, and 20 μg/mL showed lower ΔΨm in bovine oocytes when compared to oocytes matured in the absence of EOSA. • Viability of cumulus cells derived from oocytes matured with EOSA at 20 μg/mL was higher than in oocytes matured in the absence of EOSA. [ABSTRACT FROM AUTHOR]

In vitro culture of fibroblasts is a technique based on cell isolation, physiological characterization, and cryopreservation. This technique has not been described for Galea spixii , therefore, it can be used to learn about its cellular biology and genetic diversity. We established fibroblast lines of six G. spixii individuals from several passages (second, fifth, eighth, and tenth) and cryopreserved them. Fibroblasts recovered from skin biopsies were identified based on morphology, immunocytochemistry, and karyotyping. The cells were analyzed for morphology, ultrastructure, viability, proliferation, metabolism, oxidative stress, bioenergetic potential, and apoptosis before and after cryopreservation. After the eighth passage, the fibroblasts showed morphological and karyotypic changes, although their viability, metabolism, and proliferation did not change. An increase in oxidative stress and bioenergetic potential from the fifth to the eighth passages were also observed. Post cryopreservation, cell damage with respect to the ultrastructure, viability, proliferative rate, apoptotic levels, oxidative stress, and bioenergetic potential were verified. Fibroblasts up to the tenth passage could be cultured in vitro. However, cells at the fifth passage were of better quality to be used for reproductive techniques. Additionally, optimization of the cryopreservation protocol is essential to improve the physiological parameters of these cells. [Display omitted] • The G. spixii can contribute as a wild rodent model for biological, cellular, and molecular studies. • Fibroblastic lines of G. spixii up to the fifth passage show fewer morphological and physiological alterations. • Although the cryopreservation of the fibroblastic lines has been used efficiently, the optimization is necessary. [ABSTRACT FROM AUTHOR]

To optimize the protocols for assisted reproductive techniques (ARTs) in collared peccary (Pecari tajacu Linnaeus, 1758), we evaluated various conditions for oocyte in vitro maturation (IVM) and chemical activation. Initially, we assessed the IVM rates, cumulus -oocyte complex (COC) quality, and oocyte morphometry in the absence or presence of epidermal growth factor (EGF). There was no difference between the COCs matured in absence or presence of EGF for the expansion of cumulus cells (97.6% ± 1.2 vs. 100% ± 0.0), presence of first polar body (65.9% ± 1.2 vs. 70.5% ± 1.8), nuclear status in second metaphase (62.5% ± 11.6 vs. 68.4% ± 4.9), cytoplasmic maturation (100.0% ± 0.7 vs. 75.0% ± 0.7), reactive oxygen species levels (0.5 ± 0.2 vs. 0.3 ± 0.1), and mitochondrial membrane potential (1.1 ± 0.2 vs. 1.1 ± 0.1). However, the zona pellucida thickness of matured COCs was reduced in the presence of EGF. Thus, the EGF group was used for further experiments. The oocytes were artificially activated with ionomycin and four secondary activator combinations [6-dimethylaminopurine (6D), 6D and cytochalasin B (6D + CB), cycloheximide (CHX), and CHX and CB (CHX + CB)]. The effect of immature COCs based on cumulus cell layers and cytoplasm homogeneity (GI and GII or GIII COCs) on embryonic development and quality was evaluated. There was no difference in the cleavage rates among the groups of secondary activators. The cleavage rates of embryos derived from GI/GII and GIII COCs were greater than 72.2% and 25.0%, respectively. Moreover, treatment with CHX showed a reduction in the cleavage rate of embryos derived from GIII COCs when compared to the cleavage rate of embryos derived from GI/GII COCs (P < 0.05). Nevertheless, higher rates of blastocyst/total GI and GII COCs were observed in the 6D group (27.6% ± 0.3) compared to CHX group (6.9% ± 0.3). Additionally, only 6D treatment resulted in the production of embryos derived from GIII COCs (25.0% ± 0.2). The percentage of the ICM/total cell ratio was also greater in blastocysts derived from 6D (42.5% ± 19.0), 6D + CB (37.9% ± 21.9), and CHX + CB (43.8% ± 19.6) groups when compared to CHX (3.6% ± 0.1) group. Thus, the combination of ionomycin and 6D could produce collared peccary embryos by activation of both GI/GII COCs and GIII COCs. These optimized IVM conditions using EGF and chemical activation using ionomycin and 6D in collared peccaries form the first steps for establishing ARTs to conserve this species. • Ionomycin with 6-DMAP could produce collared peccary blastocysts by activation of both GI/GII COCs and GIII COCs. • Cytochalasin B was beneficial for collared peccary oocyte activation in the presence of cycloheximide. • EGF can be used to supplement the maturation medium for a greater quality of matured collared peccary oocytes. [ABSTRACT FROM AUTHOR]

Focusing on its application in reproductive biotechnology, we evaluated the effects of the essential oil of Syzygium aromaticum (EOSA) on bovine epididymal sperm quality variables, including morphology, membrane functional integrity, membrane structural integrity, mitochondrial activity, metabolic activity, motility and oxidative stress by reactive oxygen species (ROS) levels. Bovine spermatozoa from eight males were incubated into the following groups: EOSA0 (without EOSA), EOSA10 (10 μg/ml of EOSA), EOSA15 (15 μg/ml of EOSA) and EOSA20 (20 μg/ml of EOSA); the incubation time with and without the EOSA was 1 or 6 hr. None of the sperm quality variables presented difference among the EOSA concentrations. However, the incubation time had a significant effect on the membrane functional integrity, membrane structural integrity, mitochondrial activity, progressive motility and some kinetic parameters. The effect of interaction among EOSA and incubation time was significant only on ROS levels. Spermatozoa incubated in the presence of 15 μg/ml of the EOSA for 1 hr had significantly reduced ROS levels compared with all other groups in the same time. In conclusion, the EOSA at a concentration of 15 µg/ml has antioxidant effects and protects bovine epididymal spermatozoa; hence, the EOSA may potentially be used in the field of reproductive biotechnology. [ABSTRACT FROM AUTHOR]