Serum starvation is as efficient as roscovitine on the cycle synchronization in G0/G1 of red-rumped agouti fibroblasts.

Fibroblast cycle synchronization in G0/G1 is an essential step for nuclear reprogramming by cloning or induced cells to pluripotency. Considering the diversity among rodents and the ecological and scientific importance of these animals, we compared the contact inhibition, serum starvation, and 10 µM of roscovitine as methods of synchronization of red-rumped agouti fibroblasts. The effects of each protocol were evaluated on the percentage of cycle phase, morphology, viability, and apoptosis levels. The results showed that culturing the cells to serum starvation for 24 h (75.9%), 48 h (81.6%), 72 h (86.2%), 96 h (84.0%), and 120 h (83.7%) yielded a significantly higher percentage of cells arrested in the G0/G1 (P < 0.05) phase than cells not subjected to any cell cycle synchronization method (31.4%). Also, this effect was not different between the times of 48 and 120 h (P > 0.05). A similar response was observed for cells cultured with roscovitine for 12 h (86.9%), 24 h (74.8%), and 48 h (81.7%), with a higher percentage of synchronized cells in G0/G1 compared to cells not submitted to any synchronization treatment (52.2%). Nevertheless, this effect was best evidenced at 12 h (P < 0.05). Also, the contact inhibition for 24-120 h could not synchronize cells in G0/G1, with values ranging from 70.9 to 77.9% (P > 0.05). Moreover, no difference was observed for morphology, viability, and apoptosis levels in any synchronization method (P > 0.05). Therefore, serum starvation is as efficient as roscovitine on cycle synchronization in G0/G1 of red-rumped agouti fibroblasts.

Long-term preservation of established fibroblast lines from six-banded armadillos (Euphractus sexcintus, Linnaeus, 1758) by extended passage and cryopreservation.

Establishing new somatic cell cultures has raised significant attention as an effective and convenient way to preserve genetic samples for different applications. Although many lines have been established in model animals, none derived from six-banded armadillo species is currently available. We report the successful isolation and characterization of fibroblasts from six-banded armadillos, evaluating the cell quality after extended culture and cryopreservation. Initially, we collected ear skin from five captive adult individuals and identified fibroblast lines by morphology, karyotyping, and immunophenotyping assays. The isolated fibroblasts were evaluated after several passages (fourth, seventh, and tenth passages) and cryopreservation by slow freezing. Cell morphology, viability, metabolism, proliferative activity, mitochondrial membrane potential, and apoptosis levels were analyzed. The skin explants had great adhesion, and cell outgrowth could be seen after 3-6 d. The cells were verified as fibroblasts at the fourth passage by vimentin expression and normal karyotype (2n = 58). The viability remained high (> 87%) and constant from the fourth to the tenth passage (p > 0.05). The passages did not change the cell morphology and metabolic and growth rates. Moreover, cryopreservation did not affect most evaluated parameters; post-thawed cells maintained their viability, growth, metabolism, and apoptosis levels. Nevertheless, cryopreservation increased mitochondrial membrane permeability and cell population doubling time compared to non-cryopreserved cells (p < 0.05). In summary, viable fibroblasts can be obtained from six-banded armadillo skin while conserving their quality as the number of passages increases and featuring few changes after cryopreservation.

Full confluency, serum starvation, and roscovitine for inducing arrest in the G0/G1 phase of the cell cycle in puma skin-derived fibroblast lines.

The puma population is constantly decreasing, and cloning by somatic cell nuclear transfer can be used to conserve the species. One of the factors determining the success of the development of cloned embryos is the cell cycle stage of the donor cells. We evaluated the effects of full confluency (~100%), serum starvation (0.5% serum), and roscovitine (15 µM) treatments on the cell cycle synchronization in G0/G1 of puma skin-derived fibroblasts by flow cytometric analysis. Also, we assessed the effects of these synchronization methods on morphology, viability, and apoptosis levels using microscopy tools. The results showed that culturing the cells to confluence for 24 h (84.0%), 48 h (84.6%), and 72 h (84.2%) and serum starvation for 96 h (85.4%) yielded a significantly higher percentage of cells arrested in the G0/G1 (P 0.05) phase than cells not subjected to any cell cycle synchronization method (73.9%). Nevertheless, while serum starvation reduced the percentage of viable cells, no difference was observed for the full confluence and roscovitine treatments (P 0.05). Moreover, roscovitine for 12 h (78.6%) and 24 h (82.1%) was unable to synchronize cells in G0/G1 (P 0.05). In summary, full confluency induces puma fibroblast cell cycle synchronization at the G0/G1 stage without affecting cell viability. These outcomes may be valuable for planning donor cells for somatic cell nuclear transfer in pumas.

Ex Situ Approaches for the Conservation of Genetic Resources in the Superorder Xenarthra.

Xenarthra-a superorder of placental mammals endemic to the Neotropics-is represented by armadillos, anteaters, and sloths. Considering their long history in the Americas, extant xenarthrans represent an important group for understanding the impact of past environmental changes on species diversification and serve key ecological functions as ecosystem engineers. Unfortunately, most wild xenarthran populations are at risk, due primarily to anthropogenic activities, necessitating urgent conservation efforts. Moreover, the paucity of information on some species has rendered population estimation and, consequently, conservation management challenging. In addition, relatively few groups are researching this superorder, perhaps because fieldwork with armadillos, anteaters, or sloths and their captive care are challenging tasks. Nevertheless, dedicated research and efforts to ensure the long-term conservation of these animals are deemed essential. In this context, cryobanks are a practical approach for breeding and maintaining genetic diversity in wildlife, and they are important tools for assisting and improving both ex situ and in situ conservation strategies. Therefore, cryopreservation of biological resources may be a promising strategy for conserving xenarthrans. Specifically, semen cryopreservation, which has already been applied in some species, may be the most effective strategy for this group. The present article provides an overview of ex situ conservation of xenarthrans, which will contribute to the development and implementation of additional strategies for protecting these unique mammals.

Full confluency, serum starvation, and roscovitine for inducing arrest in the G0/G1 phase of the cell cycle in puma skin-derived fibroblast lines

The puma population is constantly decreasing, and cloning by somatic cell nuclear transfer can be used to conserve the species. One of the factors determining the success of the development of cloned embryos is the cell cycle stage of the donor cells. We evaluated the effects of full confluency (~100%), serum starvation (0.5% serum), and roscovitine (15 µM) treatments on the cell cycle synchronization in G0/G1 of puma skin-derived fibroblasts by flow cytometric analysis. Also, we assessed the effects of these synchronization methods on morphology, viability, and apoptosis levels using microscopy tools. The results showed that culturing the cells to confluence for 24 h (84.0%), 48 h (84.6%), and 72 h (84.2%) and serum starvation for 96 h (85.4%) yielded a significantly higher percentage of cells arrested in the G0/G1 (P < 0.05) phase than cells not subjected to any cell cycle synchronization method (73.9%). Nevertheless, while serum starvation reduced the percentage of viable cells, no difference was observed for the full confluence and roscovitine treatments (P > 0.05). Moreover, roscovitine for 12 h (78.6%) and 24 h (82.1%) was unable to synchronize cells in G0/G1 (P > 0.05). In summary, full confluency induces puma fibroblast cell cycle synchronization at the G0/G1 stage without affecting cell viability. These outcomes may be valuable for planning donor cells for somatic cell nuclear transfer in pumas.(AU)

Establishment, characterization, and cryopreservation of cell lines derived from red-rumped agouti (Dasyprocta leporina Linnaeus, 1758) - A study in a wild rodent.

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.

Bancos de recursos somáticos: Como eles contribuem para a conservação de grandes felídeos? / Somatic resource bank: How they contribute to the conservation of large felines?

Distintas estratégias de conservação têm sido adotadas visando a manutenção e recuperação da biodiversidade, especialmente de espécies que se encontram em diferentes níveis de ameaça à extinção. Neste grupo de espécies, encontram-se os grandes felídeos, os quais em virtude das ações antrópicas, como a destruição e a fragmentação de habitat, necessitam de esforços voltados para a conservação de seu material genético. Uma estratégia empregada para essa finalidade consiste nos bancos de recursos somáticos, os quais são definidos como a criopreservação de tecidos e células somáticas oriundas de diferentes populações. Essas amostras biológicas, quando adequadamente conservadas, são elementoschave para a multiplicação das espécies por meio de seu emprego na clonagem por transferência nuclear e indução de células à pluripotência. Assim, o objetivo desta revisão é abordar os aspectos relevantes envolvidos na formação de bancos de recursos somáticos em grandes felídeos, destacando os estudos desenvolvidos pelo Laboratório de Biotecnologia Animal (LBA), da Universidade Federal Rural do Semi-Árido (UFERSA) em onças-pintadas e onças-pardas.

Different conservation strategies have been adopted to maintain and recover biodiversity, especially for species that are at different levels of threat to extinction. In this group of species are the large felids, which, due to anthropic actions, such as habitat destruction and fragmentation, require efforts aimed at the conservation of their genetic material. A strategy employed for this purpose consists of somatic resource banks, which are defined as the cryopreservation of tissues and somatic cells from different populations. These biological samples, when properly preserved, are key elements for the multiplication of species through their use in cloning by nuclear transfer and induction of cells to pluripotency. Thus, the aim of this review is to address the relevant aspects involved in the formation of somatic resource banks in large felids, highlighting the studies carried out by the Laboratory of Animal Biotechnology (LBA) of the Federal Rural University of the Semi-Arid (UFERSA) in jaguars and pumas.

A Comparative Approach of Cellular Reprogramming in the Rodentia Order.

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.

Effect of supplementation of Aloe vera extracts in cold storage media and cryopreservation of domestic cat epididymal spermatozoa.

This study evaluated the effect of the extract of Aloe vera at concentrations of 10% and 20% on the cryopreservation of sperm from the epididymis of domestic cats. Epididymal spermatozoa were recovered using the flotation technique and used in the treatments: control (TRIS-egg yolk at 20%), T10% (TRIS plus 10% of A. vera extract), and T20% (TRIS plus 20% of A. vera extract). The spermatozoa were subjected to 4ºC for 60 minutes, followed by 20 minutes in nitrogen vapors, and stored in a cryogenic cylinder. The samples were thawed at 37°C for 30 seconds. The sperm motility decreased (P<0.05) after thawing in the three treatments. Only the spermatozoa in the control treatment maintained post-thawing vigor. The viability of spermatozoa decreased in the treatments with A. vera (P<0.05). According to the hypoosmotic test, all treatments maintained the sperm membrane functionality (P>0.05) during freezing; however, after thawing, it decreased (P<0.05) in the T10% and T20% treatments. The morphology and chromatin condensation of spermatozoa did not differ, regardless of the treatments and time of evaluation (P>0.05). The effect of the crude A. vera extract was not satisfactory on the cryopreservation of epididymal spermatozoa of domestic cats after thawing; although the motility of spermatozoa was similar to that found with the use of egg yolk, and it presented maintenance of the chromatin integrity. However, it is necessary to understand the action of the substances present in A. vera with the feline spermatozoa, well as the standardization and adjustment of physicochemical characteristics aiming at the future application of the vegetal extract.

Production of collared peccary (Pecari tajacu Linnaeus, 1758) parthenogenic embryos following different oocyte chemical activation and in vitro maturation conditions.

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% ±â€¯1.2 vs. 100% ±â€¯0.0), presence of first polar body (65.9% ±â€¯1.2 vs. 70.5% ±â€¯1.8), nuclear status in second metaphase (62.5% ±â€¯11.6 vs. 68.4% ±â€¯4.9), cytoplasmic maturation (100.0% ±â€¯0.7 vs. 75.0% ±â€¯0.7), reactive oxygen species levels (0.5 ±â€¯0.2 vs. 0.3 ±â€¯0.1), and mitochondrial membrane potential (1.1 ±â€¯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.

Produção de embriões partenogenéticos de cutias usando Cloreto de Estrôncio e Citocalasina B / Production of parthenogenetic embryos from agoutis using strontium chloride and cytochalasin B

The study aimed to evaluate the effect of strontium chloride (SrCl2) with cytochalasin B (CB) on the activation of agouti oocytes matured in vitro for embryo production. Thus, ovaries were used for oocyte recovery by slicing. Subsequently, viable oocytes were destined for in vitro maturation (IVM) and after 24 h evaluated for the expansion and viability of the cumulus cells and presence of the first polar body (1PB). After IVM, oocytes were activated with a combination of 10 mM SrCl2 and 5 μg/mL CB for 6 h and evaluated for embryo development kinetics. Hence, 93.3% of cumulus cell expansion was observed, with 91.2% viability and 37.3% of oocytes with the presence of 1PB. Regarding embryonic development, 43.2% (19/44) of cleaved structures and 6.8% (3/44) of morulae were observed in relation to the number of oocytes and 18.8% (3/16) morulae in relation to the number of cleaved structures. Thus, the combination of SrCl2 with CB promoted the activation of oocytes matured in vitro from agouti resulting in morulae. Finally, with this study, fundamental steps for in vitro conservation through reproductive biotechniques were developed in agoutis.(AU)

Effect of supplementation of Aloe vera extracts in cold storage media and cryopreservation of domestic cat epididymal spermatozoa

This study evaluated the effect of the extract of Aloe vera at concentrations of 10% and 20% on the cryopreservation of sperm from the epididymis of domestic cats. Epididymal spermatozoa were recovered using the flotation technique and used in the treatments: control (TRIS-egg yolk at 20%), T10% (TRIS plus 10% of A. vera extract), and T20% (TRIS plus 20% of A. vera extract). The spermatozoa were subjected to 4ºC for 60 minutes, followed by 20 minutes in nitrogen vapors, and stored in a cryogenic cylinder. The samples were thawed at 37°C for 30 seconds. The sperm motility decreased (P<0.05) after thawing in the three treatments. Only the spermatozoa in the control treatment maintained post-thawing vigor. The viability of spermatozoa decreased in the treatments with A. vera (P<0.05). According to the hypoosmotic test, all treatments maintained the sperm membrane functionality (P>0.05) during freezing; however, after thawing, it decreased (P<0.05) in the T10% and T20% treatments. The morphology and chromatin condensation of spermatozoa did not differ, regardless of the treatments and time of evaluation (P>0.05). The effect of the crude A. vera extract was not satisfactory on the cryopreservation of epididymal spermatozoa of domestic cats after thawing; although the motility of spermatozoa was similar to that found with the use of egg yolk, and it presented maintenance of the chromatin integrity. However, it is necessary to understand the action of the substances present in A. vera with the feline spermatozoa, well as the standardization and adjustment of physicochemical characteristics aiming at the future application of the vegetal extract.(AU)

Produção de embriões partenogenéticos de cutias usando Cloreto de Estrôncio e Citocalasina B / Production of parthenogenetic embryos from agoutis using strontium chloride and cytochalasin B

The study aimed to evaluate the effect of strontium chloride (SrCl2) with cytochalasin B (CB) on the activation of agouti oocytes matured in vitro for embryo production. Thus, ovaries were used for oocyte recovery by slicing. Subsequently, viable oocytes were destined for in vitro maturation (IVM) and after 24 h evaluated for the expansion and viability of the cumulus cells and presence of the first polar body (1PB). After IVM, oocytes were activated with a combination of 10 mM SrCl2 and 5 μg/mL CB for 6 h and evaluated for embryo development kinetics. Hence, 93.3% of cumulus cell expansion was observed, with 91.2% viability and 37.3% of oocytes with the presence of 1PB. Regarding embryonic development, 43.2% (19/44) of cleaved structures and 6.8% (3/44) of morulae were observed in relation to the number of oocytes and 18.8% (3/16) morulae in relation to the number of cleaved structures. Thus, the combination of SrCl2 with CB promoted the activation of oocytes matured in vitro from agouti resulting in morulae. Finally, with this study, fundamental steps for in vitro conservation through reproductive biotechniques were developed in agoutis.

Effect of supplementation of Aloe vera extracts in cold storage media and cryopreservation of domestic cat epididymal spermatozoa

This study evaluated the effect of the extract of Aloe vera at concentrations of 10% and 20% on the cryopreservation of sperm from the epididymis of domestic cats. Epididymal spermatozoa were recovered using the flotation technique and used in the treatments: control (TRIS-egg yolk at 20%), T10% (TRIS plus 10% of A. vera extract), and T20% (TRIS plus 20% of A. vera extract). The spermatozoa were subjected to 4ºC for 60 minutes, followed by 20 minutes in nitrogen vapors, and stored in a cryogenic cylinder. The samples were thawed at 37°C for 30 seconds. The sperm motility decreased (P0.05) during freezing; however, after thawing, it decreased (P0.05). The effect of the crude A. vera extract was not satisfactory on the cryopreservation of epididymal spermatozoa of domestic cats after thawing; although the motility of spermatozoa was similar to that found with the use of egg yolk, and it presented maintenance of the chromatin integrity. However, it is necessary to understand the action of the substances present in A. vera with the feline spermatozoa, well as the standardization and adjustment of physicochemical characteristics aiming at the future application of the vegetal extract.

Antioxidant effects of the essential oil of Syzygium aromaticum on bovine epididymal spermatozoa.

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.

Effects of cryopreservation techniques on the preservation of ear skin - An alternative approach to conservation of jaguar, Panthera onca (Linnaeus, 1758).

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.

Syzygium aromaticum essential oil supplementation during in vitro bovine oocyte maturation improves parthenogenetic embryonic development.

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.

Estado atual da conservação a 4°C de tecidos somáticos derivados da pele em mamíferos / Current status of conservation at 4°C of somatic tissues derived from the skin in mammals

A clonagem por transferência nuclear de células somáticas consiste em uma atraente ferramenta para a conservação e multiplicação de espécies. A eficiência desta biotécnica depende da obtenção e seleção de células doadoras de núcleo derivadas da pele de indivíduos de interesse. Em alguns mamíferos encontrados em regiões de difícil acesso ou distantes de laboratórios especializados, o armazenamento a 4°C de tecidos somáticos da pele seria uma alternativa para a conservação do material genético desses animais. Contudo, o emprego desta técnica depende de alguns fatores, como os períodos e as condições de armazenamento a 4°C das amostras, os quais podem influenciar na recuperação das células após cultivo in vitro dos tecidos. Em mamíferos domésticos, estudos têm mostrado variações quanto ao período de estocagem e a presença de meio nutritivo. Já em mamíferos silvestres, apenas são relatados o uso da refrigeração como ferramenta de transporte em curto prazo. Assim, o objetivo desta revisão é apresentar as diferentes condições de armazenamento a 4°C de tecidos somáticos, evidenciando a importância dessa técnica para a conservação da biodiversidade.(AU)

Cloning by somatic cell nuclear transfer is an attractive tool for conservation and multiplication of species. The efficiency of this biotechnique depends on the obtaining and selection of nucleus donor cells derived from the skin of individuals of interest. In some mammals found in regions difficult to access or distant from specialized laboratories, the storage at 4°C of somatic tissues of the skin would be an alternative for the conservation of the genetic material of these animals. Nevertheless, the use of this technique depends on some factors, such as periods and storage conditions at 4°C of the samples, which may influence the recovery of the cells after tissue culture in vitro. In domestic mammals, studies have shown variations regarding the period of storage and the presence of nutrient medium. Already, in wild mammals, only is related the use of refrigeration as transportation tool in the short term. Thus, the aim of this review is to present the different conditions of storage at 4°C of somatic tissues, evidencing the importance of this technique for the conservation of biodiversity.(AU)

Estado atual da conservação a 4°C de tecidos somáticos derivados da pele em mamíferos / Current status of conservation at 4°C of somatic tissues derived from the skin in mammals

A clonagem por transferência nuclear de células somáticas consiste em uma atraente ferramenta para a conservação e multiplicação de espécies. A eficiência desta biotécnica depende da obtenção e seleção de células doadoras de núcleo derivadas da pele de indivíduos de interesse. Em alguns mamíferos encontrados em regiões de difícil acesso ou distantes de laboratórios especializados, o armazenamento a 4°C de tecidos somáticos da pele seria uma alternativa para a conservação do material genético desses animais. Contudo, o emprego desta técnica depende de alguns fatores, como os períodos e as condições de armazenamento a 4°C das amostras, os quais podem influenciar na recuperação das células após cultivo in vitro dos tecidos. Em mamíferos domésticos, estudos têm mostrado variações quanto ao período de estocagem e a presença de meio nutritivo. Já em mamíferos silvestres, apenas são relatados o uso da refrigeração como ferramenta de transporte em curto prazo. Assim, o objetivo desta revisão é apresentar as diferentes condições de armazenamento a 4°C de tecidos somáticos, evidenciando a importância dessa técnica para a conservação da biodiversidade.

Cloning by somatic cell nuclear transfer is an attractive tool for conservation and multiplication of species. The efficiency of this biotechnique depends on the obtaining and selection of nucleus donor cells derived from the skin of individuals of interest. In some mammals found in regions difficult to access or distant from specialized laboratories, the storage at 4°C of somatic tissues of the skin would be an alternative for the conservation of the genetic material of these animals. Nevertheless, the use of this technique depends on some factors, such as periods and storage conditions at 4°C of the samples, which may influence the recovery of the cells after tissue culture in vitro. In domestic mammals, studies have shown variations regarding the period of storage and the presence of nutrient medium. Already, in wild mammals, only is related the use of refrigeration as transportation tool in the short term. Thus, the aim of this review is to present the different conditions of storage at 4°C of somatic tissues, evidencing the importance of this technique for the conservation of biodiversity.

Uso de bancos somáticos visando a clonagem por transferência nuclear na conservação de mamíferos silvestres uma revisão / Use of somatic banks for cloning by nuclear transfer in the conservation of wild mammals a review

Embora amostras gonadais e embriões sejam os bancos biológicos de primeira escolha para o desenvolvimento de estratégias in vitro de conservação, células e tecidos somáticos têm surgido como interessantes alternativas, especialmente para a conservação de mamíferos silvestres criticamente ameaçados de extinção. Assim, para a multiplicação destas espécies, bancos somáticos têm sido empregados como fonte de carioplasto na clonagem por transferência nuclear de células somáticas, em pesquisas voltadas para a reprogramação nuclear e obtenção de células induzidas à pluripotência. Nesse sentido, para atender a essas finalidades, é necessário inicialmente conhecer as condições adequadas de criopreservação e manipulação desses tecidos e células, visando a manutenção adequada de todas as características biológicas importantes para o estabelecimento das posteriores biotecnologias. Assim, o objetivo desta revisão é conceituar e apresentar as principais etapas técnicas fundamentais no estabelecimento da criopreservação e manipulação in vitro de células e tecidos somáticos, visando o emprego promissor e adequado destes recursos biológicos na conservação de mamíferos silvestres.(AU)

Although gonadal samples and embryos are the first-choice biological banks for the development of in vitro conservation strategies, somatic cells and tissues have emerged as interesting alternatives, especially for the conservation of wild mammals critically endangered. Thus, for the multiplication of these species, somatic banks have been used as karyoplast source in the cloning by somatic cell nuclear transfer, in research aimed at nuclear reprogramming and obtaining cells induced by pluripotency. In this sense, in order to meet these purposes, it is necessary to initially know the proper conditions for cryopreservation and manipulation of these tissues and cells, aiming at the adequate maintenance of all biological characteristics for the establishment of subsequent biotechnologies. Thus, the aim of this review is to conceptualize and present the crucial technical steps in the establishment of cryopreservation and in vitro manipulation of somatic tissues and cells, aiming at the promising and adequate use of these biological resources in the conservation of wild mammals.(AU)