Heterologous in vitro fertilization and embryo production for assessment of jaguar (Panthera onca Linnaeus, 1758) frozen-thawed semen in different extenders.

Heterologous in vitro fertilization (IVF) is an important tool for assessing fertility of endangered mammals such as the jaguar, considering difficult access to females for artificial insemination and to obtain homologous oocytes. We aimed to evaluate the fertility of jaguar sperm cryopreserved with different extenders, using domestic cat oocytes to assess the development of hybrid embryos. Semen from four captive jaguars was obtained by electroejaculation. Samples were cryopreserved in powdered coconut water (ACP-117c) or Tris extender containing 20% egg yolk and 6% glycerol. Thawed spermatozoa were resuspended (2.0 × 106 spermatozoa/mL) in IVF medium and co-incubated with cat oocytes matured in vitro for 18 h. Presumptive zygotes were cultured for 7 days. After 48 h, cleavage rate was evaluated, and non-cleaved structures were stained for IVF evaluation. On days 5 and 7, the rate of morula and blastocyst formation was assessed. Data were analyzed using the Fisher exact test (p < 0.05). No difference was observed between ACP-117c and Tris extenders, respectively, for oocytes with 2nd polar body (2/51, 3.9 ± 2.9% vs. 2/56, 3.6 ± 3.1%), pronuclear structures (5/51, 9.8 ± 4.7% vs. 8/56, 14.3 ± 8.0%), and total IVF rates (7/36, 19.4 ± 5.0% vs. 10/37, 27.0 ± 13.8%). All the samples fertilized the oocytes, with 22.9 ± 3.2% (16/70) and 16.7 ± 3.6% (12/72) cleavage of mature oocytes for ACP-117c and Tris extenders, respectively. Morula rates of 4.3 ± 2.3% (3/70) and 5.6 ± 2.2% (4/72) were observed for ACP-117c and Tris, respectively. Only the Tris extender demonstrated blastocyst production (2/12, 16.7 ± 1.5% blastocyst/cleavage). We demonstrated that jaguar ejaculates cryopreserved using ACP-117c and Tris were suitable for IVF techniques, with blastocyst production by ejaculates cryopreserved in Tris. This is a first report of embryos produced in vitro using jaguar sperm and domestic cat oocytes through IVF.

Heterologous in vitro fertilization and embryo production for assessment of jaguar (Panthera onca Linnaeus, 1758) frozen-thawed semen in different extenders

Heterologous in vitro fertilization (IVF) is an important tool for assessing fertility of endangered mammals such as the jaguar, considering difficult access to females for artificial insemination and to obtain homologous oocytes. We aimed to evaluate the fertility of jaguar sperm cryopreserved with different extenders, using domestic cat oocytes to assess the development of hybrid embryos. Semen from four captive jaguars was obtained by electroejaculation. Samples were cryopreserved in powdered coconut water (ACP-117c) or Tris extender containing 20% egg yolk and 6% glycerol. Thawed spermatozoa were resuspended (2.0 × 106 spermatozoa/mL) in IVF medium and co-incubated with cat oocytes matured in vitro for 18 h. Presumptive zygotes were cultured for 7 days. After 48 h, cleavage rate was evaluated, and non-cleaved structures were stained for IVF evaluation. On days 5 and 7, the rate of morula and blastocyst formation was assessed. Data were analyzed using the Fisher exact test (p < 0.05). No difference was observed between ACP-117c and Tris extenders, respectively, for oocytes with 2nd polar body (2/51, 3.9 ± 2.9% vs. 2/56, 3.6 ± 3.1%), pronuclear structures (5/51, 9.8 ± 4.7% vs. 8/56, 14.3 ± 8.0%), and total IVF rates (7/36, 19.4 ± 5.0% vs. 10/37, 27.0 ± 13.8%). All the samples fertilized the oocytes, with 22.9 ± 3.2% (16/70) and 16.7 ± 3.6% (12/72) cleavage of mature oocytes for ACP-117c and Tris extenders, respectively. Morula rates of 4.3 ± 2.3% (3/70) and 5.6 ± 2.2% (4/72) were observed for ACP-117c and Tris, respectively. Only the Tris extender demonstrated blastocyst production (2/12, 16.7 ± 1.5% blastocyst/cleavage). We demonstrated that jaguar ejaculates cryopreserved using ACP-117c and Tris were suitable for IVF techniques, with blastocyst production by ejaculates cryopreserved in Tris. This is a first report of embryos produced in vitro using jaguar sperm and domestic cat oocytes through IVF.(AU)

Isolation, characterization, and cryopreservation of collared peccary skin-derived fibroblast cell lines.

BACKGROUND: Biobanking of cell lines is a promising tool of support for wildlife conservation. In particular, the ability to preserve fibroblast cell lines derived from collared peccaries is of significance as these wild mammals are unique to the Americas and play a large role in maintaining the ecosystem. We identified collared peccary fibroblasts by immunofluorescence and evaluated their morphology, growth and adherence capacity. Further, we monitored the viability and metabolic activity of the fibroblasts to determine the effects of passage number and cryopreservation on establishment of cell lines. METHODS: Skin biopsies were collected from the peripheral ear region from five adult animals in captivity. Initially, cells were isolated from fragments and cultured in the Dulbecco's modified Eagle medium supplemented with 10% fetal bovine serum and 2% antibiotic-antimycotic solution under a controlled atmosphere (38.5 °C, 5% CO2). We evaluated the maintenance of primary cells for morphology, adherence capacity of explants, explants in subconfluence, cell growth and absence of contamination. Moreover, we identified the fibroblast cells by immunofluorescence. Additionally, to evaluate the influence of the number of passages (first, third and tenth passage) and cryopreservation on establishment of cell lines, fibroblasts were analysed for the viability, metabolic activity, population doubling time (PDT), levels of reactive oxygen species (ROS), and mitochondrial membrane potential (ΔΨm). RESULTS: All explants (20/20) adhered to the dish in 2.4 days ± 0.5 with growth around the explants in 4.6 days ± 0.7, and subconfluence was observed within 7.8 days ± 1.0. Moreover, by morphology and immunocytochemistry analyses, cells were identified as fibroblasts which presented oval nuclei, a fusiform shape and positive vimentin staining. No contamination was observed after culture without antibiotics and antifungals for 30 days. While there was no difference observed for cell viability after the passages (first vs. third: P = 0.98; first vs. tenth: P = 0.76; third vs. tenth: P = 0.85), metabolic activity was found to be reduced in the tenth passage (23.2 ± 12.1%) when compared to that in the first and third passage (100.0 ± 24.4%, P = 0.006). Moreover, the cryopreservation did not influence the viability (P = 0.11), metabolic activity (P = 0.77), or PDT (P = 0.11). Nevertheless, a greater ΔΨm (P = 0.0001) was observed for the cryopreserved cells (2.12 ± 0.14) when compared to that in the non-cryopreserved cells (1.00 ± 0.05). Additionally, the cryopreserved cells showed greater levels of intracellular ROS after thawing (1.69 ± 0.38 vs. 1.00 ± 0.22, P = 0.04). CONCLUSIONS: This study is the first report on isolation, characterization and cryopreservation of fibroblasts from collared peccaries. We showed that adherent cultures were efficient for obtaining fibroblasts, which can be used as donor cells for nuclei for species cloning and other applications.

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.

Efeitos da suplementação do meio com diferentes antioxidantes durante a maturação in vitro de oócitos de bovinos sobre o status oxidativo / Effects of medium supplementation with different antioxidants during in vitro maturation of bovine oocytes on oxidative status

Although widely used as reproductive biotechnology in cattle, in vitro embryo production (IVEP) has variable efficiency. During in vitro maturation (IVM), supplementing the medium with antioxidant potential could be an affordable alternative to increase the efficiency of IVEP, requiring the evaluation of new components, such as eugenol, β-caryophyllene, and acetyl eugenol. Thus, bovine oocytes were matured according to different antioxidants. Metaphase II oocytes were identified by Hoechst staining. The oxidative status was measured by evaluation of reactive oxygen species (ROS), and glutathione (GSH). After eight repetitions, no difference was observed among groups containing antioxidants, being all these groups superior to negative control (p<0.05). The ROS levels decreased and GSH increased in oocytes matured with EUG (p<0.05). These results indicate apositive effect of eugenol on the oxidative status of oocytes matured with this compound, showing that eugenol can be an efficient supplement in the IVM of bovine oocytes.(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.(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.

Efeitos da suplementação do meio com diferentes antioxidantes durante a maturação in vitro de oócitos de bovinos sobre o status oxidativo / Effects of medium supplementation with different antioxidants during in vitro maturation of bovine oocytes on oxidative status

Although widely used as reproductive biotechnology in cattle, in vitro embryo production (IVEP) has variable efficiency. During in vitro maturation (IVM), supplementing the medium with antioxidant potential could be an affordable alternative to increase the efficiency of IVEP, requiring the evaluation of new components, such as eugenol, β-caryophyllene, and acetyl eugenol. Thus, bovine oocytes were matured according to different antioxidants. Metaphase II oocytes were identified by Hoechst staining. The oxidative status was measured by evaluation of reactive oxygen species (ROS), and glutathione (GSH). After eight repetitions, no difference was observed among groups containing antioxidants, being all these groups superior to negative control (p<0.05). The ROS levels decreased and GSH increased in oocytes matured with EUG (p<0.05). These results indicate apositive effect of eugenol on the oxidative status of oocytes matured with this compound, showing that eugenol can be an efficient supplement in the IVM of bovine oocytes.

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.

Uso da clonagem por transferência nuclear na conservação e multiplicação de mamíferos silvestres / Use of cloning by nuclear transfer in the conservation and multiplication of wild mammals

A clonagem por transferência nuclear de células somáticas (TNCS) desempenha um importante papel na conservação e na multiplicação de mamíferos silvestres, especialmente quando associada à TNCS interespecífica. Contudo, a clonagem ainda possui uma baixa eficiência, sendo necessária uma extensa busca por elucidações mais precisas das metodologias empregadas em suas etapas de preparo do carioplastos (células doadoras de núcleo), obtenção dos citoplastos (oócitos receptores), e reconstrução embrionária. Assim, o objetivo desta revisão é abordar os aspectos relevantes das etapas da clonagem aplicadas a mamíferos silvestres, descrevendo seus resultados promissores e avanços alcançados.

Cloning by somatic cell nuclear transfer (SCNT) plays an important role in the conservation and multiplication of wild mammals, especially when associated with interspecific SCNT. Nevertheless, cloning still has a low efficiency, requiring an extensive search for more precise elucidations of the methodologies used in its stages of preparation of the karyoplasts (nucleus donor cells), production of cytoplasts (receptor oocytes), and embryonic reconstruction. Thus, the purpose of this review is to address the relevant aspects of the cloning steps applied to wild mammals, describing their promising results and advances.

Uso da clonagem por transferência nuclear na conservação e multiplicação de mamíferos silvestres / Use of cloning by nuclear transfer in the conservation and multiplication of wild mammals

A clonagem por transferência nuclear de células somáticas (TNCS) desempenha um importante papel na conservação e na multiplicação de mamíferos silvestres, especialmente quando associada à TNCS interespecífica. Contudo, a clonagem ainda possui uma baixa eficiência, sendo necessária uma extensa busca por elucidações mais precisas das metodologias empregadas em suas etapas de preparo do carioplastos (células doadoras de núcleo), obtenção dos citoplastos (oócitos receptores), e reconstrução embrionária. Assim, o objetivo desta revisão é abordar os aspectos relevantes das etapas da clonagem aplicadas a mamíferos silvestres, descrevendo seus resultados promissores e avanços alcançados.(AU)

Cloning by somatic cell nuclear transfer (SCNT) plays an important role in the conservation and multiplication of wild mammals, especially when associated with interspecific SCNT. Nevertheless, cloning still has a low efficiency, requiring an extensive search for more precise elucidations of the methodologies used in its stages of preparation of the karyoplasts (nucleus donor cells), production of cytoplasts (receptor oocytes), and embryonic reconstruction. Thus, the purpose of this review is to address the relevant aspects of the cloning steps applied to wild mammals, describing their promising results and advances.(AU)

Influence of storage time and nutrient medium on recovery of fibroblast-like cells from refrigerated collared peccary (Pecari tajacu Linnaeus, 1758) skin.

Animal cloning is a promising technology for biodiversity conservation, and its success depends on the recovery of nucleus donor cells. Specifically for collared peccaries, found sometimes in regions that are difficult to access, the storage at 4-6°C of skin tissues would be an alternative for the conservation of genetic material. Therefore, we aimed to evaluate different storage periods and the presence of a nutrient medium at 4-6°C on the recovery of somatic cells from the skin of collared peccaries. To analyze cell recovery rates, ear explants were distributed in non-refrigerated samples and samples refrigerated for 10, 30, and 50 d in the absence or presence of nutrient medium. All explants were analyzed by histologically and cultured. Only the fragments stored for 50 d without medium showed an increase in the total thickness of skin. Moreover, increased storage period, regardless of the presence of medium, increased the halo number and reduced the metabolic activity. After culture, only the fragments stored without medium for 50 d did not yield any somatic cells. Cells recovered from explants stored for 10 d showed similar characteristics to these recovered from non-refrigerated explants, regardless of the presence of medium, including the day at which explants achieved attachment and the total time to reach subconfluence. In conclusion, viable cells can be recovered from somatic tissues of collared peccaries stored for up to 50 d in the presence of medium, and tissues refrigerated for up to 10 d in the presence of medium yielded more viable cells.

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.

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)

Use of natural antioxidants in in vitro mammalian embryo production / Uso de antioxidantes naturais na produção in vitro de embriões de mamíferos

In vitro embryo production (IVEP) contributes to the quantitative and qualitative aspects of animal reproduction. Nevertheless, inherent technical factors such as oxidative stress can negatively influence the result and this can impair cell metabolism, thus decreasing the rates of in vitro development, and necessitating the supplementation of culture medium with antioxidants. In this context, compounds of natural origin with this property have been highlighted because of the positive results obtained at different stages of IVEP. Thus, this review aims to present the results obtained by using natural antioxidants to minimize the effects of oxidative stress on gametes and embryos. A variety of natural isolated substances and mixtures (essential oils and extracts) have been studied for supplementation of IVEP media, at stages of in vitro maturation, sperm capacitation, in vitro fertilization, and in vitro development of embryos in different mammalian species. Generally, beneficial effects are observed according to the concentration used, thus demonstrating the potential of several natural antioxidants. Therefore, the main challenges in using these compounds as antioxidants during IVEP include proving their efficiency against free radicals and determining the best concentration at each stage. In addition, understanding the mechanisms of action of such antioxidants is crucial...(AU)

A produção in vitro de embriões (PIVE) contribui para os aspectos quantitativos e qualitativos da reprodução animal. Contudo, fatores inerentes da técnica, como o estresse oxidativo, podem influenciar negativamente o resultado e isso pode prejudicar o metabolismo celular, diminuindo assim as taxas de desenvolvimento in vitro, e exigindo a suplementação do meio de cultivo com antioxidantes. Neste contexto, os compostos de origem natural com essa propriedade têm se destacado devido aos resultados positivos obtidos em diferentes estágios da PIVE. Assim, esta revisão pretende apresentar os resultados obtidos usando antioxidantes naturais para minimizar os efeitos do estresse oxidativo em gametas e embriões. Uma variedade de substâncias isoladas e de misturas naturais (óleos essenciais e extratos) tem sido estudada para a suplementação de meios de PIVE, nas etapas de maturação in vitro, capacitação espermática, fecundação in vitro e desenvolvimento in vitro de embriões em diferentes espécies de mamíferos. Geralmente, os efeitos benéficos são observados de acordo com a concentração utilizada, demostrando assim o potencial positivo de vários antioxidantes naturais. Portanto, os principais desafios para o uso desses compostos como antioxidantes durante a PIVE incluem provar sua eficiência contra os radicais livres e determinar a melhor concentração em cada etapa. Além disso...(AU)

Use of natural antioxidants in in vitro mammalian embryo production / Uso de antioxidantes naturais na produção in vitro de embriões de mamíferos

In vitro embryo production (IVEP) contributes to the quantitative and qualitative aspects of animal reproduction. Nevertheless, inherent technical factors such as oxidative stress can negatively influence the result and this can impair cell metabolism, thus decreasing the rates of in vitro development, and necessitating the supplementation of culture medium with antioxidants. In this context, compounds of natural origin with this property have been highlighted because of the positive results obtained at different stages of IVEP. Thus, this review aims to present the results obtained by using natural antioxidants to minimize the effects of oxidative stress on gametes and embryos. A variety of natural isolated substances and mixtures (essential oils and extracts) have been studied for supplementation of IVEP media, at stages of in vitro maturation, sperm capacitation, in vitro fertilization, and in vitro development of embryos in different mammalian species. Generally, beneficial effects are observed according to the concentration used, thus demonstrating the potential of several natural antioxidants. Therefore, the main challenges in using these compounds as antioxidants during IVEP include proving their efficiency against free radicals and determining the best concentration at each stage. In addition, understanding the mechanisms of action of such antioxidants is crucial...

A produção in vitro de embriões (PIVE) contribui para os aspectos quantitativos e qualitativos da reprodução animal. Contudo, fatores inerentes da técnica, como o estresse oxidativo, podem influenciar negativamente o resultado e isso pode prejudicar o metabolismo celular, diminuindo assim as taxas de desenvolvimento in vitro, e exigindo a suplementação do meio de cultivo com antioxidantes. Neste contexto, os compostos de origem natural com essa propriedade têm se destacado devido aos resultados positivos obtidos em diferentes estágios da PIVE. Assim, esta revisão pretende apresentar os resultados obtidos usando antioxidantes naturais para minimizar os efeitos do estresse oxidativo em gametas e embriões. Uma variedade de substâncias isoladas e de misturas naturais (óleos essenciais e extratos) tem sido estudada para a suplementação de meios de PIVE, nas etapas de maturação in vitro, capacitação espermática, fecundação in vitro e desenvolvimento in vitro de embriões em diferentes espécies de mamíferos. Geralmente, os efeitos benéficos são observados de acordo com a concentração utilizada, demostrando assim o potencial positivo de vários antioxidantes naturais. Portanto, os principais desafios para o uso desses compostos como antioxidantes durante a PIVE incluem provar sua eficiência contra os radicais livres e determinar a melhor concentração em cada etapa. Além disso...

Influence of commercially available follicle stimulating hormone on the in vitro maturation of bovine oocytes / Influência do hormônio folículo-estimulante disponível comercialmente sobre a maturação in vitro de oócitos bovinos

The work aimed (Experiment I) to compare commercial representations of porcine follicle stimulating hormone (FSH, Pluset® vs. Folltropin®) in concentration (10 μg/mL) and time (24 h) standard (more used in protocols of in vitro maturation, IVM); (Experiment II) to evaluate the best incubation time (6 h vs. 16 h vs. 24 h) and, (Experiment III) to analyze varying concentrations (1.0 μg/mL vs. 2.5 μg/mL vs. 10.0 μg/mL) of representations of FSH on the IVM of bovine oocytes. Thus, oocytes were recovered and submitted to IVM under appropriate conditions. After the IVM, oocytes were evaluated for expansion of cumulus cells (CCs), presence of the first polar body (1PB) and metaphase plate (MII). All the data were analyzed by the Fisher exact test (P < 0.05). Initially, in Experiment I, no difference (P > 0.05) was observed in maturation rates of the oocytes incubated with FSH Pluset® or Folltropin®, assessed by expansion of CCs (97.6% vs. 94.3%), presence of 1PB (76.6% vs. 69.4%) and MII (70.0% vs. 68.6%). In Experiment II, when the incubation time with FSH was evaluated, both Pluset® as Folltropin® showed lower rate of expansion of CCs when they were present only in the first 6 h of IVM. As for presence of 1PB, differences were observed in relation to Pluset® while Folltropin® showed similar results in all incubation times. Regarding the MII, no difference was observed between the incubation times with FSH Pluset® and Folltropin®. In Experiment III, no difference was observed in the expansion of CCs, presence of 1PB and MII for concentrations evaluated FSH Pluset® and Folltropin®. Therefore, the FSH Pluset® and Folltropin® have the same efficiency in IVM of bovine oocytes. Regarding the incubation time, it is recommended to maintain FSH (Pluset® or Folltropin®) throughout the period of IVM, since there was no difference in the results of presence of MII...(AU)

O trabalho objetivou (Experimento I) comparar representações comerciais do hormônio folículo estimulante suíno (FSH, Pluset® vs. Folltropin®) na concentração (10 μg/mL) e tempo (24 h) padrão (mais utilizados nos protocolos de maturação in vitro, MIV); (Experimento II) avaliar o melhor tempo de incubação (6 h vs. 16 h vs. 24 h) e, (Experimento III) analisar concentrações variáveis (1,0 μg/mL vs. 2,5 μg/mL vs. 10,0 μg/mL) das representações de FSH sobre a MIV de oócitos bovinos. Para tanto, oócitos foram recuperados e submetidos a MIV em condições adequadas. Após a MIV, oócitos foram avaliados quanto à expansão das células do cumulus (CCs), presença do primeiro corpúsculo polar (1CP) e placa metafásica (MII). Todos os dados foram analisados pelo teste exato de Fisher (P < 0,05). Inicialmente, no Experimento I, nenhuma diferença foi observada (P > 0,05) nas taxas de maturação de oócitos incubados com FSH Pluset® ou Folltropin®, avaliadas pela expansão das CCs (97,6% vs. 94,3%), presença de 1CP (76,6% vs. 69,4%) e MII (70,0% vs. 68,6%). No Experimento II, quanto ao tempo de incubação com FSH foi avaliado, tanto Pluset® quanto Folltropin® mostraram uma menor taxa de expansão de CCs somente nas primeiras 6 h de MIV. Quanto à presença de 1CP, diferenças foram observadas em relação ao Pluset® enquanto o Folltropin® mostrou resultados similares em todos os tempos de incubação. Em relação à MII, nenhuma diferença foi observada entre os tempos de incubação com o FSH Pluset® e Folltropin®. No Experimento III, nenhuma diferença foi observada na expansão das CCs, presença do 1CP e MII para as concentrações avaliadas de FSH Pluset® e Folltropin®. Portanto, FSH Pluset® e Folltropin® tem a mesma eficiência na MIV de oócitos bovinos. Em relação ao tempo de incubação, é recomendado manter o FSH (Pluset® or Folltropin®) ao longo do período da MIV, uma vez que nenhuma diferença foi observada nos resultados da presença de MII...(AU)

Influence of commercially available follicle stimulating hormone on the in vitro maturation of bovine oocytes / Influência do hormônio folículo-estimulante disponível comercialmente sobre a maturação in vitro de oócitos bovinos

The work aimed (Experiment I) to compare commercial representations of porcine follicle stimulating hormone (FSH, Pluset® vs. Folltropin®) in concentration (10 μg/mL) and time (24 h) standard (more used in protocols of in vitro maturation, IVM); (Experiment II) to evaluate the best incubation time (6 h vs. 16 h vs. 24 h) and, (Experiment III) to analyze varying concentrations (1.0 μg/mL vs. 2.5 μg/mL vs. 10.0 μg/mL) of representations of FSH on the IVM of bovine oocytes. Thus, oocytes were recovered and submitted to IVM under appropriate conditions. After the IVM, oocytes were evaluated for expansion of cumulus cells (CCs), presence of the first polar body (1PB) and metaphase plate (MII). All the data were analyzed by the Fisher exact test (P 0.05) was observed in maturation rates of the oocytes incubated with FSH Pluset® or Folltropin®, assessed by expansion of CCs (97.6% vs. 94.3%), presence of 1PB (76.6% vs. 69.4%) and MII (70.0% vs. 68.6%). In Experiment II, when the incubation time with FSH was evaluated, both Pluset® as Folltropin® showed lower rate of expansion of CCs when they were present only in the first 6 h of IVM. As for presence of 1PB, differences were observed in relation to Pluset® while Folltropin® showed similar results in all incubation times. Regarding the MII, no difference was observed between the incubation times with FSH Pluset® and Folltropin®. In Experiment III, no difference was observed in the expansion of CCs, presence of 1PB and MII for concentrations evaluated FSH Pluset® and Folltropin®. Therefore, the FSH Pluset® and Folltropin® have the same efficiency in IVM of bovine oocytes. Regarding the incubation time, it is recommended to maintain FSH (Pluset® or Folltropin®) throughout the period of IVM, since there was no difference in the results of presence of MII...

O trabalho objetivou (Experimento I) comparar representações comerciais do hormônio folículo estimulante suíno (FSH, Pluset® vs. Folltropin®) na concentração (10 μg/mL) e tempo (24 h) padrão (mais utilizados nos protocolos de maturação in vitro, MIV); (Experimento II) avaliar o melhor tempo de incubação (6 h vs. 16 h vs. 24 h) e, (Experimento III) analisar concentrações variáveis (1,0 μg/mL vs. 2,5 μg/mL vs. 10,0 μg/mL) das representações de FSH sobre a MIV de oócitos bovinos. Para tanto, oócitos foram recuperados e submetidos a MIV em condições adequadas. Após a MIV, oócitos foram avaliados quanto à expansão das células do cumulus (CCs), presença do primeiro corpúsculo polar (1CP) e placa metafásica (MII). Todos os dados foram analisados pelo teste exato de Fisher (P 0,05) nas taxas de maturação de oócitos incubados com FSH Pluset® ou Folltropin®, avaliadas pela expansão das CCs (97,6% vs. 94,3%), presença de 1CP (76,6% vs. 69,4%) e MII (70,0% vs. 68,6%). No Experimento II, quanto ao tempo de incubação com FSH foi avaliado, tanto Pluset® quanto Folltropin® mostraram uma menor taxa de expansão de CCs somente nas primeiras 6 h de MIV. Quanto à presença de 1CP, diferenças foram observadas em relação ao Pluset® enquanto o Folltropin® mostrou resultados similares em todos os tempos de incubação. Em relação à MII, nenhuma diferença foi observada entre os tempos de incubação com o FSH Pluset® e Folltropin®. No Experimento III, nenhuma diferença foi observada na expansão das CCs, presença do 1CP e MII para as concentrações avaliadas de FSH Pluset® e Folltropin®. Portanto, FSH Pluset® e Folltropin® tem a mesma eficiência na MIV de oócitos bovinos. Em relação ao tempo de incubação, é recomendado manter o FSH (Pluset® or Folltropin®) ao longo do período da MIV, uma vez que nenhuma diferença foi observada nos resultados da presença de MII...