RESUMO
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.
RESUMO
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)
Assuntos
Animais , Masculino , Sêmen , Blastocisto , Inseminação Artificial , Fertilização in vitro , Panthera , Técnicas In VitroRESUMO
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.
RESUMO
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.
Assuntos
Adenina/análogos & derivados , Artiodáctilos , Técnicas de Maturação in Vitro de Oócitos/veterinária , Ionomicina/farmacologia , Oócitos/efeitos dos fármacos , Partenogênese/efeitos dos fármacos , Adenina/farmacologia , Animais , Artiodáctilos/embriologia , Células Cultivadas , Células do Cúmulo/citologia , Células do Cúmulo/efeitos dos fármacos , Células do Cúmulo/fisiologia , Desenvolvimento Embrionário/efeitos dos fármacos , Fator de Crescimento Epidérmico/farmacologia , Feminino , Técnicas de Maturação in Vitro de Oócitos/métodos , Técnicas de Transferência Nuclear/veterinária , Oócitos/citologia , Oócitos/fisiologia , Oogênese/efeitos dos fármacos , Oogênese/fisiologia , Partenogênese/fisiologiaRESUMO
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)
Assuntos
Animais , Masculino , Bovinos , Oócitos/efeitos dos fármacos , Técnicas de Maturação in Vitro de Oócitos/métodos , Técnicas de Maturação in Vitro de Oócitos/veterináriaRESUMO
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)
Assuntos
Animais , Feminino , Roedores/embriologia , Desenvolvimento Embrionário/efeitos dos fármacosRESUMO
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.
Assuntos
Feminino , Animais , Desenvolvimento Embrionário/efeitos dos fármacos , Roedores/embriologiaRESUMO
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.
Assuntos
Masculino , Animais , Bovinos , Oócitos/efeitos dos fármacos , Técnicas de Maturação in Vitro de Oócitos/métodos , Técnicas de Maturação in Vitro de Oócitos/veterináriaRESUMO
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.
Assuntos
Óleos Voláteis/farmacologia , Espermatozoides/efeitos dos fármacos , Syzygium , Animais , Antioxidantes/análise , Bovinos , Avaliação Pré-Clínica de Medicamentos , Masculino , Óleos Voláteis/química , Espécies Reativas de Oxigênio/metabolismo , Espermatozoides/metabolismoRESUMO
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.