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.

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.