Publications by authors named "Alexsandra Fernandes Pereira"

17 Publications

  • Page 1 of 1

Effects of somatic tissue cryopreservation on puma (Puma concolor L, 1771) tissue integrity and cell preservation after in vitro culture.

Cryobiology 2021 Aug 10;101:52-60. Epub 2021 Jun 10.

Laboratory of Animal Biotechnology, Federal Rural University of Semi-Arid (UFERSA), Mossoro, RN, Brazil. Electronic address:

Somatic resource banks play a crucial role in the conservation of genetic diversity, allowing for the preservation of biological samples from different populations. Puma somatic cells can be recovered from these banks and used in assisted techniques toward enhancing their multiplication and conservation. In response to the population reduction of this ecologically importance species, we aimed to evaluate the capacity of cryopreservation of somatic tissues on the maintenance of the integrity and quality of the cells recovered after culture, with the aim of establishing a somatic tissue bank that will allow for the safeguarding of a wide genetic sampling of pumas. Cryopreservation increased the thickness of the corneum layer in the tissues, and the number of perinuclear halos and empty gaps. Nevertheless, cryopreservation was able to maintain normal fibroblast patterns, even showing an increase in the percentage of collagen fibers. Cryopreservation maintained the proliferative potential of the tissues and the parameters evaluated during in vitro culture, mainly regarding the viability, proliferative activity, and apoptosis levels. Nevertheless, cells from cryopreserved tissues showed decreased metabolism and mitochondrial membrane potential when compared to cells from non-cryopreserved tissues. In summary, we demonstrated for the first time that puma somatic tissues subjected to cryopreservation are viable and maintain tissue integrity, featuring minimal changes after warming. Although viable somatic cells are obtained from these tissues, they undergo alterations in their metabolism and mitochondrial membrane potential. Improvements in the conservation conditions of somatic samples are needed to increase the quality of somatic tissue banks in this species.
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http://dx.doi.org/10.1016/j.cryobiol.2021.06.003DOI Listing
August 2021

Influence of freezing techniques and glycerol-based cryoprotectant combinations on the survival of testicular tissues from adult collared peccaries.

Theriogenology 2021 Jun 26;167:111-119. Epub 2021 Mar 26.

Laboratory of Animal Germplasm Conservation, Federal Rural University of Semi-Arid - UFERSA, Mossoró, RN, Brazil. Electronic address:

The objective of the study was to evaluate the effects of different cryopreservation techniques including glycerol-based cryoprotectant combinations on the structure and viability of testicular tissues from adult collared peccaries. Tissue biopsies (3.0 mm³) from 5 different individuals were allocated to 10 different groups: fresh control; slow freezing (SF), conventional vitrification (CV), or solid-surface vitrification (SSV); each of them using three different combinations of cryoprotectants [dimethyl sulfoxide (DMSO) + ethylene glycol (EG); DMSO + Glycerol; and EG + Glycerol]. After thawing/warming, samples were evaluated for histomorphology, viability, proliferative capacity potential, and DNA integrity. Most effective preservation of testicular histomorphology was achieved using SF and CV with DMSO + EG. However, the use of glycerol-based cryoprotectant combinations increased the occurrence of tubular cell swelling, tubular cell loss and shrinkage from the basal membrane. Cell viability was comparable among cryopreservation methods and cryoprotectant combinations. Regarding cell proliferative capacity, the use of SF with EG + Glycerol and SSV with DMSO + Glycerol impaired the conservation of spermatogonia proliferative potential compared to other treatments. Moreover, CV with DMSO + EG was better than SF with EG + Glycerol for Sertoli cell proliferation potential. Regarding DNA integrity, less damage occurred when using SF with DMSO + EG while more fragmentations were observed when using CV with EG + Glycerol or DMSO + Glycerol as well as SSV with EG + Glycerol or DMSO + Glycerol. In sum, SF and CV appeared to be the most suitable methods for the cryopreservation of adult peccary testicular tissues. Additionally, the use of glycerol-based cryoprotectant combinations did not improve testicular tissues preservation with DMSO + EG being the most efficient option.
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http://dx.doi.org/10.1016/j.theriogenology.2021.03.013DOI Listing
June 2021

Cryopreservation of Spix's yellow-toothed cavy epididymal sperm using Tris- and coconut water-based extenders supplemented with egg yolk or Aloe vera.

Cryobiology 2021 04 27;99:40-45. Epub 2021 Jan 27.

Laboratory of Animal Germplasm Conservation, Federal Rural University of Semi-Arid, Mossoro-RN, Brazil. Electronic address:

Addressing the establishment of biobanks for the conservation of wild hystricomorph rodents' germplasm, we verified the effects of different extenders and distinct concentrations of non-permeant cryoprotectants on the sperm parameters of Spix's yellow-toothed cavies. Nine testis-epididymis complexes were used for sperm collection by retrograde washing using Tris or a powdered coconut water extender (ACP®-116c). Spermatozoa were diluted and frozen with the same extenders supplemented with egg yolk or Aloe vera at a 10% or 20% concentration. After recovery and cryopreservation, all samples were evaluated for sperm kinetic parameters, morphology, membrane integrity, osmotic response, and sperm-binding capability using an egg yolk perivitelline membrane assay. After recovery, no differences were observed between Tris and ACP®-116c that provided 515.4 × 10 sperm/mL and 561.6 × 10 sperm/mL, presenting >65% motile sperm, respectively. After cryopreservation, most effective preservation of sperm kinetic parameters (68.1 ± 5.9% motile sperm) and membrane integrity (48.2 ± 7.4%) was provided by Tris extender supplemented with 10% egg yolk. However, both extenders supplemented with any concentration of egg yolk or Aloe vera presented similar preservation of osmotic response and sperm-binding ability after cryopreservation. In summary, we suggest the use of a Tris extender supplemented of 10% egg yolk for cryopreservation of Spix's yellow-toothed cavy epidydimal sperm.
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http://dx.doi.org/10.1016/j.cryobiol.2021.01.016DOI Listing
April 2021

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

Cryobiology 2021 02 24;98:63-72. Epub 2020 Dec 24.

Laboratory of Animal Biotechnology, Federal Rural University of Semi-Arid (UFERSA), Mossoro, RN, Brazil. Electronic address:

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.
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http://dx.doi.org/10.1016/j.cryobiol.2020.12.006DOI Listing
February 2021

A Comparative Approach of Cellular Reprogramming in the Rodentia Order.

Cell Reprogram 2020 10 11;22(5):227-235. Epub 2020 Aug 11.

Laboratory of Animal Biotechnology, Federal Rural University of the Semi-Arid Region, Mossoró, Brazil.

Cellular reprogramming mainly involves induction of reactivation of genes responsible for nuclear plasticity, a process that can be performed 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.
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http://dx.doi.org/10.1089/cell.2020.0024DOI Listing
October 2020

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

PeerJ 2020 3;8:e9136. Epub 2020 Jun 3.

Laboratory of Animal Biotechnology, Universidade Federal Rural do Semi-Árido, Mossoró, Rio Grande do Norte, Brazil.

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% CO). 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: = 0.98; first vs. tenth: = 0.76; third vs. tenth: = 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%, = 0.006). Moreover, the cryopreservation did not influence the viability ( = 0.11), metabolic activity ( = 0.77), or PDT ( = 0.11). Nevertheless, a greater ΔΨm ( = 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, = 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.
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http://dx.doi.org/10.7717/peerj.9136DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7275682PMC
June 2020

Cryopreservation and Culture of Testicular Tissues: An Essential Tool for Biodiversity Preservation.

Biopreserv Biobank 2020 Jun 13;18(3):235-243. Epub 2020 Apr 13.

Laboratory of Animal Germplasm Conservation, Federal Rural University of Semi-Arid, Mossoró, Brazil.

Systematic cryo-banking of reproductive tissues could enhance reproductive management and ensure sustainability of rare mammalian genotypes. Testicular tissues contain a vast number of germ cells, including at early stages (spermatogonia and spermatocytes), that can potentially develop into viable spermatozoa after grafting or culture , and the resulting sperm cells then can be used for assisted reproductive techniques. The objective of this review was to describe current advances, limitations, and perspectives related to the use of testicular tissue preservation as a strategy for the conservation of male fertility. Testes can be obtained from mature or prepubertal individuals, immediately postmortem or by orchiectomy, but testicular biopsies could also be an alternative to collect samples from living individuals. Testicular fragments can be then cryopreserved by using slow or ultra-rapid freezing, or even vitrification methods. The composition of cryopreservation media can vary according to species-specific characteristics, especially regarding the cryoprotectant type and concentration. Finally, spermatozoa have been usually obtained after xenografting of testicular fragments into severely immunodeficient mice, while this method still has to be optimized after culture conditions.
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http://dx.doi.org/10.1089/bio.2020.0010DOI Listing
June 2020

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

Theriogenology 2020 Jan 14;142:320-327. Epub 2019 Oct 14.

Laboratory of Animal Biotechnology, Federal Rural University of Semi-Arid, Mossoro, RN, Brazil. Electronic address:

To optimize the protocols for assisted reproductive techniques (ARTs) in collared peccary (Pecari tajacu Linnaeus, 1758), we evaluated various conditions for oocyte in vitro maturation (IVM) and chemical activation. Initially, we assessed the IVM rates, cumulus-oocyte complex (COC) quality, and oocyte morphometry in the absence or presence of epidermal growth factor (EGF). There was no difference between the COCs matured in absence or presence of EGF for the expansion of cumulus cells (97.6% ± 1.2 vs. 100% ± 0.0), presence of first polar body (65.9% ± 1.2 vs. 70.5% ± 1.8), nuclear status in second metaphase (62.5% ± 11.6 vs. 68.4% ± 4.9), cytoplasmic maturation (100.0% ± 0.7 vs. 75.0% ± 0.7), reactive oxygen species levels (0.5 ± 0.2 vs. 0.3 ± 0.1), and mitochondrial membrane potential (1.1 ± 0.2 vs. 1.1 ± 0.1). However, the zona pellucida thickness of matured COCs was reduced in the presence of EGF. Thus, the EGF group was used for further experiments. The oocytes were artificially activated with ionomycin and four secondary activator combinations [6-dimethylaminopurine (6D), 6D and cytochalasin B (6D + CB), cycloheximide (CHX), and CHX and CB (CHX + CB)]. The effect of immature COCs based on cumulus cell layers and cytoplasm homogeneity (GI and GII or GIII COCs) on embryonic development and quality was evaluated. There was no difference in the cleavage rates among the groups of secondary activators. The cleavage rates of embryos derived from GI/GII and GIII COCs were greater than 72.2% and 25.0%, respectively. Moreover, treatment with CHX showed a reduction in the cleavage rate of embryos derived from GIII COCs when compared to the cleavage rate of embryos derived from GI/GII COCs (P < 0.05). Nevertheless, higher rates of blastocyst/total GI and GII COCs were observed in the 6D group (27.6% ± 0.3) compared to CHX group (6.9% ± 0.3). Additionally, only 6D treatment resulted in the production of embryos derived from GIII COCs (25.0% ± 0.2). The percentage of the ICM/total cell ratio was also greater in blastocysts derived from 6D (42.5% ± 19.0), 6D + CB (37.9% ± 21.9), and CHX + CB (43.8% ± 19.6) groups when compared to CHX (3.6% ± 0.1) group. Thus, the combination of ionomycin and 6D could produce collared peccary embryos by activation of both GI/GII COCs and GIII COCs. These optimized IVM conditions using EGF and chemical activation using ionomycin and 6D in collared peccaries form the first steps for establishing ARTs to conserve this species.
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http://dx.doi.org/10.1016/j.theriogenology.2019.10.016DOI Listing
January 2020

Combination of intracellular cryoprotectants preserves the structure and the cells proliferative capacity potential of adult collared peccary testicular tissue subjected to solid surface vitrification.

Cryobiology 2019 12 31;91:53-60. Epub 2019 Oct 31.

Laboratory of Animal Germplasm Conservation, Federal Rural University of Semi-Arid, Mossoró, RN, Brazil. Electronic address:

The objective was to evaluate different permeating cryoprotectants to vitrify testicular tissue biopsies from adult collared peccaries. Five pairs of testicles were dissected into fragments (9 mm³) that were allocated to non-vitrified (control) and vitrified groups using a solid-surface method following exposure to different cryoprotectants (3.0 M dimethyl sulfoxide (DMSO), 3.0 M ethylene glycol (EG) or 1.5 M DMSO + 1.5 M EG). After warming, samples were evaluated for histomorphology, ultrastructure, viability, and proliferative capacity potential. The appropriate conservation of the ultrastructural organization of the seminiferous tubule in terms of lumen presence and cell junctions was only observed at the use of DMSO/EG combination. Regardless of the cryoprotectant, the vitrification effectively preserved cell nuclear visualization and condensation similarly as observed at the non-vitrified group. Moreover, DMSO/EG combination provided a better preservation of basal membranes of seminiferous tubules than DMSO (P < 0.05). The occurrence of cell swelling was more evident in the use of DMSO than EG (P < 0.05), but both isolate cryoprotectants were similar to the DMSO/EG combination. Only the DMSO/EG combination maintained the proliferative capacity potential for spermatogonia (3.69 NORs/cell) and Sertoli cell (3.19 NORs/cell) similar to controls (3.46 and 3.31 NORS/cell, respectively). Moreover, ~40% cell viability was found after vitrification independent of cryoprotectant. In conclusion, DMSO/EG in combination is better than DMSO or EG alone for SSV of testicular tissue biopsies from adult collared peccaries.
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http://dx.doi.org/10.1016/j.cryobiol.2019.10.199DOI Listing
December 2019

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

Andrologia 2019 Dec 22;51(11):e13448. Epub 2019 Oct 22.

Laboratory of Animal Biotechnology, Federal Rural University of Semi-Arid, Mossoro, Brazil.

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.
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http://dx.doi.org/10.1111/and.13448DOI Listing
December 2019

Potential role of intraspecific and interspecific cloning in the conservation of wild mammals.

Zygote 2019 Jun 11;27(3):111-117. Epub 2019 Jun 11.

SummaryIntraspecific and interspecific cloning via somatic cell nuclear transfer (iSCNT) is a biotechnique with great possibilities for wild mammals because it allows the maintenance of biodiversity by recovering species, nuclear reprogramming for the production of pluripotency-induced cells, and studies related to embryonic development. Nevertheless, many areas in cloning, especially those associated with wild mammals, are still in question because of the difficulty in obtaining cytoplasmic donor cells (or cytoplasts). Conversely, donor cell nuclei (or karyoplasts) are widely obtained from the skin of living or post-mortem individuals and often maintained in somatic cell banks. Moreover, the creation of karyoplast-cytoplast complexes by fusion followed by activation and embryo development is one of the most difficult steps that requires further clarification to avoid genetic failures. Although difficult, cloning different species, such as wild carnivores and ungulates, can be successful via iSCNT with embryo development and the birth of offspring. Thus, novel research in the area that contributes to the conservation of biodiversity and knowledge of the physiology of species continues. The present review presents the failures and successes that occurred with the application of the technique in wild mammals, with the goal of helping future work on cloning via iSCNT.
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http://dx.doi.org/10.1017/S0967199419000170DOI Listing
June 2019

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

Cryobiology 2019 06 25;88:15-22. Epub 2019 Apr 25.

Laboratory of Animal Biotechnology, Federal Rural University of Semi-Arid (UFERSA), Mossoro, RN, Brazil. Electronic address:

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.
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http://dx.doi.org/10.1016/j.cryobiol.2019.04.007DOI Listing
June 2019

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

Theriogenology 2019 Apr 2;128:74-80. Epub 2019 Feb 2.

Laboratory of Animal Biotechnology, Federal Rural University of Semi-Arid, Mossoro, RN, Brazil. Electronic address:

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.
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http://dx.doi.org/10.1016/j.theriogenology.2019.01.031DOI Listing
April 2019

Ultrastructural description of fresh and frozen/thawed sperm derived from collared peccaries (Pecari tajacu Linnaeus, 1,758).

Microsc Res Tech 2018 Nov 8;81(11):1301-1309. Epub 2018 Oct 8.

Laboratory of Animal Germplasm Conservation, Universidade Federal Rural do Semiárido - UFERSA, Rio Grande do Norte, Brazil.

The aim was to describe, through scanning electron microscopy (SEM) and transmission electron microscopy (TEM), the ultrastructure of peccaries' fresh and frozen-thawed sperm. For that, semen derived from three mature males was obtained by electroejaculation and evaluated for motility, membrane integrity, membrane functionality, chromatin integrity, and morphology through light microscopy. Samples were frozen using a Tris extender plus egg yolk (20%) and glycerol (6%). Then, fresh and frozen-thawed semen samples were mixed in different sperm pools that were processed for SEM and TEM. Sperm motility, membrane integrity, and functionality were impaired (p < .05) by freezing-thawing procedures, but sperm morphology, and chromatin integrity evaluated by light microscopy were not significantly affected. The SEM revealed that peccaries' sperm presents a flattened head in a paddle format, measuring 6.07 μm in length and 3.84 μm in width, with a vastus acrosome (4.46 μm). Normal tails measure 38.11 μm, being formed by an extensive midpiece with 15.52 μm in length. In frozen-thawed samples, both SEM and TEM provide us information about damage undetected through light microscopy as the presence of vesicles in the acrosome, loose plasma membrane, vacuolized mitochondria, dense fibers disorganized, and decondensed chromatin. In conclusion, we provide the first description of the sperm ultrasctruture in collared peccaries. Moreover, SEM and TEM help us to identify some nanometric damage provoked by freezing-thawing procedures, thus providing valuable information for the improvement of such important protocols used for biobanking formation.
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http://dx.doi.org/10.1002/jemt.23138DOI Listing
November 2018

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

In Vitro Cell Dev Biol Anim 2018 Aug 19;54(7):486-495. Epub 2018 Jun 19.

Laboratory of Animal Biotechnology, Federal Rural University of Semi-Arid, Av. Francisco Mota, 572, Mossoró, RN, 59625 900, Brazil.

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.
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http://dx.doi.org/10.1007/s11626-018-0270-6DOI Listing
August 2018

Conservation of somatic tissue derived from collared peccaries (Pecari tajacu Linnaeus, 1758) using direct or solid-surface vitrification techniques.

Cytotechnology 2017 Aug 4;69(4):643-654. Epub 2017 Mar 4.

Laboratory of Animal Biotechnology, Federal Rural University of Semiarid, Mossoro, 59625900, Brazil.

Cryopreservation of somatic tissue can be applied in biodiversity conservation, especially for wild species as collared peccary. We aimed to evaluate the effect of vitrification techniques of ear tissue of collared peccary [direct vitrification in cryovials (DVC) or solid-surface vitrification (SSV)] on the layers of epidermis and dermis by conventional histology and cell ability during the in vitro culture. Thus, both the vitrification methods were able to maintain normal patterns of the epidermis as the cornea and granular layers, furthermore the intercellular space and dermal-epidermal junction of the spinous layer when compared to fresh control. Nevertheless, DVC and SSV percentage of normality decreased in the morphological integrity of cytoplasm (37.5 and 25.0%) of spinous layer, respectively, as compared to the fresh fragments (100%, p < 0.05). Moreover, other differences between the fresh control (100%) and DVC tissues were verified in the intra-epidermal cleavage of the spinous (37.5%) and basal (37.5%) layers. In general, DVC and SSV techniques were efficient for the recovery of the somatic cells according to most of the evaluated parameters for the in vitro culture (p > 0.05). In addition, only at time of 72 h (D3), in the growth curve, DVC fragments showed a reduced cell concentration than fresh control. In conclusion, SSV was found to be a more efficient method for vitrifying collared peccary skin tissue when compared to DVC. These results are relevant for the tissue cryopreservation from collared peccary and could also be useful for mammals with phylogenetic relationships.
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http://dx.doi.org/10.1007/s10616-017-0074-7DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5507844PMC
August 2017

Estimating the binding ability of collared peccary (Pecari tajacu Linnaeus, 1758) sperm using heterologous substrates.

Theriogenology 2017 Apr 5;92:57-62. Epub 2017 Jan 5.

Laboratory of Animal Germplasm Conservation, Federal Rural University of Semi-Arid (UFERSA), BR 110, Km 47, Mossoro, RN, 59625-900, Brazil. Electronic address:

In collared peccaries, the development of artificial insemination (AI) is scarce, requiring search for alternative methods for the evaluation of sperm fertilizing ability. Thus, the aims of this study were to estimate the binding capability of collared peccaries sperm, using swine oocytes and the egg perivitelline membrane, and to evaluate the prognostic value of sperm parameters on the in vitro interactions among sperm and heterologous substrates. Eleven ejaculates were collected by eletroejaculation and evaluated for viability and morphology by light microscopy, for functionality by hypo-osmotic swelling test, for plasma membrane integrity by epifluorescence microscopy, and for sperm motility by computerized analysis. Subsequently, for analysis of the in vitro interactions, sperm samples were cultured in an incubation medium with swine oocytes and egg perivitelline membrane for 18 h and 20 min, respectively, at 38.5 °C and humidified atmosphere. The sperm-oocyte interaction rate was 100% with sperm penetrating 19.8+ 5.5% of oocytes. The average values of bound sperm and penetrated sperm per oocyte were 39.4 + 4.6 and 2.5 + 0.7, respectively. Already for perivitelline membrane binding assay, all samples presented sperm bound (100%) with average of 140.6 ± 19.4 bound sperm (range 33.9-308.7). Moreover, positive correlations were observed for the number of sperm bound to swine oocytes and osmotic response (r = 68.5%; P = 0.02), membrane integrity (r = 65.1%; P = 0.03), and straightness (r = 66.5%; (P = 0.03), as weel as for the number of sperm bound to egg perivitelline membrane and sperm viability (r = 74.0%; P = 0.01), total motility (r = 63.6%; P = 0.04), and linearity (r = 70.5%; P = 0.02). Finally, a negative correlation among slow (r = -80.5%; P = 0.01) and static (r = -84.3%; P = 0.01) sperm with the egg perivitelline membrane was observed. In conclusion, swine oocytes and perivitelline membrane can be used as indicators for the functional evaluation of the binding capability of sperm derived from collared peccaries. These tests could be incorporated into the routine of semen technologies.
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http://dx.doi.org/10.1016/j.theriogenology.2017.01.008DOI Listing
April 2017
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