Assisted Reproductive Techniques on South American Wild Mammals

For South American wild mammals, assisted reproductive techniques (ARTs) have been established in accordance with the procedures used for domestic mammals. However, often, it is not possible to infer the experimental conditions from one species to another. In general, the use of these ARTs aims the conservation of the animal genetic material, using gamete manipulation, embryo technology by in vivo or in vitro methods, and cloning. In all animals, the previous knowledge of reproductive physiology becomes the starting point for the use of ARTs in the expansion of all species of interest. In this sense, the purpose of this chapter is to develop an approach of theoretical, technical, and applied aspects of the ARTs in South American wild mammals, with emphasis on the state-of-the-art and its progress and perspectives

Estrus Cycle Monitoring in Wild Mammals: Challenges and Perspectives

The knowledge of reproductive physiology is of paramount importance to guide reproductive management and to make possible future application of assisted reproduction techniques (ARTs) aiming ex situ conservation of wild mammals. Nevertheless, information on the basic reproductive aspects of wild mammals remain scarce, and appropriate management practices have not yet been developed for all the species. This chapter discusses the methods most currently used for reproductive monitoring in wild females. Additionally, the difficulties regarding their use in different species and the possibilities of these procedures in captivity or in free-living mammals are addressed

In vitro maturation of collared peccary (Pecari tajacu) oocytes after different incubation times

ABSTRACT: Oocyte in vitro maturation (IVM) is the first step of the in vitro reproductive technologies that enables mature oocytes to be generated ex vivo and after used for embryo production. In this sense, the establishment of culture environment, as oocyte incubation time, is essential for the success of the IVM. Therefore, the study was carried out to investigate the relationship between the meiotic potential and the IVM times of collared peccary oocytes, wild mammals of great commercial and ecological interest. Thus, ovaries were collected of females derived from captivity and transported to the laboratory within 1 hour of slaughtering. The oocytes derived from follicles (3-6mm in diameter) were recovered by aspirated and sliced. Good quality oocytes (evenly granulated cytoplasm with a least one layer of surrounding cumulus cells) were selected and subjected to culture in TCM 199 supplemented with 10µg/mL FSH, 10% FBS and 100µM cysteamine at 38.5°C, 5% CO2 and maximum humidity for 24 or 48 hours. After the incubation period, the nuclear status, the presence of first polar body and the expansion of cumulus cells of oocytes were assessed. The data obtained were analyzed by Fisher exact test (P<0.05). A total of four sessions (2-3 females per session) were performed, resulting in eighteen aspirated and sliced ovaries with normal morphological characteristics. An oocyte recovery rate of about 83.1% (59/71) was obtained with 3.3 oocytes/ovary and 2.3 viable oocytes/ovary. After different incubation times, differences (P<0.05) were observed in 24 and 48 hours for expansion of the cumulus cells (38.1% vs. 100%), presence of first polar body (52.4% vs. 90.5%) and nuclear status in second metaphase (19.0% vs. 76.2%), respectively. In conclusion, 48 hours is suitable time for the in vitro maturation of oocytes derived from collared peccaries when compared to the time of 24 hours, according to the meiotic potential observed. Additional studies should be conducted to improve the quality of the oocyte culture environment, as medium composition, aiming to obtain viable mature oocytes for other in vitro biotechnologies

Combination of ethylene glycol with sucrose increases survival rate after vitrification of somatic tissue of collared peccaries (Pecari tajacu Linnaeus, 1758)

ABSTRACT: The cryopreservation of somatic tissue in collared peccaries promotes an alternative source of genetic material of this specie. The solid-surface vitrification (SSV) is a great option for tissue conservation; nevertheless, the optimization of SSV requirements is necessary, especially when referred to cryoprotectants that will compose the vitrification solution. Therefore, the aim was to evaluate the effect of the presence of 0.25 M sucrose in addition to different combinations (only or association) and concentrations (1.5 M or 3.0 M) of ethylene glycol (EG) and/or dimethyl sulfoxide (DMSO) in the somatic tissue vitrification of collared peccaries. Subsequently, we tested six combinations of cryoprotectants with or without sucrose in Dulbecco modified Eagle medium (DMEM) plus 10% fetal bovine serum (FBS). Thus, 3.0 M EG with sucrose was able to maintain normal tissue characteristics compared with non-vitrified (control), especially for the volumetric ratio of epidermis (61.2 vs. 58.7%) and dermis (34.5 vs. 36.6%), number of fibroblast (90.3 vs. 127.0), argyrophilic nucleolar organizer region (AgNOR) ratio (0.09 vs. 0.17%) and nucleus area (15.4 vs. 14.5 μm2) respectively. In conclusion, 3.0 M EG with 0.25 M sucrose and 10% FBS resulted in a better cryoprotectant composition in the SSV for somatic tissue of collared peccaries

In vitro culture of somatic cells derived from ear tissue of collared peccary (Pecari tajacu Linnaeus, 1758) in medium with different requirements

ABSTRACT: The maintenance of metabolic activities during the in vitro culture of somatic cells of wild animals, especially collared peccary (Pecari tajacu), is an interesting step in conservation of these cells for the use in nuclear transfer. In this context, it is necessary to optimize the culture conditions of somatic cells by the establishment of appropriate supplementation to the media. Therefore, this study aimed to analyze the composition of the culture means of somatic cell derived from ear tissue of collared peccaries, evaluating concentrations of fetal bovine serum (FBS; 10% vs. 20%) and epidermal growth factor (EGF; 5ng/mL vs. 10ng/mL). Tissues were submitted to primary culture and subcultures for 40 days and cells were analyzed for morphology, adhesion, subconfluence, and proliferative activity to develop the growth curve and to determine the population doubling time (PDT), viability, and functional/metabolic activity. No difference was observed between the concentrations of FBS for several parameters, except for viability [FBS10: 85.6% vs. FBS20: 98.2%], PDT [FBS10: 155.4h vs. 77.2h], and functional/metabolic assay [FBS10: 0.57-0.55 vs. FBS20: 0.82-0.99 (D5-D7)]. For the EGF in culture, no difference was observed in the evaluated parameters. In all experiments, the growth curves were typical S-shape and the cells passed through a lag, logarithmic, and plateau phase. In conclusion, 20% FBS is suitable for the recovery of somatic cells; nevertheless, EGF does not improve the quality of growing these cells. To our knowledge, this is the first study culturing somatic cells of collared peccaries