RESUMEN
In this study, we aimed to evaluate the efficacy of cryopreserving canine ovarian tissue using vitrification and slow freezing methods while investigating potential differences in cryotolerance based on follicular type and cryopreservation technique. Twenty-eight ovaries were collected from 14 anoestrus bitches of various breeds, aged between 2 and 5 years, and undergoing elective ovariohysterectomy. The ovaries were sectioned into small fragments and randomly assigned to three groups: vitrification, slow freezing, and a control group (fresh tissue). Vitrification was performed using cryotubes containing DAP 213 solution (2M DMSO, 1M acetamide, 3M propylene glycol) in two stages, while slow freezing involved cryotubes with 1.5M DMSO solution inserted into a programmable machine. The effects of cryopreservation were evaluated by histology and immunohistochemistry (cleaved caspase-3), to determine the percentage of cells undergoing apoptosis. Histological examination revealed that the slow freezing group exhibited a significantly higher percentage of intact follicles (45.75 %) compared to those subjected to vitrification (38.17 %; P = 0.01). Immunohistochemical evaluation further indicated that 84.21 % of the follicles in the slow freezing group did not express caspase-3, suggesting the absence of apoptosis. Conversely, vitrified samples exhibited significantly more apoptotic cells compared to other groups (P < 0.001). Furthermore, early antral follicles displayed a higher susceptibility to degeneration regardless of the cryopreservation method employed. Nevertheless, when comparing the cryopreserved groups, early antral follicles showed greater degeneration in slow freezing group, while preantral follicles were the most affected in the vitrification group. In conclusion, slow freezing demonstrated superior preservation of viable follicles compared to vitrification and emerged as the preferred technique for cryopreserving canine ovarian tissue. These findings contribute valuable insights into optimizing cryopreservation methods for canine ovarian tissue, potentially benefiting reproductive technologies and fertility preservation in canines.
RESUMEN
Direct detection of Mycoplasma hyopneumoniae through molecular tools is a growing trend for early diagnosis, highlighting the importance of knowing M. hyopneumoniae dynamics in the respiratory tract upon infection. This study focused on monitoring the infection level and its effects in different anatomic sites of the respiratory tract of experimentally infected swine in four time-points post-infection. To this end, 24 pigs were allocated to either non-inoculated group (n = 8) or inoculated group (n = 16). On day 0 post-infection (dpi), animals of the inoculated group were intratracheally inoculated with M. hyopneumoniae. Nasal swabs were collected weekly for qPCR detection of bacterial shedding. At 14, 28, 42, and 56 dpi, four animals from the inoculated group and two from the control group were necropsied. Bronchoalveolar lavage fluid (BALF) and samples from three different anatomical tracheal sections (cranial - CT, medium - MT, lower - LT) were collected for qPCR and histopathology. Bacterial loads (qPCR) in tracheal samples were: 4.47 × 102 copies∕µL (CT), 1.5 × 104- copies∕ µL (MT) and 1.4 × 104 copies∕µL (LT samples). M. hyopneumoniae quantification in BALF showed the highest load at 28 dpi (2.0 × 106 copies∕ µL). Microscopic lesions in LT samples presented the highest scores at 56 dpi and were significantly correlated with the pathogen load on 14 dpi (0.93) and 28 dpi (0.75). The greatest bacterial load of M. hyopneumoniae in CT samples and BALF was registered at 28 dpi, and it remained high in BALF and LT throughout the 56 dpi. The pathogen was able to persist during the whole experimental period, however higher estimated quantification values were registered in the lower parts of the respiratory tract, especially at 56 dpi. These findings are important for improving diagnostics, treatment, and control measures of M. hyopneumoniae infection in swine herds.
