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Containers used to cryopreserve sperm are made with non-biodegradable plastic compounds, having a high monetary and environmental cost. Therefore, the development of biodegradable alternative containers for cell cryopreservation is necessary. Thus, this study aimed to evaluate the efficiency of hard-gelatin and hard-hydroxypropyl methylcellulose (HPMC) capsules as low-cost and biodegradable alternative containers for sperm cryopreservation. Sperm from twelve South American silver catfish Rhamdia quelen were individually cryopreserved in plastic straws 0.25 mL (as control), hard-gelatin, and hard-HPMC capsules. The quality of post-thaw sperm cryopreserved in the different containers was checked by measuring spermatozoa membrane integrity, kinetic parameters, mitochondrial activity, fertilization, hatching, and normal larvae rates. The samples cryopreserved in straws showed a higher percentage of membrane integrity (68%) than those frozen in hard-gelatin (40%) and hard-HPMC capsules (40%). However, we did not observe differences between the samples stored in straws and hard capsules for the rest of tested sperm parameters. Thus, based on the high sperm fertility capability, both capsules were efficient as cryopreservation containers for maintaining sperm functionality.

Piracanjunba (Brycon orbignyanus) is an endangered South American fish, and ovarian tissue cryopreservation is an alternative method for preserving maternal germplasm and genetic diversity. Therefore, our aim was to test a vitrification protocol for ovarian tissue containing primary growth (PG) oocytes of B. orbignyanus as a strategy to avoid the threat of extinction. Two vitrification solutions were evaluated (VS1: 1.5 M methanol + 4.5 M propylene glycol and VS2: 1.5 M methanol + 5.5 M Me2SO) and compared using control/fresh ovarian tissue. After vitrification, the following factors were analyzed: membrane integrity using trypan blue, morphology using a histological assessment, oxidative stress (total reactive antioxidant potential (TRAP) and reduced thiol [-SH]), mitochondrial activity using MTT, and DNA damage using a comet assay. The vitrified oocytes (VS1 = 24.3 ± 0.49% and VS2 = 24.8 ± 0.69%) showed higher DNA damage than the control group (control = 20.7 ± 1.03%) (P = 0.004). In contrast, in most evaluations (membrane integrity, membrane damage, oxidative stress, and mitochondrial activity), there were no discernible differences between the control group and the vitrified samples. In addition, oocyte (P = 0.883) and nuclear diameter (P = 0.118) did not change after vitrification. VS2 treatment resulted in higher nuclear damage (15.7 ± 1.45%) than in the control treatment (3.5 ± 1.19%); however, VS1 treatment did not result in significantly more damage (9.5 ± 3.01%) than in the control (P = 0.015). Therefore, the protocol for ovarian tissue vitrification tested in this study resulted in high maintenance of PG oocyte cell integrity, making it a promising alternative for B. orbignyanus maternal genome preservation.

Zebrafish is an important animal model, thousands lines have been developed, thus having a great need for their preservation. However, the cryopreservation of fish oocytes is still limited and needs improvement. The sodium alginate hydrogel, in addition to providing support for the cells, has been shown to be a potential cryoprotectant. Therefore, the aim of this study was to evaluate the sodium alginate hydrogel encapsulation technique efficiency during zebrafish ovarian tissue vitrification. The encapsulation methodology was standardized in the first experiment. In Experiment 2, we evaluated four vitrified groups: standard protocol without encapsulation (VS); encapsulated with cryoprotectants (VS1-A); encapsulated with half the cryoprotectants concentration (VS2-A); encapsulated without cryoprotectants (VA). VS treatment (54.6 ± 12.3%; 23.7 ± 9.9%; 12.6 ± 5.0%) did not differ from the VS1-A and VA showed a lower membrane integrity percentage (1.2 ± 1.4%; 0.3 ± 0.6%; 0.5 ± 1.5%). Mitochondrial activity was significantly greater in non-encapsulated treatment (VS) when compared to the encapsulated treatments. VS1-A and VS obtained the lowest lipid peroxidation (39.4 ± 4.4 and 40.5 ± 3.3 nmol MDA/mg respectively) in which VS was not significantly different from the VS2-A treatment (63.6 ± 3.1 nmol MDA/mg), unlike, VA obtained the highest lipid peroxidation level (124.7 ± 7.9 nmol MDA/mg). The results obtained in this study demonstrate that the sodium alginate hydrogel encapsulation technique did not have a cryoprotective action, but maintained the membrane integrity when used the standard concentration of cryoprotectants. However, halving the cryoprotectant concentration of fragments encapsulated in alginate hydrogel did not cause an increase in lipid peroxidation. In addition, it provided support and prevented the oocytes from loosening from the tissue during the vitrification process, being an interesting alternative for later in vitro maturation.

Made available in DSpace on 2022-04-29T08:40:18Z (GMT). No. of bitstreams: 0 Previous issue date: 2022-05-15 Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES) The use of technologies in assisted reproduction, such as the use of centrifugation, has been reported as a tool that allows separating the best portion of sperm from cryopreserved milt. The main of this work was to compare two separation techniques, differential and density gradient, on thawed Rhamdia quelen milt, based on sperm functionality and fertilization capacity. Three treatments were compared: control, differential centrifugation or pelletization (T1), and density gradient centrifugation (T2). Motility percentage was better for differential (42.58 ± 6.50%) than for the control treatment (32.44 ± 6.03%). Membrane integrity and kinetic parameters was not different between treatments. The presence of normal sperm was higher in T2 treatment (13.55 ± 5.82%) when compared to the control (10.72 ± 3.53%) and T1 (3.97 ± 1.95%). There was a lower rate of morphological tail damage in the T1 (26.91 ± 7.13%) treatment compared to the T2 (37.48 ± 10.81%) and control (45.88 ± 4.84%). For the head morphologies, the lowest percentages were for the control (35.72 ± 5.89%) and T2 (4.89 ± 14.94%) treatments compared to the T1 treatment (62.59 ± 9.67%). In the MTT (3- (4, 5-dimethylthiazolyl-2)-2, 5-diphenyltetrazolium bromide) test (AU/100 million sptz), the centrifuged treatments resulted in values of 1.58 ± 0.79 (T1) and 2.02 ± 1.88 (T2), better than the treatment control (0.20 ± 0.08). Likewise, the fertilization and hatching rates, respectly, were better for the T1 (76.68 ± 15.22% and 54.62 ± 17.39%) and T2 (69.85 ± 17.85% and 50.87 ± 20.59%) treatments, than the control treatment (40.03 ± 11.67% and 24.14 ± 13.97%). The comparison of differential centrifugation with density gradient centrifugation was carried out for the first time with cryopreserved milt of R. quelen. In summary, both centrifugation techniques proved to be efficient in obtaining a better portion of functional sperm from thawed milt. This is based on the high rates of embryonic survival and hatching obtained after both differential and density gradient separation. Animal Science Research Program of Federal University of Rio Grande do Sul, Bento Gonçalves, 7712, RS Aquaculture Center Fisheries Engineering Department Paulista State University Júlio de Mesquita Filho, SP Biotechnology Laboratory University of Vale of Taquari - Univates, Avelio Tallini Street, 171, RS Faculty of Veterinary Medicine Swine Sector Program of Federal University of Rio Grande do Sul, Bento Gonçalves, 9090, RS Department of Agricultural and Aquacultural Sciences Faculty of Natural Resources Catholic University of Temuco Aquaculture Center Fisheries Engineering Department Paulista State University Júlio de Mesquita Filho, SP CAPES: CAPES/PROEX N° 88882.346090/2015-01

The aim was to evaluate the effect of a post-thaw dilution of Rhamdia quelen sperm in 1.1% NaCl (325 mOsm kg

Endocrine cells (ECs) act as a luminal surveillance system responding to either the presence or absence of food in the gut through the secretion of peptide hormones. The aim of this study was to analyze the effects of feeding and fasting on the EC peptide-specific distribution along the intestine of Nile tilapia. We assessed the density of ECs producing gastrin (GAS), cholecystokinin-8 (CCK-8), neuropeptide Y (NPY), and calcitonin gene-related peptide (CGRP) in nine segments of the intestine using immunohistochemistry. Our results show that ECs immunoreactive to CCK-8, GAS, NPY, and CGRP can be found along all the intestinal segments sampled, from the midgut to hindgut, although differences in their distribution along the gut were observed. Regarding nutrient status, we found that the anterior segments of the midgut seem to be the main site responding to luminal changes in Nile tilapia. The NPY+ and CGRP+ EC densities increased in the fasted group, while the amount of CCK-8+ ECs were higher in the fed group. No effects of fasting or feeding were found in the GAS+ EC densities. Changes in ECs density were found only at the anterior segments of the intestine which may be due to the correlation between vagus nerve anatomy, EC location, and peptide turnover. Lastly, ECs may need to be considered an active cell subpopulation that may adapt and respond to different nutrient status as stimuli. Due to the complexity of the enteroendocrine system and its importance in fish nutrition, much remains to be elucidated and it deserves closer attention.