Semen Cryopreservation in Brazilian Freshwater Fish: Advances and Main Challenges

Studies on semen cryopreservation in Brazilian freshwater fish have been growing in number of publications and investigated species. Despite this apparent increase in research, standardization of cryoprotocols is still missing, making it clear that the grounds on the quality of cryopreserved semen has not yet reached a level that guarantee satisfactory results for its replication. This chapter aims to make a critical and reflective analysis on the ways cryopreservation of freshwater fish semen has been conducted in Brazil. The difficulties in standardizing protocols, broodstock, and selection of genetically superior animals; the barriers in transferring technology from laboratory benches to the field and make feasible the use of cryopreserved semen on a commercial scale; the formation of germplasm banks and the responsible use of cryopreserved material are also discussed. We have no intention to point out the successes and mistakes that may have been committed in pursuing development of cryopreservation protocols, but a reflection on the future directions considering what should be pondered on this subject with objectivity and scientific consolidation

Viability of zebrafish (Danio rerio) ovarian follicles after vitrification in a metal container.

Cryopreservation of ovarian tissue has been studied for female germline preservation of farm animals and endangered mammalian species. However, there are relatively few reports on cryopreservation of fish ovarian tissue and especially using vitrification approach. Previous studies of our group has shown that the use of a metal container for the cryopreservation of bovine ovarian fragments results in good primordial and primary follicle morphological integrity after vitrification. The aim of this study was to assess the viability and in vitro development of zebrafish follicles after vitrification of fragmented or whole ovaries using the same metal container. In Experiment 1, we tested the follicular viability of five developmental stages following vitrification in four vitrification solutions using fluorescein diacetate and propidium iodide fluorescent probes. These results showed that the highest viability rates were obtained with immature follicles (Stage I) and VS1 (1.5 M methanol + 4.5 M propylene glycol). In Experiment 2, we used VS1 to vitrify different types of ovarian tissue (fragments or whole ovaries) in two different carriers (plastic cryotube or metal container). In this experiment, Stage I follicle survival was assessed following vitrification by vital staining after 24 h in vitro culture. Follicular morphology was analyzed by light microscopy after vitrification. Data showed that the immature follicles morphology was well preserved after cryopreservation. Follicular survival rate was higher (P < 0.05) in vitrified fragments, when compared to whole ovaries. There were no significant differences in follicular survival and growth when the two vitrification devices were compared

A study on the vitrification of stage III zebrafish (Danio rerio) ovarian follicles

AbstractAttempts to cryopreserve fish embryos have been conducted over the past three decades, nevertheless successful cryopreservation protocol for long-term storage still remains elusive. Fish oocytes offer some advantages when compared to embryos, which may help in improving the chances of cryopreservation. In the present study, a series of cryo-solutions were designed and tested for their vitrifying ability using different devices (0.25ml plastic straw, vitrification block and fibreplug™). Toxicity of vitrification solutions was evaluated by assessing follicle membrane integrity with trypan blue staining. In addition, the effect of vitrification protocol on stage III zebrafish ovarian follicles was investigated by measuring the cytoplasmic ATP content and the mitochondrial distribution and activity using JC-1 probe and confocal microscopy. After vitrification, follicles showed membrane integrity of 59.9±18.4% when fibreplug and V16 (1.5M methanol+4.5M propylene glycol) solution were employed. When vitrified in V2 (1.5M methanol+5.5M Me2SO) the membrane integrity decreased to 42.0±21.0%. It was observed that follicles located in the middle of the fragments were more protected from injuries and some of them showed good morphological appearance 2h post-warming. Mitochondria integrity of granulosa cells layer was clearly damaged by the vitrification protocol and ATP level in the follicles declined significantly after warming. Vitrification of zebrafish follicles in ovarian tissue fragments and its effect at sub-cellular level is reported here for the first time. Information gained from this study will help in guiding development of optimal protocol for cryopreservation of fish oocytes

Cooling of pacu (Piaractus mesopotamicus) embryos at various stages of development for 6 or 10 hours

The objective of this research was to verify the effects of cooling embryos of pacu, Piaractus mesopotamicus, in four stages of development during two stocking periods. The stages of embryo development were at: blastoderm, similar to 64 cells-1.4 h after fertilization (haf); 25% of the epiboly movement-5.2 haf; blastoporous closing-8.0 haf; and optical vesicle appearing-13.3 haf. Embryos were exposed to a cryoprotectant solution containing methanol (10%) and sucrose (0.5 M). Thereafter, embryos were submitted to a cooling curve until they reached -8 degrees C, and then kept cooled for 6 or 10 h. In addition, for each stage of embryonic development, a control group with uncooled embryos was used to compare hatching rates. The total number of larvae from the first two stages of ontogenetic development (1.4 and 5.2 haf) was lower compared to the other stages (0.0 and 8.0 haf). There was no significant difference between stages 8.0 and 13.3 haf for the total number of larvae (49.9 +/- 6.7% and 55.2 +/- 6.7%, respectively). Embryo diameter varied according to embryonic stage, providing evidence of differences in membrane permeability. There was a negative correlation between embryo diameter and the total number of larvae (r = -0.372). In conclusion, use of embryonic stages 8.0 and 13.3 haf were recommended for maintaining cooled pacu embryos at -8 degrees C for 6 or 10 h. (C) 2011 Elsevier B.V. All rights reserved.Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq

Assessing the Viability of Reintroduction of Locally Extinct Migratory Fish Brycon orbignyanus: Successful Growth, Dispersal and Maturation

The reintroduction of threatened fish species in areas where wild populations have been depleted due to anthropogenic impacts is an increasingly popular conservation tool and mitigation policy. Despite the importance of fish reintroduction for conservation purposes, little is known about its efficiency. Here, we assessed the viability of reintroduction of the endangered migratory fish, Brycon orbignyanus, in an area of the Upper Uruguay River basin where the species has not been reported for more than 30 years. We released 4000 yearling juveniles in the Pelotas River in 2014 and maintained 400 juveniles in captivity as a control population. After three years, a total of 13 individuals was recaptured, of which, 10 were considered sexually mature with first maturation being recorded in animals larger than 42 cm in total body length. The age&ndash;length comparison with a control population growth curve showed that recaptured fish were slightly bigger than those in captivity. Furthermore, important ecological attributes as schooling behavior and dispersal capacity were recorded for all recaptured individuals. Combined, our results suggest that the re-establishment of a self-sustained population of locally extinct species B. orbignyanus in the Pelotas River may be successful if sustained over time and supported by conservation policies

Caracterización genética de lotes de "Brycon orbignyanus" utilizados en programas de repoblamiento

Objetivo. Caracterizar geneticamente dos lotes y una progenie de Brycon orbignyanus destinados para programas de repoblamiento, utilizando la tecnica molecular de RAPD (Random amplified polymorphic DNA). Materiales y metodos. Se analizaron 58 reproductores originarios de dos piscicolas ubicadas en las ciudades de Castillo (A:30 individuos) y Porto Ferreira (C:28 individuos), mantenidos en cautiverio hace seis anos en la estacion de acuicultura e hidrologia de la Duke Energy Internacional (Geracao Paranapanema) (Sao Paulo-Brasil). Treinta larvas de la progenie del lote A (B) tambien se analizaron. Resultados. Los 14 primers usados produjeron 87 fragmentos de los cuales 70.11% fueron polimorficos. Fueron observadas diferencias (p.0.05) en la frecuencia de 31 fragmentos, con tres exclusivos para el lote A. Los valores de divergencia, distancia e identidad genetica mostraron que la diversidad genetica del lote A fue mantenida en la progenie y que existe una baja diferenciacion entre los lotes de reproductores. El analisis de variancia molecular mostro que la mayor parte de la variacion esta dentro de cada lote (87.45%) y no entre ellos (12.55%). Este resultado se corroboro con los valores de FST (0.125) y con el dendrograma, que indicaron una moderada diferenciacion genetica, sin la formacion de agrupamientos. Conclusiones. La diversidad genetica fue preservada en la progenie debido al manejo eficiente de la reproduccion. No hubo una diferenciacion genetica entre los lotes de reproductores, debido posiblemente a que el origen natural de ambos fue el rio Parana.Objective. To genetically characterize two Brycon orbignyanus stocks and one progeny intended for restocking programs using the molecular technique RAPD (Random Amplified Polymorphic DNA). Materials and methods. Fifty eight broodstocks native to two fish farms in the cities of Castilho (A:30 individuals) and Porto Ferreira (C:28 individuals) were analyzed. The fish had been maintained for six years in captivity in the aquaculture and hydrology station of Duke Energy International (Geracao Paranapanema. Sao Paulo - Brazil); thirty larvae of progeny from the stock A (B) were also analyzed. Results. The fourteen primers used produced 87 fragments of which 70.11% were polymorphic. Differences were observed (p.0.05) in the frequency of 31 fragments, with three exclusive from stock A. The values for divergence, distance and genetic identity showed that genetic diversity of stock A was maintained in progeny and that a low differentiation exists among reproductive stocks. Analysis of Molecular variance showed that most of the variation is inside each stock (87.45%) and not between them (12.55%). This result was corroborated with FST (0.125) values and with the dendrogram indicating a moderate genetic differentiation, without cluster formation. Conclusions. Genetic variability was maintained in progeny, possibly because both were native to the Parana rive

Morphological and morphometric analysis of skeletal muscle between male and female young adult Colossoma macropomum (Characiformes: Serrasalmidae)

ABSTRACT This study aimed to evaluate muscle organization in tambaqui in order to describe the muscle growth process. We analyzed the morphometric pattern of fibers from white muscle of young-adults (300 days) by smaller diameter. The organization of white muscle exhibited a typical morphological pattern found in other fish species. Heavier animals showed higher frequency of larger diameter fibers (>50 μm ) and smaller animals had higher frequency of smaller diameter fibers (50 μm ) than males. However, there was no difference between body weight and sex (P =0.8). Our results suggest that muscle growth is by hypertrophy and hyperplasia due to a mosaic appearance from different diameters fibers, which is characteristic of large size fish species