Alexandre Nizio Maria

Out-of-season sperm cryopreserved in different media of the Amazonian freshwater fish pirapitinga (Piaractus brachypomus)

The pirapitinga (Piaractus brachypomus) is a freshwater fish that inhabits the Amazon and Orinoco River basins. The use of cryopreserved sperm has been considered to facilitate procedures during the artificial reproduction. The aim of the present study was to develop a freezing protocol for pirapitinga sperm collected outside the spawning season. Sperm samples were diluted in four freezing media prepared by a combination of two extenders (glucose and BTS-Beltsville Thawing Solution) and two cryoprotectant agents (DMSO and methylglycol) loaded into 0.5-mL straws, frozen in a nitrogen-vapor shipping dewar (dry-shipper) and stored in liquid nitrogen at -196 degrees C. Post-thaw sperm motility was evaluated both subjectively using a light microscope and by a computer-assisted sperm analyzer (CASA). Curvilinear, average path and straight-line velocities were also determined. There were no differences (P>0.05) in post-thaw sperm motility between evaluations performed subjectively and using the CASA. Sperm samples cryopreserved in glucose-methylglycol yielded the greatest post-thaw sperm motility (81%) and fastest sperm velocities when compared to the samples frozen in the other three media (P<0.05). Out-of-season sperm cryopreserved in glucose and methylglycol under the conditions described above is of high quality and can therefore be used to facilitate artificial reproduction procedures, as only females will need handling for hormonal induction and gamete collection during the spawning season. Although the CASA system provides precise data on sperm motility, the subjective evaluation is practical and can be conducted by well-trained personnel at commercial fish farms as an acceptable evaluation of sperm quality.

Hormonal induction and semen characteristics of tambaqui Colossoma macropomum.

In the hatchery-bred tambaqui Colossoma macropomum, spontaneous semen release does not occur, and hand-stripping produces reduced semen volume. The goal of this work is to evaluate the effects of hormonal induction with carp pituitary extract (CPE) on both qualitative (visual aspect, pH, motility, viability and morphological abnormalities) and quantitative (volume, concentration and number of spermatozoa per ejaculate) traits of tambaqui semen. Eleven males were treated with CPE (induced), and 11 were left untreated as a control (non-induced). All analysed parameters except motility and percentage of viable spermatozoa presented significant differences (p < 0.05) between the induced and non-induced treatments. CPE induction resulted in a 25-fold increase in semen volume and a 10-fold increase in the number of spermatozoa collected. However, both sperm concentration and the frequency of sperm with morphological abnormalities (commonly detached heads or bent tails) were significantly lower in CPE-induced fish. The hormonal induction of tambaqui males with CPE is efficient and positively influences some qualitative and quantitative properties of semen. Additionally, semen collection via gentle abdominal massage occurs more readily in CPE-induced fish.

Use of cryotubes for the cryopreservation of tambaqui fish semen (Colossoma macropomum).

Tambaqui (Colossoma macropomum) is a freshwater fish of great importance to aquaculture in several South American countries. Recent studies have developed a protocol for semen cryopreservation in 0.25 and 0.5 mL straws; however, this technique has limitations for fingerling production at a large scale due to the high fecundity of tambaqui. The main objective of this study was to evaluate the feasibility of using cryotubes (1.6 and 4.5 mL) for tambaqui semen cryopreservation. Semen samples were diluted in freezing solution (5% glucose solution, 10% methylglycol, 5% egg yolk), stored in 1.6 and 4.5 mL cryotubes, frozen in liquid nitrogen vapor at -175°C and transferred to a cryogenic container at -196°C. The cryotubes were thawed in a water bath at 60°C for 70 or 90 s and the motility (total motility - TM; progressive motility - PM; curvilinear velocity - VCL; straight line velocity - VSL and average path velocity - VAP) and the viability of sperm were evaluated. There was no significant difference in sperm motility and viability post-thawing between 1.6 and 4.5m L cryotubes, except for TM (47% and 40%, respectively). Thawing for 90 s provided better results, being used in fertilization trials. Although the fertilization rate did not differ between the cryotubes (41-45%), it was significantly lower than that for fresh semen (74%). A strong positive correlation was observed between the sperm motility and fertilization rate (r=0.69-0.89). We conclude that 1.6 and 4.5 mL cryotubes have high potential for tambaqui semen cryopreservation when thawed for a minimum time of 90 s at 60°C.

Histochemical and morphological features of biopsied and stripped oocytes from the Brazilian endangered teleost pirapitinga, Brycon nattereri (Characiformes)

The aim of the present study was to characterize biopsied and stripped oocytes from the Brazilian endangered teleost pirapitinga (Brycon nattereri) using histochemical and morphological analyses. Biopsied oocytes had a mean diameter of 2.225 mm (modal diameter: 2.312 mm), complete vitellogenesis and a central or slightly eccentric nucleus. Neutral polysaccharides were detected in the follicular cells, zona radiata and yolk globules, while acidic polysaccharides were detected in the follicular cells and cortical alveoli. Ten out of the 19 females treated with two doses of carp pituitary extract (cPE) released oocytes, which were also analysed. Stripping occurred 292 ± 39 degree-hours after the second dose of cPE and led to a mean spawning weight of 36.2 g, 10% spawning index, 241 oocytes/g of ova, 8222 oocytes/female and 23 oocytes/g of body weight. Stripped oocytes had a mean diameter of 2.33 mm and a mode at 2.375 mm, were weakly adhesive and coloration ranged from wine to brown. Under scanning electron microscopy, stripped oocytes exhibited a single funnel-shaped micropyle located at the animal pole and a zona radiata that measured 7.7 μm in thickness with eight pore canals/μm2. Oocyte morphology in Brycon nattereri is similar to that found in other species of the genus, except for the larger size and weaker adhesiveness. These findings provide essential information for a better understanding of the reproductive biology of B. nattereri and the establishment of conservation measures for this threatened species.

Embryonic development and larval growth of Brycon nattereri Günther, 1864 (Characidae) and its implications for captive rearing

The aim of this study was to describe, for the first time, the embryogenesis and larval growth of the Paraitinga Brycon nattereri Günther, 1864 reared in captivity. After artificial fertilization, eggs were incubated at constant temperature (~19°C) and collected every 15 min during the first 3 h and then every 3 h until hatching. Five larvae were collected daily over 15 days for evaluation of the length, yolk sac volume and specific growth rate. The following stages of embryonic development were identified: zygote, cleavage, gastrula, segmentation and larval. The hatching occurred after 50-54 h, with larvae poorly developed and fully depigmented, devoid of mouth and swimming capacity, presenting 6.32 mm total length and 3.64 mm3 yolk sac volume. The mouth opening was observed between days 3-4 after hatching. The yolk sac absorption was slow during the first 3 days, increasing sharply after this period, being completed on the day 11. During this period there was a decrease in the larval growth rate. After yolk sac absorption, an increase in the growth rate was observed that coincided with the start of exogenous feeding. Cannibalism was not observed during the 15 days of evaluation. The initial development of B. nattereri was slow and poorly developed larvae in relation to other Brycon species, certainly due to the lower temperature required for egg incubation and larval rearing. Other studies are needed in order to develop techniques to improve the methods of incubating eggs and feeding larvae.

Distillation methods affect the chemical composition of Varronia curassavica Jacq. essential oil

The objective of this work was to evaluate the chemical composition of essential oil from Varronia curassavica Jacq. obtained by microwave (MI) and hydrodistillation (HD) extraction methods. The MI method tested three powers (500, 600, and 700W), three distillation times (20, 30, and 40 min.), and three water volumes (0, 25, and 50 mL per sample). The HD method tested three distillation times (100, 120, and 140 min.) and three water volumes (1.0, 1.5, and 2.0 L per 3-liter flask). The essential oils were analyzed by GC/MS-FID. The optimal condition for the essential oil extraction by the MI method was 700W for 40 min. (3.28%), regardless of the volume of water. In its turn, the best condition for essential oil extraction by the HD method was 120 min. with 1.0 L of water per flask (3.34%). The most abundant compounds for MI (700 W for 40 min. without water) were shyobunol (26.53%) and bicyclogermacrene (4.96%); and the most abundant compounds for HD (120 min. with 1.0 L of water/flask) were shyobunol (24.00%) and germacrene D-4-ol (10.23%). Methyl farnesoate (2E, 6E) and farnesyl acetate (2Z, 6E) were not detected in the essential oil extracted by HD; however, they were identified by the MI method. By increasing the distillation time and/or volume of water in HD, a reduction was observed for the content of the chemical compounds -elemene (from 1.23 to 0.97%), Ecaryophyllene (from 5.49 to 4.35%), -humulene (from 1.80 to 1.43%), alloaromadendrene (from 1.78 to 1.44%), bicyclogermacrene (from 5.63 to 4.55%), and germacrene D-4-ol (from 11.40 to 9.86%). Power, extraction time, and their interactions influenced the content of essential oil obtained by microwave extraction (MI). Within each power, the highest essential oil content was extracted at the longest distillation time (40 min.), except for 600W, where no significant difference was detected between 30 and 40 min. The optimal essential oil contents for both extraction methods were statically similar by the t-test for dependent samples. However, the MI method presents advantages, such as shorter distillation time and less energy and water consumption.