Post-thaw dilution of Rhamdia quelen sperm improves the reproductive success.

The aim was to evaluate the effect of a post-thaw dilution of Rhamdia quelen sperm in 1.1% NaCl (325 mOsm kg-1; pH 7.6; 24 °C) solution on the quality and reproductive capacity. Sperm from eight males were cryopreservation in nitrogen vapor at - 170 °C for 18 h in 0.25 mL straws in a freezing medium containing 5% fructose, 5% Powdered milk, and 10% methanol. The samples were thawed and post-thaw diluted (1:20) in NaCl solution or not (control). The higher spermatozoa velocities were observed in the post-thaw diluted samples (curvilinear (VCL) - 69 ± 11 µm s-1; average path (VAP) - 45 ± 8 µm s-1; straight-line (VSL) - 43 ± 8 µm s-1) compared to the control (VCL - 47 ± 10 µm s-1; VAP - 31 ± 6 µm s-1; VSL - 30 ± 6 µm s-1). Greater straightness (STR), progression (PROG), and beat cross frequency (BCF) were observed in the post-thaw diluted samples (STR - 96 ± 7%; PROG - 666 ± 128 µm; BCF - 42 ± 2 Hz) than in control (STR - 95 ± 5%; PROG - 463 ± 92 µm; BCF - 40 ± 2 Hz). The strongly curled tail was the only morphology change that differ between the post-thaw diluted (5 ± 2%) and control (2 ± 1%). Membrane integrity, mitochondrial activity, and normal larvae rate were not different between treatments. Fertilization and hatching were higher in the post-thaw diluted sperm (93 ± 3%; 82 ± 9%) when compared to control samples (65 ± 13%; 55 ± 17%). Were used oocytes from one female, limiting these results. The post-thaw dilution improved the sperm kinetics and reproductive parameters. Thus, this methodology can be included in the sperm cryopreservation protocol for R. quelen.

Sperm selection of cryopreserved milt of catfish (Rhamdia quelen) by density gradient centrifugation with AllGrad® 90.

Density gradient centrifugation is a technique used to wash or separate samples of cryopreserved milt, mainly in humans and bovines allowing, for example, reducing the concentration of cryoprotectants or choosing the best portion of sperm. The proposed method seeks to reduce the presence of cryoprotectant in the cryopreserved milt of the Rhamdia qhelen and to obtain a fraction of better quality sperm. Gradient centrifugation was formed from 90% AllGrad® and different centrifugation times and forces were compared. The separated sperm presented a low increase in motility and decreased head damage and presence of gout, however, it was better compared to the non-separated samples. The speed of 1000 × g for 10 min, 4 °C, allowed 22.25 ± 4.64% of normal spermatozoa, that is, 9.25% more than the non-centrifuged milt (p = 0.0013).•The centrifugation method allows a fraction of spermatozoa morphologically less affected by cryopreservation.•Density gradient centrifugation with AllGrad® 90% is proposed as a tool of easy adaptation and application for the separation of cryopreserved sperm of R. quelen.•Density gradient centrifugation method at 1000 × g for 10 min allows obtaining a better fraction of normal sperm.

Urine, feces, and blood contamination of frozen and fresh tambaqui (Colossoma macropomum Cuvier, 1818) sperm.

Contamination of fish milt during collection can have an important effect on the quality of fresh and frozen samples. The aim of this study was to evaluate the effects of biological contaminants (urine, feces, and blood) on the sperm of Colossoma macropomum. After hormonal induction, contaminated and contaminant-free milt samples from thirteen males (6.48 ± 2.82 kg) were collected and frozen. The sperm motility was evaluated in fresh and frozen-thawed sperm. Membrane and DNA integrity and mitochondrial functionality were evaluated only in frozen samples. The results revealed lower motility for contaminated sperm in both fresh and frozen-thawed samples [urine (76.15 ± 19.38% and 8.08 ± 6.63%), feces (78.85 ± 26.07% and 1.67 ± 3.26%), and blood (79.62 ± 20.96% and 2.69 ± 4.39%), respectively] than for contaminant-free sperm (95.77 ± 6.07% and 40.00 ± 12.25%, respectively). Motility was different between contaminant-free (118.50 ± 52.08 s) and feces-contaminated (77.00 ± 42.54 s) fresh samples. However, in frozen samples, there was no difference in motility among the groups. The membrane integrity was lower in the contaminated (urine: 72.38 ± 15.55%, blood: 77.00 ± 11.50%, and feces: 68.00 ± 13.64%) than in the contaminant-free (91.46 ± 5.12%) sperm. DNA integrity and mitochondrial functionality were greater in the contaminant-free (82.85 ± 12.19% and 87.15 ± 9.01%, respectively) than in the feces-contaminated (93.38 ± 5.49% and 94.92 ± 6.73%, respectively) samples. C. macropomum sperm contaminated with urine, blood, or feces should not be used for cryopreservation, as these contaminants have detrimental effects on sperm quality.

Effects of anesthetic MS-222 on stress and reproduction of South American silver catfish (Rhamdia quelen) males.

Anesthesia is a common practice used in fish research and aquaculture. It is important to understand anesthetic effects on the animal and tissues of interest to ensure validity of data and to improve animal welfare in research and fish production endeavors. The production of some captive fish species is only possible by imposing artificial reproduction procedures, and manipulation of fish for these purposes is a stressor. The purpose of this study, therefore, was to evaluate effects of different concentrations (100, 200, and 300 mg/L) of the anesthetic MS-222 (tricaine methanesulfonate) on cortisol concentrations and effects on sperm quality in Rhamdia quelen. After hormonal induction of gamete production, 28 sexually mature males were randomly assigned to treatments, and milt and blood samples were collected. Anesthesia induction time, motility rate, sperm concentration and morphology, plasma cortisol concentrations, and reproductive hormone concentrations (testosterone, 17-α-hydroxyprogesterone, and estradiol) were evaluated. Sperm motility was greater in the control than 300 mg/L treatment group but did not differ among the control, 100, and 200 mg/L groups. The estradiol concentration was greater in non- anesthetized than anesthetized Rhamdia quelen, but plasma cortisol concentrations did not differ among treatment groups (182.50 ±â€¯42.03 ng/mL). The anesthetic MS-222 at concentrations of 100, 200, and 300 mg/L did not inhibit the stress response due to handling of Rhamdia quelen males. In addition, treatment with MS-222 was not effective in inhibiting detrimental effects on sperm quality because this treatment was associated with impaired sperm motility and lesser concentrations of plasma estradiol.