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.

Effect of melatonin supplementation to a cytoprotective medium on post-thawed Brycon orbignyanus sperm quality preserved during different freezing times.

The aim of the present study is to verify the viability of frozen B. orbignyanus sperm cells after freezing with dilution media containing different concentrations of the melatonin and after different freezing times. Semen from 15 males was collected and pooled as five pools from three random animals. Oocytes (100) from three females were separately used for fertilization. There were three treatments: (C) Control Medium: 90% of extender Beltsville thawing solution (5% concentration) + 10% methylglycol (MG); (M1) Control Medium + 1 mM melatonin; and (M2) Control Medium + 2 mM melatonin. Sperm samples were diluted in media at a final proportion of 1:4 [125 µl sperm (25% V/V) + 337.5 µl BTS (65% V/V) + 37.5 µl MG (10% V/V)]. Melatonin was added at final solution. Three Dry shipper freezing times were used: T1 (15 min), T2 (12 h) and T3 (24 h). The samples were transferred, stored in a cryobank and thawed in a water bath at 60 °C for 5 s and evaluated concerning viability, morphology and fertilization rate. B. orbignyanus semen frozen in M2 presented the highest fertilization rate (8.40 ± 2.54%). The highest vitality (85.2 ± 2.8%), motility (64.63 ± 8.3%), motility duration (84.22 ± 11.4 s) and progressive motility (17.01 ± 1.2%) rates were maintained for M2. The highest number of altered cells was observed in C (57.4 ± 5.9%). Melatonin at 2 mmol L-1 associated with the cryoprotectant methylglycol in cryopreservation could be used to improve a cryobank for endangered Brycon orbignyanus populations.