Fish sperm cryopreservation using biodegradable containers: New low-cost and environment-friendly methodology.

In brief: The containers used in cell cryopreservation are essential to maintain cell integrity and viability after thawing. This paper reveals the methodology of using biodegradable containers for fish sperm cryopreservation. Cryopreserved sperm in biodegradable containers showed high fertility capability. Biodegradable capsules could be alternative containers to plastic straws for sperm cryopreservation. Abstract: 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 12South 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 the tested sperm parameters. Thus, based on the high sperm fertility capability, both capsules were efficient as cryopreservation containers for maintaining sperm functionality.

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.

Morphological abnormalities in zebrafish cryopreserved sperm.

The aim of the present study was to evaluate the effect of cryopreservation on the morphology of zebrafish sperm (Danio rerio). Sperm from 30 males were collected and divided in two treatments: fresh and cryopreserved semen. The following were measured sperm morphology, motility and membrane integrity. Cryopreservation reduced motility, the number of normal cells and the membrane integrity, as well as increased the percentage of sperm abnormalities. The most frequent types of morphological changes found in cryopreserved semen were macrocephaly, loose head, degenerated head, proximal gout, curled tail and short tail. This study opens the way for further investigations on morphological changes and for a new classification of these changes in fish semen due to cryopreservation.