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.

A simulated marine heatwave impacts European sea bass sperm quantity, but not quality.

Rapid environmental changes will be the major challenge that most biota will have to deal with in the near future. Extreme events, such as marine heatwaves, are becoming more frequent and could be spatially uniform at a regional scale for a relatively long period of time. To date, most research studies on heatwaves have focused on sessile organisms, but these extreme events can also impact mobile species. Here, a 3-week marine heatwave was simulated to investigate its effects on the male reproductive performance of a Mediterranean Sea emblematic species, the European sea bass Dicentrarchus labrax. Males from the control condition (c. 13°C) produced significantly more sperm than those exposed to a relatively warm thermal treatment (c. 16°C). Nonetheless, neither the percentage of motile spermatozoa nor most of the other sperm motility parameters were significantly affected by the rearing temperature over the whole period. Overall, the results of this study suggest only moderated effects of a potential winter heatwave on the reproductive performance of male European sea bass.

Vitrification protocol for immature Brycon orbignyanus ovarian tissue as an extinction escape strategy.

Piracanjunba (Brycon orbignyanus) is an endangered South American fish, and ovarian tissue cryopreservation is an alternative method for preserving maternal germplasm and genetic diversity. Therefore, our aim was to test a vitrification protocol for ovarian tissue containing primary growth (PG) oocytes of B. orbignyanus as a strategy to avoid the threat of extinction. Two vitrification solutions were evaluated (VS1: 1.5 M methanol + 4.5 M propylene glycol and VS2: 1.5 M methanol + 5.5 M Me2SO) and compared using control/fresh ovarian tissue. After vitrification, the following factors were analyzed: membrane integrity using trypan blue, morphology using a histological assessment, oxidative stress (total reactive antioxidant potential (TRAP) and reduced thiol [-SH]), mitochondrial activity using MTT, and DNA damage using a comet assay. The vitrified oocytes (VS1 = 24.3 ± 0.49% and VS2 = 24.8 ± 0.69%) showed higher DNA damage than the control group (control = 20.7 ± 1.03%) (P = 0.004). In contrast, in most evaluations (membrane integrity, membrane damage, oxidative stress, and mitochondrial activity), there were no discernible differences between the control group and the vitrified samples. In addition, oocyte (P = 0.883) and nuclear diameter (P = 0.118) did not change after vitrification. VS2 treatment resulted in higher nuclear damage (15.7 ± 1.45%) than in the control treatment (3.5 ± 1.19%); however, VS1 treatment did not result in significantly more damage (9.5 ± 3.01%) than in the control (P = 0.015). Therefore, the protocol for ovarian tissue vitrification tested in this study resulted in high maintenance of PG oocyte cell integrity, making it a promising alternative for B. orbignyanus maternal genome preservation.

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.

The Reproductive Biology of Puye (Galaxias maculatus) under Experimental Culture.

This study determines the reproductive patterns of puye (Galaxias maculatus) under culture conditions. A population of 567 wild fish was caught in the Cautín River, Chile, and held in captivity for four years. Mortality, sex ratio, gonadosomatic index (GSI), sexual maturity stages, spawning period, type and frequency of spawning, and fecundity were measured. The fish grew throughout the experimental period, with the fastest rate during the first half of the first year of life. The highest mortality occurred during the first three months of the experiment and during the spawning season. The sex ratio was almost 1:1 (female:male). First sexual maturity was reached at one year of age, with an average weight of 0.85 ± 0.01 g, total length of 4.85 ± 0.16 cm, and condition factor 0.0074. The highest GSI in both females (12.14 ± 0.74) and males (17.7 ± 2.70) was recorded in August. Nevertheless, the females spawned 3 to 10 times between September and February, with the highest reproductive peak between September and October. The number of embryos per female per day varied from 1 to 429, while the total number of embryos per female during the entire season evaluated varied from 163 to 1044. There was a high correlation (r = 0.82) between absolute fecundity and body weight. Although further studies are needed in this field, these results are basic for establishing future reproductive programs in captivity as a strategy for sustainable fisheries and aquaculture management.

Preparation and extraction of chorion proteins from Salmo salar embryos at the pigmented eye stage for electrophoresis with SDS-polyacrylamide gel.

The chorion fulfills important functions in fish embryos, including protecting the embryo during development. The characterization of the protein profile of this envelope could be used as a bioindicator in the evaluation of the quality of embryonic development. The object of this work was to validate a standardized protocol for protein extraction from chorion of Salmo salar embryos at 280 accumulated thermal units (ATU) by comparing and combining existing methods. The protocol consists of consecutive washing of the chorion samples followed by protein extraction with the solution that was named SDS solution (Tris-HCl 100 mM (pH 8), Urea 8 M, 1% SDS, ß-mercaptoethanol 300 mM and EGTA 10 Mm, and 1% protease inhibitor cocktail) and mechanical methods. Protein extraction is enhanced by a working temperature of 75 °C and use of a disperser. The protein concentration was quantified by Bradford Assay. After extraction, the samples were diluted (dilution factor 10) before reading against the calibration curve. After gel electrophoresis with a load of 3 µg of protein, staining showed more than 4 bands, with molecular weights between 25 kDa and 180 kDa.•The protein profile of fish chorion was between 25 kDa and 180 kDa.•Solution containing 1% SDS allows a higher extraction of proteins from the chorion of Atlantic salmon embryos with 280 ATU.•Chorion protein identification is a valuable tool in determining gamete and embryo quality in fish.

Hydrogel encapsulation as a handling and vitrification tool for zebrafish ovarian tissue.

Zebrafish is an important animal model, thousands lines have been developed, thus having a great need for their preservation. However, the cryopreservation of fish oocytes is still limited and needs improvement. The sodium alginate hydrogel, in addition to providing support for the cells, has been shown to be a potential cryoprotectant. Therefore, the aim of this study was to evaluate the sodium alginate hydrogel encapsulation technique efficiency during zebrafish ovarian tissue vitrification. The encapsulation methodology was standardized in the first experiment. In Experiment 2, we evaluated four vitrified groups: standard protocol without encapsulation (VS); encapsulated with cryoprotectants (VS1-A); encapsulated with half the cryoprotectants concentration (VS2-A); encapsulated without cryoprotectants (VA). VS treatment (54.6 ± 12.3%; 23.7 ± 9.9%; 12.6 ± 5.0%) did not differ from the VS1-A and VA showed a lower membrane integrity percentage (1.2 ± 1.4%; 0.3 ± 0.6%; 0.5 ± 1.5%). Mitochondrial activity was significantly greater in non-encapsulated treatment (VS) when compared to the encapsulated treatments. VS1-A and VS obtained the lowest lipid peroxidation (39.4 ± 4.4 and 40.5 ± 3.3 nmol MDA/mg respectively) in which VS was not significantly different from the VS2-A treatment (63.6 ± 3.1 nmol MDA/mg), unlike, VA obtained the highest lipid peroxidation level (124.7 ± 7.9 nmol MDA/mg). The results obtained in this study demonstrate that the sodium alginate hydrogel encapsulation technique did not have a cryoprotective action, but maintained the membrane integrity when used the standard concentration of cryoprotectants. However, halving the cryoprotectant concentration of fragments encapsulated in alginate hydrogel did not cause an increase in lipid peroxidation. In addition, it provided support and prevented the oocytes from loosening from the tissue during the vitrification process, being an interesting alternative for later in vitro maturation.

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.

Reservas de carbono del pasto Cenchrus clandestinus (Poaceae) en los sistemas de manejo tradicional y silvopastoril, en diferentes relieves

El aumento de las emisiones de Gases de Efecto Invernadero (GEI) derivadas de las actividades humanas, son consideradas el principal responsable del cambio climático actual y el sector ganadero es responsable del 18 % de las emisiones de GEI en CO2 equivalente. El pasto kikuyo puede optimizar tanto la captura como la fijación del carbono. El objetivo del trabajo fue identificar las existencias de carbono en el pasto kikuyo en sus diferentes compartimentos, biomasa aérea (BA) y biomasa radicular (BR), a 20 y 40 cm de profundidad del suelo, bajo los sistemas tradicional y silvopastoril en diferentes relieves. Se realizaron seis muestreos (M) sucesivos de acuerdo al sistema de pastoreo (tradicional y silvopastoril), la geoforma del terreno (flanco cóncavo (FCC), flanco convexo (FCX), flanco rectilíneo (FR) y relieve plano (RP)). Se muestrearon la biomasa aérea (BA) y de raíces (BR). El método estadístico utilizado fue un diseño en bloques incompletos aleatorizados, se evaluaron dos tratamientos (T) (T1: tradicional y T2: silvopastoril) con cuatro bloques (B) en cada uno (B1: FCC, B2: FCX, B3: FR, B4: RP). El trabajo se realizó entre junio 2016 y junio 2017 en San Pedro de los Milagros, Antioquia Colombia. Los resultados permitieron determinar que las raíces a 20 cm de profundidad, el colchón muerto y las hojas, fueron los compartimentos con mayores existencias de carbono (4.52, 3.58 y 1.9 ton de C ha-1 respectivamente). Se encontraron diferencias (P < 0.05) entre relieve plano y el relieve rectilíneo para la biomasa de hojas, y entre el relieve plano con los demás relieves evaluados para la variable raíces gruesas a 20 cm de profundidad. La biomasa producida por la planta es directamente proporcional al carbono incorporado. La biomasa radicular, tanto para raíces finas como gruesas, contribuye a capturar en promedio 2 820 y 655 kg ha-1 de carbono a 20 y 40 cm de profundidad respectivamente. El pasto kikuyo contribuye a mantener reservas de carbono en las praderas. Por la alta producción de biomasa radicular, de colchón y la alta capacidad de rebrote en condiciones adversas, se concluye que este pasto juega un papel importante en la disminución de GEI y la conservación de los suelos del trópico alto bajo sistemas de lechería especializada.

The increase of Greenhouse Gases (GHG) emissions derived from human activities are considered the main cause of current climate change and the livestock sector is responsible for 18 % of the GHG emissions in CO2 equivalent. Kikuyu grass can optimize both carbon capture and carbon fixation. The aim of this paper was to identify carbon stocks in the kikuyu grass in its different compartments, above-ground biomass (AB) and below-ground biomass (BB) at 20 and 40 cm soil depth, under the traditional and silvopastoral systems in different reliefs. Six successive samplings (M) were taken according to the grazing system (traditional and silvopastoral system), and the geoform of the terrain (concave flank (CCF), convex flank (CXF), rectilinear flank (RF) and flat relief (FR)). The above-ground biomass and below-ground biomass were sampled. The statistical method used was a design in incomplete randomized blocks, two treatments were evaluated (T) (T1: traditional system and T2: silvopastoral system) with four blocks (B) in each one (B1: CCF, B2: CXF, B3: RF, B4: FR). This experiment was done from June 2016 to June 2017 in San Pedro de los Milagros, Antioquia, Colombia. The results allowed to determine that the roots at 20 cm depth, the dead creeping stems, and the leaves were the compartments with the highest carbon stocks (4.52, 3.58 and 1.9 ton of C ha-1, respectively). Differences were found (P < 0.05) between flat and rectilinear relief for the biomass of leaves, and between the flat relief with the other reliefs evaluated for the variable thick roots at 20 cm depth. The biomass produced by the plant is directly proportional to the incorporated carbon. The root biomass, both fine and thick roots, contributes to capture on average 2 820 kg and 655 kg of carbon per hectare at of 20 and 40 cm depth respectively. Kikuyu grass contributes to keep carbon reserves in the grasslands. Due to the high production of below-ground biomass and creeping stems, and its high capacity of regrowth under adverse conditions, this grass plays an important role in the reduction of GHG and the conservation of high tropical soils under specialized dairy systems.