Fig 3 - uploaded by Victor Mauricio Medina Robles
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Scanning Electron Microscope images of Piaractus orinoquensis spermatozoa. A) fresh spermatozoa (10.0 KV; SP= 9.0; WD=12.4; x 5.0 K). B: Spermatozoa cryostored for 12 months (10.0 KV; SP= 12.0; WD=12.3; x 5.0 K). Spermatozoa with cryo-damage at head level (white arrows). KV: voltage acceleration; SP: spot size; WD: working distance; K: 1000.
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To date, little attention has been paid to identifying the effects of long-term cryopreservation on sperm quality for Piaractus orinoquensis. The object of this study was therefore to evaluate the effect of long-term cryopreser-vation (24 h, 1, 6 and 12 months) on sperm motility, viability, DNA integrity, ATP content, total antioxidant capacity (TA...
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... iii) destruction of mitochondrial cristae; iv) loss of the electron-dense chromatin of the nucleus; and v) degeneration in the middle region of the flagellum with uncoupling or loss of the flagellum in some cases (Fig. 2). The main structural damages at the head level in spermatozoa of P. orinoquensis cryopreserved for 12 months are shown in Fig. ...
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Understanding freezing is essential for stable cryopreservation of sperm. In the freezing process, ice crystal is developed from a change of matter from liquid water to solid water. Unfortunately, they tend to make damage of membrane in frozen sperm. From a perspective, sperm membrane damage due to ice crystal formation, magnetized f...
Citations
... Milt cryopreservation in fish has been tried for different species, especially those of commercial and/or environmental interest [125][126][127][128][129][130][131]. When using cryopreserved milt from Brycon insignis (Characiformes), sperm motility of cryopreserved milt was 54% compared to 100% in fresh milt. ...
To meet growing fish demand, aquaculture must develop sustainable, cost‐effective, and high‐performance fish feed formulations for industry advancement. Plant‐based lipids are a viable alternative to forage fish ingredients in finfish diets. However, most plant‐derived lipids lack long‐chain polyunsaturated fatty acids (LC‐PUFA) like EPA and DHA, which are nutritionally essential for fish. This review summarizes recent findings on the nutritional aspects of fish brood stock and sperm quality, emphasizing the effects of fatty acids on reproductive outcomes. Studies indicate that substituting fish oil with plant oils alters the fatty acid profiles in fish tissues and gametes, potentially compromising sperm quality, cryopreservation success, and progeny viability. Sperm membrane lipids, including EPA, DHA, and ARA, are vital for motility and fertilization rates post‐thaw. Additionally, cryopreservation has been linked to molecular and epigenetic alterations in sperm, which may negatively affect offspring quality. The role of mRNA and noncoding RNA in regulating sperm function and embryonic development is well known, and thus any damage to these molecules as a consequence of cryopreservation might induce long‐term effects on offspring. Proteomic analyses reveal that cryopreservation can lead to significant protein loss in sperm, diminishing their functional and fertilization capabilities. Optimizing cryoprotectant protocols and freezing techniques is crucial to reduce damage, while dietary fatty acids play a key role in preserving sperm quality during cryopreservation stress. Further research with omic technologies is crucial to fully understand sperm tolerance to cryopreservation, which will improve reproductive outcomes and enhance sustainability and quality in aquaculture.
... Duarte-Trujillo et al. (2021) también encontraron un aumento significativo de anormalidades espermáticas en semen crioconservado de P. mariae, en comparación al semen fresco. En cuanto a daños a la ultraestructura espermática causado por la crioconservación, Medina-Robles et al. (2023a), reportó para P. orinoquensis, anormalidades como: aumento en el espesor de la membrana plasmática y ruptura de la misma, así como una disminución en el número de mitocondrias y en las remanentes presentaron daños en sus crestas, el núcleo también se vio afectado, ya que se mostró una cromatina menos densa, igualmente se identificaron desarreglos en las piezas medias, desacoplamiento o pérdida del flagelo también fueron observados. Estos defectos están relacionados al entorno físico-químico al que son expuestos los gametos en procesos de crioconservación (Ozkavukcu et al., 2008). ...
La acuicultura a nivel mundial cada vez toma más relevancia y se ha convertido en una fuente de alimento e ingresos económicos de muchas comunidades alrededor del mundo. Sin embargo, actividades como la sobreexplotación del recurso hídrico y pesquero, el aumento de la población humana, la contaminación ambiental, el cambio climático, entre otros factores, están poniendo en riesgo no solo la seguridad alimentaria del planeta, sino también la biodiversidad de organismos, entre ellos los peces. Una de las herramientas para salvaguardar los recursos biológicos acuáticos, es la crioconservación, que en peces se enfoca principalmente en la conservación de semen en nitrógeno líquido y esta, pesar de ser una técnica muy útil, es susceptible a mejoramiento, ya que se ha reportado que las bajas temperaturas usadas en la crioconservación pueden causar efectos negativos en las células espermáticas, debido a esto es necesario establecer protocolos especie específicos para sacarle todo el potencial a esta forma de conservación. Por lo tanto, el objetivo de esta revisión es recopilar información sobre los principales parámetros de calidad espermática que se ven afectados en el momento de crioconservar el semen, haciendo énfasis en las especies dulceacuícolas.
Milt cryopreservation facilitates native fish’s constant production of commercial interest. However, it can damage spermatozoa (sptz) due to the osmotic stress of diluents and low temperatures. This study compares spermatic quality in fresh (SF) and cryopreserved (CS) milt in B. amazonicus for 24 h, 1 month, and 3 months. Eight sexually mature males were induced with carp hypophysis extract. Milt was diluted in one proportion in a glucose solution (5.5%), egg yolk (12%), and dimethyl sulfoxide (10%). An evaluation of parameters such as motility, viability, morphology, total antioxidant capacity (TAC), and adenosine triphosphate (ATP) contents was performed at 24 h, 1 month, and 3 months, and the fertility test was conducted at 24 months of cryopreservation. The motility percentage was higher ( p ≤ 0.05) in SF (95.5 ± 2.9%). Motility duration of SF was significantly higher (113.6 ± 22.6 s) than that of CS at 3 months (71 ± 12.2 s). The spermatic viability of SF was higher (97.1 ± 1%) in comparison with CS treatments. Sperm anomalies in SF were lower (12.4 ± 3.2%) than in all other treatments. ATP contents of (1.74 ± 4.5 nM × 10 ⁸ sptz) were superior only ( p ≤ 0.05) in CS of 1 month (1.16 ± 1.2 nM × 10 ⁸ sptz). TAC was higher ( p ≤ 0.05) in SF (26.46 ± 1.2 mM of Trolox [6‐hydroxy‐2,5,7,8‐tetramethilcromane‐acid 2‐carboxylic]) compared to other CS treatments. However, there were not any significant differences in this parameter between cryopreservation times ( p ≤ 0.05). Fertility rates were the same for all treatments. In terms of cellular ultrastructure, it was observed that B. amazonicus sptz had an oval‐shaped head without an acrosome, middle part, and only one flagellum.
Fish sperm cryopreservation is an important technique for optimizing juvenile production in aquaculture stations and laboratories and contributing to the conservation of endangered species. Despite its benefits, the cryopreservation process can cause cellular damage, affecting spermatozoa quality and offspring viability. This study aimed to evaluate the larval development of jundiá Rhamdia quelen originating from cryopreserved sperm. Larvae were obtained from artificial reproduction using oocyte samples from four females combined with fresh (Control) or cryopreserved/thawed sperm. The semen was diluted in the cryoprotective solution (1:3 ratio) consisting of skimmed milk powder (5%), methanol (10%), and fructose (5%), and was packaged into 0.25 mL straws. The straws were then stored and cooled in liquid nitrogen vapor for 18 h. The straws were individually warmed in a water bath at 25 °C for 10 s to thaw the samples. The experiments were performed in triplicates. Sperm quality, fertilization, hatching, and larval development were evaluated. After larval hatching, six larval collections were performed (5, 10, 15, 20, and 25 days after hatching), and 15 larvae were sampled per collection per treatment. Cryopreservation reduced sperm motility (70.48 ± 7.70 fresh to 41.36 ± 4.80 cryopreserved semen), progressivity (3874 fresh to 2505 cryopreserved semen), and beat cross frequency (55.83 ± 155 fresh to 50.22 ± 190 cryopreserved semen). Increased the percentage of sperm with abnormal morphology and increased most sperm pathologies. Furthermore, the fertilization rate was lower in the cryopreserved group (63.1 ± 18, and 83.72 ± 7.59 for fresh semen), while hatching was not different between groups (65.3 ± 18.05 fresh, 48.89 ± 21.77 cryopreserved semen) Otherwise, the initial larval development morphology showed no difference in the appearance of structures such as the presence of the vitelline structure, pigmentation pattern, development of the anal pore, embryonic membrane, eye, barbells, notochord flexion, and fin rays, for both treatments. There was no significant difference in the frequency of structures between larvae from fresh and cryopreserved/thawed sperm, revealing a similar developmental pattern in both treatments. In conclusion, the cryopreservation protocol affects sperm quality; however, those sperm able to fertilize the oocytes originate normal larvae with regular larval development of R. quelen up to 25 days old.
Graphical Abstract
In this study, we developed an optimized cryopreservation protocol for goldlined seabream sperm through five small experimental groups: Diluting the sperm with a mixed cryoprotectant (containing 10 g/L glucose and 10% DMSO in an RS extender) at a ratio of 1:3, followed by equilibration at 4 °C for 30 min. Subsequently, the samples were subjected to liquid nitrogen steam fumigation at 5 cm above the surface for 5 min before drop in the liquid nitrogen. Using computer-assisted sperm analysis (CASA), we examined the motility parameters of the sperm before and after freezing for 2 days, as well as the changes in energy metabolism enzyme (T-ATP, LDH, SDH) activities and antioxidant enzyme (T-GSH, CAT, SOD, and GSH-px) activities at 0, 5, 10, and 20 days after freezing. After 250 days cryopreservation, fluorescence staining of sperm showed that most cell membranes were less damaged, and the fertilization rate of sperm after 250 days cryopreservation was 72.53 ± 7.32%. objective of this study was to select an effective cryopreservation protocol for goldlined seabream sperm and elucidate the physiological damage mechanisms before and after cryopreservation.
This study documents the effect of cryopreservation on motility, DNA integrity, and gene expression in Mugil cephalus sperm. Fresh sperm were cryopreserved using V2 extender (V2E) or 0.3 M glucose, each in combination with one of three cryoprotective agents (CPAs), i.e., 10 % of dimethylsulfoxide, ethylene glycol, or glycerol, all at once. After two different storage (7- vs 60- day) periods in liquid nitrogen, sperm samples were thawed. Single-cell gel electrophoresis was used to detect the DNA integrity. Heat shock proteins (HSPs), HSP70, HSP90 and glutathione peroxidase (GPx2) genes mRNA expression levels was documented using qRT-PCR. The results demonstrated that among 0.3 M glucose + CPAs combinations, EG recorded higher frozen-thawed motility 69 % (7- day) and 59 % (60- day). Similarly, in V2E + CPAs combinations, EG recorded higher frozen-thawed motility 31 % (7- day) and 26 % (60- day). The DNA integrity of all thawed sperm (both periods) did not differ from that of fresh sperm. The qRT-PCR results revealed that in the combination of 0.3 M glucose + CPAs, the level of HSP90 and GPx2 gene expression was found to be upregulated in frozen-thawed sperm on both periods. Whereas, the expression level of the HSP70 gene was down-regulated. On the contrary, in the combination of V2E + CPAs, the expression levels of HSP70, HSP90 and GPx2 genes could not be detected on both periods. Overall, the findings of this study demonstrate that the cryomedium (extender + cryoprotectant) has a more influential role in the motility and levels of gene expression in the frozen-thawed sperm of M. cephalus.
