Motility, viability and fertility of goldfish Carassius auratus (Pisces: Cyprinidae) post short-term cryopreservation.

BACKGRUND: Goldfish Carassius auratus is a popular ornamental fish extensively cultured worldwide. Sperm cryopreservation is a common fish breeding method that ensures sperm availability around the year. Studies on cryopreservation of goldfish sperm, especially on the suitability of cryoprotectant types and pre-freezing time, are scarcely available. OBJECTIVE: To determine the most suitable type of cryoprotectant and pre-freezing for the successful cryopreservation of goldfish sperm. MATERIALS AND METHODS: A completely randomized design with two factors was utilized in this study. The first factor is the type of cryoprotectants, which included methanol, ethanol, ethylene glycol, glycerol, and DMSO. The second is pre-freezing times of 10, 20, 30, and 40 min at each of the pre-freezing temperatures of 4 degree C, -10 degree C, and -79 degree C, meaning that the total times for the ramping down of temperature were 30, 60, 90 and 120 min, respectively. The Ringer solution and 10% egg yolk were used as extender and extracellular cryoprotectant. The sperm was stored at -179 degree C for 7 days. RESULTS: The ANOVA test showed that cryoprotectants and pre-freezing significantly affected the motility, viability, and fertility of goldfish sperm after freezing in liquid nitrogen for 7 days (P<0.05). Furthermore, 10% DMSO combined with 15% egg yolk with an pre-freezing time of 20 min can maintain sperm motility, viability, and fertility higher than other treatments, by 79%, 80%, and 33%, respectively. The agarose gel electrophoresis showed no DNA fragmentation in all samples, including fresh sperm. CONCLUSION: We conclude that 10% DMSO combined with 15% egg yolk and 20 min pre-freezing is the best treatment for goldfish sperm cryopreservation. DOI: 10.54680/fr23310110412.

The effect of Cissus quadrangularis Salisb. extract on maturation of rat mesenchymal stem cells.

Degenerative diseases, such as osteoporosis, could be treated by stem cells. The aim of this study was to identify the gene expression of bone marrow mesenchymal stem cells (BM-MSC) derived from Sprague Dawley rats and to assess the effect of Cissus quadrangularis Salisb. extract on their maturation into bone cells. The BM-MSC were divided into three groups: (a) BM-MSCs + osteoblast cell growth basal medium as the positive control; (b) BM-MSCs + Dulbecco's modified eagle's medium (DMEM) + 0.3 mg/mL methanol extract of C. quadrangularis as methanol group; and (c) BM-MSC + DMEM + 0.3 mg/mL ethyl acetate extract of C. quadrangularis as ethyl acetate group. A relative quantification approach using was used to analyze the expression of the alp (alkaline phosphatase) gene, with the beta-actin gene was used to normalize the expression of the alp gene. The intra-assay variation was calculated to validate the RT-qPCR data. Our study found that the intra-assay variation value was acceptable, with most of the coefficients of variability (CV) value <5. Ethyl acetate solvent outperformed methanol solvent in extracting the active compound C. quadrangularis. In the ethyl acetate extract group, the expression of the alp gene increased three times compared to the positive control. In methanol extract group, the expression of alp gene was lower six times compared to positive control. This study suggests that C. quadrangularis extracts using ethyl acetate could induce the maturation of BM-MSCs. However, further studies are warrant to confirm this effect using different indicators.

Improvement of sperm quality of the depik fish, rasbora tawarensis, after cryopreservation using antioxidant.

BACKGROUND: The cryopreservation of the sperm of the depik fish, Rasbora tawarensis, has previously been developed. However, the quality of the sperm post cryopreservation was not satisfactory and might be improved through the application of antioxidants. OBJECTIVE: To determine the most suitable antioxidant for the cryopreservation of the depik fish spermatozoa. MATERIALS AND METHODS: A completely randomized design with a non-factorial experiment was used and the tested antioxidants were glutathione, beta-carotene, ascorbic acid, and butylated hydroxytoluene (BHT) at 6 % concentrations. All treatments had three replications. The sperms were collected from 10 male fishes and diluted with Ringer solution in a ratio of 1: 20 (v/v, sperm: Ringer solution). Then 5% DMSO and 5 % egg yolk were added to the diluted sperms. Furthermore, 6 % of the tested antioxidants were added to the diluents, and then, cryopreservation was carried out in liquid nitrogen for 14 days. RESULTS: The ANOVA test showed that the application of antioxidants significantly affected the sperm motility, fertility, and hatching rates of the eggs (P < 0.05). Furthermore, the antioxidants also protected the sperm cells during cryopreservation, with glutathione being the best antioxidant. CONCLUSION: The application of antioxidants during the cryopreservation of depik fish sperm had a significant effect on motility, fertility and hatchability of eggs post-cryo. Furthermore, glutathione was the most suitable antioxidant. doi.org/10.54680/fr23110110312.

Effect of glutathione on sperm quality after short-term cryopreservation in seurukan fish Osteochilus vittatus (Cyprinidae).

The objective of this present study is to determine the optimum concentration for glutathione in the cryopreservation of seurukan fish (Osteochilus vittatus) spermatozoa. A completely randomized design (CRD) was used with 6 treatments and 3 replications. The Seurukan fish sperm was diluted in extender with a ratio of 1:20 (sperm: extender), then glutathione was added at a concentration of 0, 10, 20, 30, 40 and 50  mg L-1. Next, the sperm thawed at 39-40 °C for 3 min and mixed with 100 eggs which were randomly selected. The success of the fertilized egg was observed 6 h after fertilization, while the hatching rate was examined 60 h after fertilization. The ANOVA test showed that the addition of glutathione significant affected the sperms motility, fertility and hatching rate of seurukan fish Osteochilus vittatus eggs (P < 0.05). By segregation the fresh sperm, a higher sperm motility rate was recorded with an addition of 30 mgL-1 of glutathione (63.00 ±â€¯5.89), however, this value was not significantly different from using concentration of 10 and 20  mg L-1. A higher fertilization rate was produced using glutathione concentration of 50  mg L-1 (51.33 ±â€¯17.01); however, it was also not significantly different from the concentration of 20, 30 and 40 mgL-1. In addition, a higher hatching rate was also recorded using glutathione concentration of 50 mg L-1 (40.33 ±â€¯12.89), this value was not significantly different from using 40 mgL-1 glutathione. Hence, a conclusion was drawn that the optimum concentration of glutathione is 40 mg L-1 of diluent.