Effect of Different Plant Extracts on Microbial Population in The Frozen African Catfish (Clarias gariepinus) Semen.

Fish sperm cryopreservation has been attempted on roughly freshwater and marine species since 1953. This study sought to assess the potential of various plant extracts to function as natural antimicrobial agents in the frozen semen of African catfish (Clarias gariepinus). Diluted sperm was packaged in 0.25ml straws and left for 10min equilibration at 4°C. Following equilibration, the straws were exposed to liquid nitrogen vapor for 10 min and plunged into the liquid nitrogen (-196°C) and then thawed in a water bath at 35°C for 20s. Sperm samples were put into sterile 1.5 ml tubes immediately after thawing and the microbial count was detected with classical microbiological culture method. In the results of microbiological analyses, these tree plant extracts especially Echinacea purpurea were found highly effective for decreasing bacterial contamination levels of African catfish (C. gariepinus) semen. These plant extracts may have the potential for antibacterial effect, and they can be useful for the dilution of semen.

Cryoprotective Effect of Vitamin E Supplementation of Different Extenders on Quality and Fertilizing Ability of Frozen-Thawed Brown Trout Sperm.

Vitamin E is one of the most powerful antioxidants for prevention of cell damage resulting from cryopreservation, but its efficacy for cryopreserving brown trout sperm is still unclear. In this work, the protective effect of vitamin E on quality, fertilizing capacity, and DNA damage of brown trout (Salmo trutta macrostigma) sperm after cryopreservation was evaluated. Sperm samples were diluted at the ratio of 1:10 with three different extenders (E): (E-I): 300 mM glucose, 10% egg yolk; (E-II): 33.3 mM glucose, 5.1 mM NaCl, 0.5 mM NaHCO3,, 15% DMA; and (E-III): 61.6 mM NaCl, 134.2 mM KCl, 1.9 mM CaCl2, 0.8 mM MgCl2, 2.3 mM NaHCO3 in distilled water. Each extender was supplemented with 10% DMSO and different concentrations of vitamin E at 0.1, 0.5, and 1.0 mM. Spermatozoa frozen without vitamin E (0 mM, control) and fresh sperm were also used. After dilution, the sperm was aspirated into 0.25 mL straws, frozen 3 cm above the liquid nitrogen (LN2) surface, and plunged into the LN2. Cell motility, viability, fertilization, and eyeing were determined in post-thawed samples. DNA damage was determined by the comet assay after cryopreservation. Supplementation of 1 mM vitamin E to all extenders exhibited the best cryoprotective effect in terms of sperm motility, duration of motility, viability, fertility, and DNA integrity against cryopreservation damage, compared with 0.1, 0.5, and control group (0 mM) (p < 0.05). The highest post-thaw motility (62.4% ± 0.36%), fertilization (48.2 ± 0.84), and the lowest DNA damage (7.245%) were obtained with the extender-II including 1.0 mM vitamin E (p < 0.05). Consequently, vitamin E positively affected the motility parameters, fertility, and DNA integrity, and the results suggest the addition of extenders with vitamin E as an antioxidant for the cryopreservation of brown trout sperm.

An evaluation of soybean lecithin as an alternative to avian egg yolk in the cryopreservation of fish sperm.

Plant-derived lecithin has been used as a more sanitary alternative to avian egg yolk in livestock sperm cryopreservation protocols but its efficacy for cryopreserving fish sperm has not previously been tested comparatively. Here various concentrations of soybean lecithin were evaluated for the cryopreservation of carp (Cyprinus carpio) sperm. Sexually mature fish were induced to spermiation and ovulation with ovopel. The extenders were prepared by using 300 mM glucose, 10% DMSO, supplemented with different ratios of lecithin (5%, 10%, 15%, and 20%) and 10% egg yolk (control I). Negative control was made without egg yolk and soybean lecithin (control II). The pooled semen was diluted separately at ratio of 1:3 (v/v) by using egg yolk and soybean-based extenders. Diluted semen placed into 0.25 ml straws were equilibrated at 4 °C for 15 min and frozen in liquid nitrogen vapor. Fertilization was conducted using a ratio of 1 × 10(5)spermatozoa/egg. Supplementation of 10% lecithin to extender showed the best cryoprotective effect for sperm motility and duration of motility against freezing damage compared to 15%, 20% and control II groups (p<0.05). Cryopreserved sperm with extender containing 10% lecithin provided a greater result in terms of fertilization success when compared to extenders containing 20% lecithin or control II (p<0.05). It is concluded that the animal protein-free extender containing 10% soybean lecithin has a similar cryoprotective actions with conventional egg yolk-based extender against freezing damages and fertilization. Therefore, soybean lecithin is a suitable alternative to avian egg yolk for the cryopreservation of fish sperm.