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Sperm Cryopreservation of the Critically Endangered Olive Barb (sarpunti) Puntius Sarana (hamilton, 1822)

M. Nahiduzzaman
Department of Fisheries Biology and Genetics, Bangladesh Agricultural University, Mymensingh 2202, Bangladesh

Terrence R. Tiersch
Aquaculture Research Station, Louisiana Agricultural Experiment Station, Louisiana State University Agricultural Center, Baton Rouge, LA 70803, USA

Mostafa A.R. Hossain
Department of Fisheries Biology and Genetics, Bangladesh Agricultural University, Mymensingh 2202, Bangladesh

S.N.A. Mamuna
Department of Fisheries Biology and Genetics, Bangladesh Agricultural University, Mymensingh 2202, Bangladesh

U. Habiba Khanam
Department of Fisheries Biology and Genetics, Bangladesh Agricultural University, Mymensingh 2202, Bangladesh

M. Mahbubul Hassan
Department of Fisheries Biology and Genetics, Bangladesh Agricultural University, Mymensingh 2202, Bangladesh

Abstract/ Executive Summary:

The present study focused on development of a sperm cryopreservation protocol for the critically endangered olive barb Puntius sarana (Hamilton, 1822) collected from two stocks within Bangladesh and reared in the Fisheries Field Laboratory, Bangladesh Agricultural University (BAU). The sperm were collected in Alsever’s solution prepared at 296 mOsmol kg^-1. Sperm were activated with distilled water (24 mOsmol kg^-1) to characterize motility. Maximum motility (90%) was observed within 15 s after activation, and sperm remained motile for 35 s. Sperm activation was evaluated in different osmolalities and motility was completely inhibited when osmolality of the extender was P287 mOsmol kg^-1. To evaluate cryoprotectant toxicity, sperm were equilibrated with 5%, 10% and 15% each of dimethyl sulfoxide (DMSO) and methanol. Sperm motility was noticeably reduced within 10 min, when sperm were equilibrated with 15% DMSO, indicating acute toxicity to spermatozoa and therefore this concentration was excluded in further trials. Sperm were cryopreserved using DMSO at concentrations of 5% and 10% and methanol at 5%, 10% and 15%. The one-step freezing protocol (from 5 ⁰C to 80⁰C at 10⁰C/min) was carried out in a computer-controlled freezer (FREEZE CONTROL CL-3300; Australia) and 0.25-ml straws containing spermatozoa were stored in liquid nitrogen for 7–15 days at 196 ⁰C. The highest motility in thawed sperm 61 ± 8% (mean ± SD) was obtained with 10% DMSO. The fertilization and hatching rates were 70% and 37% for cryopreserved sperm, and 72% and 62% for fresh sperm. The protocol reported here can be useful for hatchery-scale production of olive barb. The use of cryopreserved sperm can facilitate hatchery operations, and can provide for long-term conservation of genetic resources to contribute in the recovery of critically endangered fish such as the olive barb.

Keywords: Sperm cryopreservation, Fertilization, Olive barb Puntius sarana, Critically endangered.

Location: Bangladesh Agricultural University

Start Date: 00-00-2008

End Date: 00-00-2009

Program Area: Variety and Species

Commodity: Aquatic animal

Objectives:

To characterize sperm motility and duration
To evaluate sperm motility at different osmotic pressures;
To evaluate the toxicity of cryoprotectants;
To evaluate the post-thaw motility at different concentrations of two cryoprotectants (DMSO and methanol), and
To evaluate the fertilization and hatching rates of eggs fertilized using cryopreserved and fresh sperm. To our knowledge, this is the first report on cryopreservation of olive barb sperm.

Methodology:

Collection and rearing of broods: Mature P. sarana (females showing swollen bellies and milt flowing freely from males with gentle abdominal pressure) were collected from two natural sources: the Chalan beel under the Natore district, and the Mogra river of the Netrokona district in January of 2008. Fish were transported to brood-rearing earthen ponds (800 m²) of the Fisheries Field Laboratory complex, Bangladesh Agricultural University (BAU) in Mymensingh and reared for 1.5 years. Water quality parameters including temperature (30.16 ± 2.0 ⁰C), pH (7.58 ± 0.52), dissolved oxygen (5.70 ± 0.55 mgl^-1) and total alkalinity (87.48 ± 26.19 mg l^-1) of the ponds during reproduction were recorded. The brood fish were fed twice daily a commercial diet (35% protein; Paragon Feeds Limited, Bangladesh) at 4–5% of body weight in the rearing ponds. Sperm collection: Mature male fishes were collected from the brood-rearing ponds during the breeding season (May–July 2009). After measuring the total length (cm) and body weight (g), the fish were held for as long as 12 h for conditioning in the tanks of the hatchery of the Faculty of Fisheries, BAU. They were injected with carp pituitary supernatant (collected and processed by local fish hatchery owners) at 2 mg kg^-1 of body weight and released in the conditioning tank. Six hours after injection, the males were captured from the tank and were laid on foam to wipe the urogenital pore. Gentle pressure was applied at the base of the pelvic fin, and sperm were collected in glass vials containing Alsever’s solution (0.7% NaCl, 0.8% sodium citrate dissolved in 100 ml water; pH 7.2) prepared at 296 mOsmol kg^-1. The samples were placed on ice (4 ⁰C) and brought to the Laboratory of Fish Biodiversity and Conservation, BAU. Sperm concentration was determined in triplicate counts by use of haemacytometer and expressed as the number of cells10⁹ ml^-1. Milt was diluted 4000-fold in distilled water, and a droplet of the diluted milt was placed within a haemacytometer (area of the smallest square = 1/400 mm², depth 0.1 mm) with cover slip (Essential Scientific Bangladesh; made by Marienfeld, Germany). The number of spermatozoa in five large squares (area mm²) of the counting chamber was counted at 400 magnification. Sperm quality: Before cryopreservation, the motility of sperm samples was evaluated by use of a light microscope (Novex K-range, Holland) at 400 magnification. Sperm motility was estimated by adding 19 M of distilled water (24 mOsmol kg^-1) as activating medium to 1 M of fresh milt on a glass slide. The motility was observed within 3–4 s after activation and was expressed as the percentage of sperm that had active forward movement. Sperm vibrating in place without forward movement were not considered to be motile. Only sperm samples showing motility above 80%, were used for cryopreservation experiments. Experiment 1: motility characteristics Sperm motility and duration were characterized by using sperm samples (P90% motility) from five fish (replicated in triplicate) after activation with distilled water. Motility was estimated at 5- s intervals until all motility in the sample ceased (no motility, some vibrating in place). Experiment 2: sperm motility in various osmolalities of sodium chloride (NaCl) Sperm samples were exposed to 10 graded dilutions of NaCl ranging in osmotic pressure from 48 to 319 mOsmol kg^-1 to evaluate the relationship between osmotic pressure and sperm motility. The motility was estimated as described above. Experiment 3: determination of cryoprotectant toxicity Cryoprotectant concentrations were prepared according to the percentage (5%, 10%, and 15%) in the final suspension with five replicates. Either DMSO or methanol was added with Alsever’s solution and further mixed with fresh spermatozoa to yield the final concentration of the cryoprotectant in the suspension. The toxicity of these cryoprotectants was evaluated by observing sperm motility at exposure times of 10, 20 and 30 min. Experiment 4: evaluation of post-thaw motility of DMSO and methanol Following the toxicity experiments, cryoprotectant concentrations were chosen and post-thaw motility was evaluated for 5% and 10% DMSO, and for 5%, 10% and 15% methanol. The cryoprotectants were mixed in Alsever’s solution and added to the samples to yield the desired final concentrations (1:1:8 = sperm:cryoprotectant: extender). The sperm samples were equilibrated for 8–10 min for 10% DMSO and 25–30 min for 5% DMSO and 5%, 10% and 15% methanol before freezing. Percent motility was observed for all samples before addition of cryoprotectant and again just prior to the initiation of freezing. Pre-labeled 0.25-ml French plastic straws (Minitüb System, Minitüb, Tiefenbach, Germany) were filled with 0.23 ml of diluted spermatozoa and sealed manually using a heated crucible tongs. After sealing, the straws were transferred to a controlled-rate freezer (FREEZE CONTROL CL-3300; Australia) programmable by computer-based software (CryoGenesis™ V5). A one-step freezing protocol (5 ⁰C to 80 ⁰C at a rate of 10 ⁰C/min) was used. After freezing, the straws were immediately plunged into liquid nitrogen (196 C) for storage of 7–15 days. Straws were removed from the liquid nitrogen and thawed at 40 ⁰C for 7 s in water bath [39] and post-thaw motility of the sperm was assessed within 3–4 s after activation with distilled water as described above. Experiment 5: assessment of fertilization and hatching Three fertilization trials were conducted to evaluate the fertility and hatching rates of fresh and cryopreserved spermatozoa. Female olive barb were injected twice with locally available carp pituitary supernatant (6 mg kg1 of body weight; 1/3 of volume as first dose, and 2/3 as second dose after a 6 h interval) and were stripped 12 h after the second injection. Eggs collected from three females were immediately fertilized at room temperature (25 ⁰C) using fresh and pooled cryopreserved spermatozoa (Alsever’s solution with 10% DMSO chosen based on the results of ‘‘Experiment 4’’). After thawing, sperm motility was estimated and 15 straws (2.3 x 10⁹ cells) of cryopreserved sperm were used for fertilization of 2 ml of eggs (1 ml =5646 eggs). The cryopreserved spermatozoa were activated with tap water (31 mOsmol kg^-1). The spermatozoa and the eggs were mixed with a hen feather and placed in hatching jars (radius 0.3 m, height 0.7 m, capacity 90 L) with 12–14 L/min of water exchange used for incubation of eggs. The fertilization rates were calculated as the percentage of fertilized eggs obtained from the total number of eggs at 22–24 h after fertilization. The hatching rates were calculated as the percentage of larvae obtained from total number of eggs for cryopreserved or fresh sperm. Statistical analysis: All percent motility values were subjected to arcsine transformation prior to statistical analysis. Treatment effects were analyzed using one factor, two-factor or repeated measures ANOVA. Means were separated by Duncan’s Multiple Range Test and a value of P < 0.05 was considered as being statistically significant. Data were expressed as mean ± SD and analyzed by Statistical Package for the Social Science (SPSS v 11.5). We did not attempt to standardize the sperm concentrations, so no statistical comparisons were made for fresh and cryopreserved samples. The fresh samples were used to evaluate the quality (fertilization and hatch potential) of the eggs.

Source of Information: Cryobiology 62 (2011) 62–67

Article Link (Online Journal):

Funding Source:

Total Budget (Tk.) :

Achievement/Findings:

This study developed a simplified cryopreservation protocol for use with the critically endangered olive barb. In summary, spermatozoa of olive barb could be cryopreserved using Alsever’s solution as extender with 10% DMSO (5–10 min equilibration) with a cooling rate of 10 C/min from 5 to 80 ⁰C.

Knowledge Management: Journal