Chilled storage of walking catfish (Clarias macrocephalus) semen

Aquaculture 296 (2009) 58–64

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Aquaculture j o u r n a l h o m e p a g e : w w w. e l s ev i e r. c o m / l o c a t e / a q u a - o n l i n e

Chilled storage of walking catfish (Clarias macrocephalus) semen Verapong Vuthiphandchai a,⁎, Itthinan Thadsri a, Subuntith Nimrat b a b

Department of Aquatic Science, Faculty of Science, Burapha University, Chonburi 20131, Thailand Department of Microbiology and Environmental Science Program, Faculty of Science, Burapha University, Chonburi 20131, Thailand

a r t i c l e

i n f o

Article history: Received 16 March 2009 Received in revised form 21 July 2009 Accepted 22 July 2009 Keywords: Walking catfish Clarias macrocephalus Sperm quality Storage

a b s t r a c t The aim of the present study was to determine changes in sperm quality of walking catfish (Clarias macrocephalus) during its spawning season, and develop a simple and efficient protocol for the chilled storage of semen at 4 °C. Semen samples were evaluated monthly during April–November in 2006. Percentage of spermiating males and sperm motility peaked during the middle of the spawning season, June–September. Sperm density and seminal plasma osmolality increased significantly (P b 0.05) throughout the spawning season, while semen pH did not change during this period. The effects of extender, dilution ratio and timing of chilled storage on successful storage period of extended semen were examined during the years 2006–2007. Fresh semen was diluted 1:1 with several sperm extenders, calcium-free Hanks' balanced salt solution (Ca–F HBSS), Hanks' balanced salt solution (HBSS), extender 7 (NaCl 5.780 g, KCl 2.558 g, CaCl2·2H2O 0.103 g, NaHCO3 0.235 g, MgCl2·6H2O 0.220 g, pyruvate 6.0 g, citric acid 0.100 g, HEPES buffer 2.380 g in 1000 mL distilled water), extender 13 (NaCl 8.760 g in 1000 mL distilled water), and modified Cortland, in tissue culture flasks. Longest successful storage period of sperm was 10 days with Ca–F HBSS. Semen diluted with Ca–F HBSS at 1:1, 1:2 or 1:4 ratios significantly lengthened (P b 0.05) the storage period over those at dilutions of 1:9 or 1:19. To determine the effect of season on the success of chilled storage, semen diluted with Ca–F HBSS at a ratio of 1:4 was prepared at three sampling intervals in the beginning (May), middle (August) and end (November) of spawning season in 2007. Extended semen prepared in August remained motile (20 ± 3.1%) for as long as 10 days, whereas those prepared in May or November were immotile on days 9 and 7 of storage, respectively. Extender-preserved semen was capable of fertilizing eggs as efficiently as fresh semen within two days of storage. A significant decrease in fertilization and hatching rates of extender-preserved semen compared to fresh semen on days 4 and 6 was due to low sperm motility. These results demonstrate that semen of walking catfish can be stored for short term at 4 °C without appreciably affecting motility and fertilization. © 2009 Elsevier B.V. All rights reserved.

1. Introduction Thai walking catfish (Clarias macrocephalus), a freshwater food fish native to Thailand, is an important aquaculture species in Thailand and Southeast Asia because of its tender flesh and acceptable flavour, high market value and aquaculture potential (Areerat, 1987). This species occurs in freshwater canals and rivers and spawns between March and November (Panprommin et al., 2008). Due to the decline of wild stocks of walking catfish, attention has focused on developing broodstocks for production of larvae. Catfish aquaculture production of about 146,482 tons has been reported in Thailand for the year 2006, surpassing wild catches of about 2994 tons (Department of Fisheries, 2008a,b). Since C. macrocephalus do not release semen by manual stripping, spermatozoa are normally collected after decapitation and

⁎ Corresponding author. Tel.: +66 38 103 093; fax: +66 38 393 491. E-mail address: [email protected] (V. Vuthiphandchai). 0044-8486/$ – see front matter © 2009 Elsevier B.V. All rights reserved. doi:10.1016/j.aquaculture.2009.07.018

removal of testis for artificial insemination of stripped eggs. Effective use of C. macrocephalus semen will reduce numbers of male broodstock sacrificed for this purpose. Asynchrony in gamete production between males and females in this species is also a major problem regulating the success of artificial propagation, especially towards the end of the spawning season (Pongthana et al., 1995). Typically at this time, females produce good quality eggs while males generate lower volumes and poorer quality of semen. The provision of adequate quantity and quality of semen is of obvious benefit (importance, value) to the artificial propagation of walking catfish. One approach to prolonging sperm quality and quantity that has proven successful is chilled storage (DeGraaf and Berlinsky, 2004; Babiak et al., 2006a). However, understanding change in sperm quality during the spawning season provides important baseline information of sperm fitness, and is prerequisite for development of chilled storage protocol of walking catfish sperm. Chilled storage of fish sperm for a few days would facilitate hatchery management during artificial insemination and solve the

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problem of sperm quality during the end of reproductive season. For example, refrigerated storage of sperm would provide sperm transfer between hatcheries for artificial insemination and allow hybridization of selective breeding programs for stock improvement. These advantages of chilled storage in fish sperm would lead to saving of manpower, time and production cost. From the commercial point of view, synchronizing the timing of obtaining good quality of eggs and sperm facilitates production procedure and hatchery management. Due to deteriorated egg quality after stripped-spawning, the storage of extender-preserved sperm in advance ensures sperm quality and faster handling of sperm for artificial insemination. Chilled storage of fish sperm could be achieved by storage in undiluted and diluted form. These approaches are easy to perform without requiring expensive equipment or specific training, and allow prolonging sperm viability for weeks (Stoss, 1983; Rana, 1995). Storage of undiluted semen at low temperature (0–4 °C) has been reported to result in decreased fertilization capacity after short time storage in some of cultured species (Stoss, 1983; Lahnsteiner et al., 1997). Storage of diluted semen with extender provides better control in physiochemical condition during storage and prolongs sperm viability compared to undiluted storage (Stoss, 1983; Harvey and Kelley, 1984; Mongkonpunya et al., 1995). Chilled storage of sperm has been successfully reported in striped bass Morone saxatilis (Walbaum) (He and Woods, 2003), channel catfish Ictalurus punctatus (Rafinesque) (Christensen and Tiersch, 1996) and several species of salmonids (Scott and Baynes, 1980; Erdahl and Graham, 1987). However, chilled storage of C. macrocephalus sperm and identification of an appropriate extender have not been developed. The purpose of this study was to (1) evaluate semen quality of walking catfish during the spawning season; (2) develop a protocol for chilled storage of semen cooled at 4 °C and (3) evaluate fertilization capacity of stored semen.

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2.2. Assessment of sperm quality Immediately after collection, fresh semen was evaluated for sperm quality parameters at room temperature (approximately 25 °C). Sperm motility was evaluated using a light microscopy (Olympus CX21FS1, Tokyo, Japan) at ×400 magnification. Fresh sample (1 µL) was mixed with 50 µL of 0.4% NaCl on a slide covered with a glass coverslip (22 mm × 22 mm; VWR, West Chester, PA, USA) to achieve the even mixing of sperm. Sperm motility was defined as the percentage of forward-moving spermatozoa. The rate of sperm motility was categorized as immotile and every 20% motile increment in a forward direction i.e. 0, 20, 40, 60, 80 and 100% motile, based on subjective estimation (Vuthiphandchai et al., 2009). Evaluation of sperm motility was carried out in triplicate within 30 s after activation. All sperm motility observations were made by the same person in order to minimize the degree of variation among observers. Immotile sperm were defined as sperm that did not show forward movement after activation. Sperm cells that vibrated in place were not considered to be motile. Sperm density was determined in duplicate and expressed as number of spermatozoa × 1010/mL. Semen was diluted 500-fold by adding 10 µL of semen to 5 mL of distilled water and then mixed on a vortex mixer. Number of spermatozoa in a known Neubauer haemocytometer volume (BOECO, Hamburg, Germany) was counted under a light microscope. For determination of testicular fluid osmolality, semen was transferred into plain micro-haematocrit tubes and centrifuged at 8000 ×g (for 10 min, 4 °C). Osmolality of the supernatant was measured by a vapor pressure osmometer (model 5520, Wescor, Logan, UT, USA). pH of seminal plasma was determined by a pH meter (Horiba, D-21, Japan). Evaluation of sperm quality parameters was made for three replicates of each sample. 2.3. Sperm quality during the spawning season

2. Materials and methods 2.1. Collection and maintenance of broodstock Sexually mature walking catfish males (0.26–0.38 kg) were collected with seine nets from earthen ponds (0.5 ha) at Chonburi province, Thailand during the spawning seasons of 2006 and 2007, and maintained in the hatchery of Department of Aquatic Science, Faculty of Science, Burapha University. After arrival to the hatchery, fish were acclimatized for 5–7 days in 5-ton rectangular cement tanks with a continuous flow of freshwater. Fish were fed once daily to satiation with a diet containing 40% protein. Prior to the beginning of experiments, fishes were anesthetized 250 ppm of 2-phenoxyethanol before handling. All male fish used in the experiments were injected with gonadotropin-releasing hormone analogue (D-Trp6-GnRHa; Sigma Chemical Corp., St Louis, MO, USA) and dopamine antagonist dissolved in physiological saline solution at dosage of 10 µg/kg and 5 mg/kg, respectively. Testes were surgically removed 8 h post injection. Dissected testes were cut in small pieces to allow sperm release after gentle squeezing. Semen was stored in sterile petri discs on ice before use. Fresh semen was used in the experiments within 10 min post collection. In the experiments requiring diluted semen, samples were pooled and diluted with extenders being stored in an incubator at 4 °C. During manipulation, semen, extenders and petri discs were kept on crushed ice. Semen contamination with blood was avoided as much as possible. Fourteen collections of semen were made in the two spawning seasons (April 2006–November 2006; May, August and November 2007) for all experiments. Semen volume from each fish was about 0.2–1.1 mL. All experiments were carried out in triplicates. All chemicals used were of reagent grade (Sigma Chemical Corp., St Louis, MO, USA).

Male broodstocks were randomly sampled (n = 15) every month from earthern ponds during the spawning period from March to November 2006. Fish were sampled for determination of sperm quality parameters. Number of spermiating males was recorded and expressed as percentage of spermiating males. After collection of testis, spermiation was evaluated based on the presence of semen from dissected testis. Within 10 min of testis collection, fresh semen was stored on ice before beginning sperm quality analysis. 2.4. Chilled storage of semen The aim of this experiment was to examine factors affecting the motility of sperm during cool storage of semen. Effects of type of extenders, dilution ratio and timing of chilled storage on sperm motility were evaluated. To evaluate the effect of type of extenders, semen samples from 48 males were collected, pooled and diluted 1:1 (1 mL semen:1 mL extender) during June, the middle of the spawning season in 2006 in one of the extender solutions, calcium-free Hanks' balanced salt solution (Ca–F HBSS), Hanks' balanced salt solution (HBSS), extender 7, extender 13, and modified Cortland. Semen samples were diluted with extenders supplemented with 0.5% of penicillin–streptomycin (10,000 units of penicillin/mL; 10,000 μg of streptomycin/mL (GIBCO 15140-122, Invitrogen, CA, USA). Broad-spectrum antibiotics (penicillin–streptomycin) at an optimal dose of 0.5% (unpublished data) was selected due to their ability for controlling bacterial growth without having a negative effect on sperm viability (Nimrat and Vuthiphandchai, 2008). Gentle mixing of extender-preserved sperm was applied to ensure the homogenousity of mixture prior to chilled storage. Diluted semen samples were stored in capped 25-mL tissue culture flasks (THOMAS Scientific 229320, NJ, USA) for 10 days at 4 °C. Diluted semen was assessed daily for determination of sperm motility. Undiluted, fresh semen samples stored in capped 25-mL tissue culture flasks at 4 °C

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served as the control. Tissue culture flasks were placed on ice before beginning of experiments. Composition of extenders was as follows: – Hanks' balanced salt solution (HBSS; NaCl 0.8000 g, KCl 0.0400 g, Na2HPO4·7H2O 0.0120 g, NaHCO3 0.0350 g, KH2PO4 0.0060 g, MgSO4·7H2O 0.0200 g, glucose 0.1000 g, CaCl2·2H2O 0.0160 g dissolved in 100 mL water, pH 7.6 (Wayman et al., 1998); – Calcium-free Hanks' balanced salt solution (Ca–F HBSS; NaCl 0.8890 g, KCl 0.0440 g, Na2HPO4·2H2O 0.0130 g, NaHCO3 0.0390 g, KH2PO4 0.0070 g, MgSO4·7H2O 0.0220 g, glucose 0.1110 g dissolved in 100 mL, pH 7.6); – Extender 7 (NaCl 5.780 g, KCl 2.558 g, CaCl2·2H2O 0.103 g, NaHCO3 0.235 g, MgCl2·6H2O 0.220 g, pyruvate 6.0 g, citric acid 0.100 g, HEPES buffer 2.380 g and 10 mL of KOH (1.27 g/100 mL). These compositions were dissolved in 1000 mL distilled water and adjusted pH 7.6) (Brown and Mims, 1995); – Extender 13 (NaCl 8.760 g dissolved in 1000 mL distilled water); – Modified Cortland solution (NaCl 1.880 g, KCl 7.200 g, NaH2PO4·H2O 0.410 g, CaCl2·2H2O 0.230 g, NaHCO3 (sodium bicarbonate) 1.000 g, MgSO4·7H2O 0.230 g, glucose 1.000 g dissolved in 1000 mL distilled water, pH 7.6) (Truscott et al., 1968). The effect of dilution ratio of semen and extender was examined in relation to storage period when an appropriate extender was identified. For this study conducted in mid spawning season, July 2006, 45 spermiating males were used. Triplicate samples of pooled semen were diluted to get a total volume of 2 mL, at dilution ratios of 1:1 (1 mL semen:1 mL extender), 1:2 (0.667 mL semen:1.333 mL extender), 1:4 (0.4 mL semen:1.6 mL extender), 1:9 (0.2 mL semen:1.8 mL extender), and 1:19 (0.1 mL semen:1.9 mL extender) in Ca–F HBSS containing 0.5% penicillin–streptomycin previously described. Diluted semen samples were transferred to capped 25-mL tissue culture flasks (Thomas Scientific 229320, NJ, USA), and stored at 4 °C. Undiluted semen from pooled samples stored in tissue culture flasks served as the control. Sperm motility was evaluated daily as described. In order to evaluate the effect of chilled storage time on sperm motility, pooled semen samples from different sampling periods were diluted 1:4 (0.4 mL semen:1.6 mL extender) with Ca–F HBSS supplemented with 0.5% penicillin–streptomycin. Male broodstocks (n = 54) were collected during the beginning, middle and end of spawning season in May, August and November of 2007, respectively. At each sampling period, extender-preserved sperm, having a total volume of 2 mL, was prepared in six replicates in 25-mL tissue culture flasks (THOMAS Scientific 229320, NJ, USA), and stored at 4 °C. Sperm motility was evaluated daily from three replicates for 10 days. Extender-preserved semen prepared in November 2007 was also used for fertilization studies. 2.5. Fertilization tests Ovulation was induced on sexually mature female walking catfish with gonadotropin-releasing hormone analogue (D-Trp6-GnRHa; Sigma Chemical Corp., St Louis, MO, USA) and dopamine antagonist at a dosage of 20 µg/kg and 5 mg/kg, respectively. They were allowed to ovulate in tanks (5 m3) maintained at 27–29 °C. We began checking ovulation at 9 h after hormonal administration. When fish ovulated, they were rinsed with hatchery water to remove anesthetic from their surface. Pooled eggs from 4 to 6 females were used for each fertilization assay. Eggs were collected by manual stripping of the abdomen and kept on crushed ice to control temperature of eggs before fertilizing with sperm. Eggs were used only when they were well-rounded and brown in color. Due to the unavailability of catfish eggs, fertilization trial was conducted only in November 2007. Pooled semen (n = 18) diluted with Ca–F HBSS from the previous experiment in November 2007 was used to fertilize eggs after chilled storage for 2, 4 or 6 days. A portion of

ca. 300 eggs (0.5 mL) for each replicate was placed into a sterile, dry 100 mm × 15 mm Petri dish using a 1 mL syringe. The tip of syringe was cut off to prevent eggs from being compressed. Predetermined volume of extender-preserved semen was added on top of the eggs to obtain a fixed sperm to egg ratio of approximately 0.5 × 106:1. After mixing eggs with extended semen by a feather during artificial insemination process for 10 s, 5 mL of 0.4% NaCl was added to activate sperm motility by gentle stirring for two min. Eggs were rinsed three times and transferred for incubation in a flow through system at 25 °C. Fertilization rate (number of fertilized eggs to total eggs × 100) was determined at the gastrula stage from the percentage of embryos developed at 10–12 h after fertilization, by randomly counting 200 eggs from each incubator under a light microscope. The number of hatched larvae was evaluated at 25 h after artificial fertilization, and expressed as hatching percentage. In the control group, fresh semen of pooled samples collected from each sampling period was used to fertilize eggs at the same sperm to egg ratio with a similar fertilization protocol. Average density of fresh sperm was 1.8 × 1010/mL. All experiments were carried out in triplicates. 2.6. Statistical analysis Data were expressed as means ± standard error of the mean (SEM). Analysis of variance (ANOVA) was conducted to determine the effects of tested factors on quantitative parameters of semen. Percentage of sperm motility, sperm density and fertilization and hatching rates were arc-sine square root transformed to normalize variance prior to statistical analysis. In the chilled storage experiments, sperm motility was evaluated over time using a repeated measures analysis of variance to determine the effects of type of extender, dilution ratio and timing of storage. Regression analysis was used to evaluate the correlation between sperm motility and fertilization rates. Means were separated using Duncan's New Multiple Range Test, and were considered to be significant at P b 0.05. Statistical analysis was performed using SPSS version 10.0 (SPSS Inc., Chicago, IL, USA). 3. Results 3.1. Sperm quality during the spawning season The spawning season of walking catfish began in April, and extended to November (Table 1). At the first sampling (March 2006, data not shown), no semen was found from any fish. In April, 20% of the males began to spermiate. All collected males produced sperm from June to September, but this declined to 53.3% in November. Sperm motility increased significantly (P b 0.05) from 66.7 ± 4.4% in April to 77.1 ± 2.5% in May. Highest motilities (89.1 ± 1.5% to 94.8 ± 1.3%) were observed between June and September, followed by a significant decline in October and November (Table 1). Sperm density increased significantly (P b 0.05) from 1.4 ± 0.2× 1010/ mL at the beginning of spawning season (April) to 3.6 ± 0.7× 1010/mL in August after which it decreased gradually thereafter reaching values of about 1.6± 0.2 × 1010/mL in November (Table 1). Osmolality of testicular fluid increased significantly (P b 0.05) from 281.3± 2.7 mOsm/kg on April to 329.7 ± 5.8 mOsm/kg on August, and did not change thereafter (Table 1). Semen pH did not change during the spawning season with pHs between 7.6 and 7.8. 3.2. Chilled storage of semen Sperm motility of diluted and undiluted semen of walking catfish, refrigerated at 4 °C, gradually decreased as storage period increased (Table 2). The initial motility of diluted and undiluted semen after activation with 0.4% NaCl was not significantly different (P N 0.05), showing average values of 86.7 ± 3.1 to 91.1 ± 3.3%. Motility of

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Table 1 Sperm quality of walking catfish (n = 120) during the spawning season. Parameters

Sampling period in 2006

Spermiating males (%) Sperm motility (%) Sperm density (x1010/mL) Osmolality (mOsm/kg) pH

April

May

June

July

August

September

October

November

20a 66.7 ± 4.4a 1.4 ± 0.2a 281.3 ± 2.7a 7.7 ± 0.2a

60b 77.1 ± 2.5b 1.7 ± 0.3a 288.5 ± 3.1a 7.8 ± 0.2a

100c 89.1 ± 1.5c 2.5 ± 0.3ab 297.1 ± 3.7ab 7.7 ± 0.1a

100c 93.5 ± 1.4c 3.3 ± 0.3b 306.2 ± 2.2b 7.6 ± 0.2a

100c 94.8 ± 1.3c 3.6 ± 0.7bc 329.7 ± 5.8c 7.6 ± 0.1a

100c 90.4 ± 1.6c 2.7 ± 0.4ab 324.7 ± 3.5c 7.8 ± 0.2a

80c 83.9 ± 1.9b 2.2 ± 0.5ab 321.3 ± 3.4c 7.8 ± 0.1a

53.3b 81.7 ± 1.7b 1.6 ± 0.2a 324.2 ± 2.8c 7.6 ± 0.1a

Different letter superscripts on the row indicate means that were significantly different (P b 0.05) over time.

Table 2 Changes in percentage of motile sperm of walking catfish (n = 48) during chilled storage for 10 days. Extenders

Days of storage

Ca–F HBSS HBSS Extender 7 Extender 13 Modified Cortland Control

0

1

2

3

4

5

6

7

8

9

10

91.1 ± 3.3 a,6 88.9 ± 3.3 a,7 86.7 ± 3.1 a,8 88.9 ± 3.3 a,6 86.7 ± 3.1 a,6

66.7 ± 6.7 bc,5 75.6 ± 5.1 a,6 62.2 ± 3.9 b,7 64.4 ± 2.8 bc,5 53.3 ± 5.8 c,5

51.1 ± 6.1 ab,4 53.3 ± 5.8 ab,5 55.6 ± 5.1 a,6 51.1 ± 6.1 ab,4 44.4 ± 5.1 b,4

48.9 ± 8.4 a,4 48.9 ± 6.1 a,5 42.2 ± 3.9 a,5 26.7 ± 5.8 b,3 42.2 ± 2.1 a,4

46.7 ± 8.2 a,4 42.2 ± 3.8 a,4 44.4 ± 2.8 a,5 17.8 ± 2.1 b,2 26.7 ± 5.8 b,3

35.6 ± 5.1 a,3 22.2 ± 3.8 b,3 26.7 ± 3.1 b,4 15.6 ± 5.1 c,2 15.6 ± 5.1 c,2

24.4 ± 2.8 a,2 11.1 ± 6.1 bc,2 17.8 ± 3.9 ab,3 4.4 ± 5.1 cd,1 13.3 ± 5.8 ab,2

22.2 ± 2.1 a,2 2.2 ± 3.9 bc,1 8.9 ± 6.1 ab,2 1.1 ± 1.9 bc,1 4.4 ± 5.1 bc,1

17.8 ± 2.1 a,1 0 2.2 ± 3.9 b,1 0 0

13.3 ± 3.1 a,1 0 1.1 ± 1.9 b,1 0 0

11.1 ± 3.3 a,1 0 1.1 ± 1.9 b,1 0 0

0

0

0

0

0

0

0

0

91.1 ± 3.3

a,3

6.7 ± 5.8

d,2

2.2 ± 3.9

c,1

Semen was diluted 1:1 with various extenders containing 0.5% penicillin–streptomycin (10,000 units of penicillin/mL; 10,000 μg of streptomycin/mL), stored in tissue culture flasks at 4 °C during June 2006. Semen samples were daily evaluated for motility. Control referred to undiluted semen that was stored in tissue culture flasks at 4 °C. Different letter superscripts on the column indicate means that were significantly different (P b 0.05) among extenders. Different number superscripts on the row indicate means that were significantly different (P b 0.05) over storage time.

undiluted semen significantly decreased (P b 0.05) to 6.7 ± 5.8% after one day of storage, while motility of all diluted samples ranged from 53.3 ± 5.8 to 75.6 ± 5.1 %. Motility of semen diluted in Ca–F HBSS and extender 7 was 11.1 ± 3.3% and 1.1 ± 1.9%, respectively, after 10 days. Sperm diluted in HBSS, extender 13 and modified Cortland solution was immotile after eight days of storage. Sperm of undiluted samples was immotile within 3 days of storage. The effects of dilution on sperm motility are shown in Table 3. At the beginning of the experiment, motility of semen diluted at ratios from 1:1 to 1:19 was not significantly different (P N 0.05) from that of undiluted semen samples. Semen diluted at 1:1, 1:2 or 1:4 after storage for 10 days retained similar motilities ranging from 13.3 ± 3.1% to 17.8 ± 2.2%, while that diluted at 1:9 or 1:19 was immotile within 8 and 6 days, respectively. Extended semen prepared in August had the longest successful storage period showing an average motility of 20 ± 3.1% on the tenth day of storage (Table 4). However, extended semen stored in May or November had a shorter successful storage period as sperm were immotile after chilled storage for 9 or 7 days, respectively.

Extended semen stored for 4 or 6 days had significantly lower fertilization rates compared with fresh sperm. Fertilization rates of extended semen after storage for 4 and 6 days decreased to 52.4 ± 3.5% and 18.5 ± 4.4%, respectively, compared with 78.5 ± 3.1% and 74.5 ± 4.2% for fresh sperm. Fertilization rates correlated significantly (P = 0.0001; r = 0.89) with motility of these sperm samples. The highest percentage of hatching (71.6 ± 3.4%) was obtained when extended semen was stored for 2 days before artificial insemination (Table 5). The lowest percentage of hatching (4.5 ± 2.1%) was observed in extended semen stored for 6 days. The mean hatching percentage when fresh sperm was used did not differ significantly (P N 0.05) with storage time of 2–6 days, and varied between 67.3 and 72.8%. 4. Discussion 4.1. Sperm quality This is the first study to develop a chilled storage protocol for walking catfish semen capable of fertilizing eggs. Sperm quality decreased towards the end of spawning season. A decline in semen quality at the end of the spawning season has been reported in rainbow trout Salmo gairdneri (Munkittrick and Moccia, 1987), Finnish landlocked salmon Salmo salar m. sebago (Piironen, 1985), Atlantic

3.3. Fertilization trials Sperm diluted with Ca–F HBSS for 2 days had a fertilization rate of 80.1 ± 3.6%, comparable to that of fresh sperm, 85.6 ± 2.9% (Table 5).

Table 3 Effect of dilution ratio on percentage of motile sperm of walking catfish (n = 45) during chilled storage for 10 days. Dilution 1:1 1:2 1:4 1:9 1:19 Control

Days of storage 0

1

2

3

4

5

6

7

8

9

10

93.3 ± 3.1 a,6 93.3 ± 3.1 a,7 91.1 ± 3.3 a,4 88.9 ± 3.3 a,5 88.9 ± 3.3 a,4 93.3 ± 3.1 a,3

77.8 ± 2.2 a,5 75.5 ± 2.2 a,6 82.2 ± 3.8 a,4 64.4 ± 2.8 b,4 42.2 ± 2.1 c,3 17.8 ± 2.2 d,2

75.5 ± 2.8 a,5 73.3 ± 3.1 a,6 77.8 ± 2.2 a,4 44.4 ± 5.1 b,3 43.3 ± 3.3 b,3 6.6 ± 3.1c,1

64.4 ± 2.8 a,4 68.9 ± 3.3 a,6 64.4 ± 2.8 a,3 26.7 ± 3.1 b,2 26.7 ± 6.7 b,2 0

57.8 ± 4.5 a,4 53.3 ± 4.2 a,5 55.6 ± 4.4 a,2 13.3 ± 4.2 b,1 15.5 ± 2.2 b,1 0

43.3 ± 3.3 a,3 44.8 ± 2.8 a,4 48.9 ± 5.9 a,2 17.8 ± 2.2 b,1 11.1 ± 2.2 c,1 0

40 ± 3.6 a,2 42.2 ± 2.1 a,4 44.4 ± 2.2 a,2 13.3 ± 4.2 b,1 0 0

37.7 ± 3.8 a,2 35.6 ± 3.1 a,3 33.3 ± 3.9 a,1 6.7 ± 3.1 b,1 0 0

22.2 ± 3.8 a,1 24.4 ± 2.8 a,2 26.7 ± 6.7 a,1 0 0 0

17.7 ± 2.1 a,1 23.3 ± 3.3 a,2 22.2 ± 3.8 a,1 0 0 0

15.5 ± 2.8 a,1 13.3 ± 3.1 a,1 17.8 ± 2.2 a,1 0 0 0

Semen was diluted with Ca–F HBSS containing 0.5% penicillin–streptomycin (10,000 units of penicillin/mL; 10,000 μg of streptomycin/mL) at 1:1, 1:2, 1:4, 1:9 and 1:19, stored in tissue culture flasks at 4 °C in July 2006. Semen samples were daily evaluated for motility. Control referred to undiluted semen that was stored in tissue culture flasks at 4 °C. Different letter superscripts on the column indicate means that were significantly different (P b 0.05) among different dilution ratios. Different number superscripts on the row indicate means that were significantly different (P b 0.05) over storage time.

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Table 4 Effect of chilled storage period of walking catfish (n = 54) semen on percentage of motile sperm during May, August and November of 2007. Storage period

Days of storage

May August November

82.2 ± 2.1 93.3 ± 3.1 80 ± 3.1

0

1 b,7 a,5 b,6

84.4 ± 2.8 88.9 ± 3.3 71.1 ± 3.3

a,7 a,5 b,5

2

3

4

5

6

7

8

9

10

71.1 ± 3.3 ab,6 77.8 ± 2.1 a,4 68.9 ± 3.3 b,5

62.2 ± 2.1 a,5 68.9 ± 4.6 a,4 55.6 ± 2.8 ab,4

51.1 ± 3.3 ab,4 60 ± 3.1 a,4 44.8 ± 2.8 b,3

37.8 ± 2.1 b,3 46.7 ± 3.1 a,3 28.9 ± 3.3 c,2

22.2 ± 2.1 b,2 44.4 ± 2.8 a,3 13.3 ± 3.1 c,1

17.8 ± 2.1 b,2 31.1 ± 4.6 a,2 0

6.7 ± 3.1 b,1 33.3 ± 3.1 a,2 0

0 28.9 ± 3.3 a,2 0

0 20 ± 3.1 0

a,1

Semen from different sampling periods was diluted 1:4 with Ca–F HBSS containing 0.5% penicillin–streptomycin (10,000 units of penicillin/mL; 10,000 μg of streptomycin/mL), stored in tissue culture flasks at 4 °C, and samples were daily evaluated for motility. Different letter superscripts on the column indicate means that were significantly different (P b 0.05) among semen sampling time. Different number superscripts on the row indicate means that were significantly different (P b 0.05) over storage time.

halibut Hippoglossus hippoglossus (Methven and Crim, 1991), winter flounder Pleuronectes americanus (Shangguan and Crim, 1999) and Atlantic cod Gadus morhua L. (Rouxel et al., 2008). These have been associated with a change in sperm plasma membrane and certain plasma C21 steroids at the end of spawning (Rana, 1995; Suquet et al., 1998; Vermeirssen et al., 2004; Rouxel et al., 2008). An increase in sperm motility and sperm density in the present study from the beginning to the middle part of the spawning season and then a decrease towards the end of spawning season was also reported in Atlantic cod (Rouxel et al., 2008). Further studies are necessary to investigate the natural changes in semen quality of walking catfish over the spawning season without hormonal administration.

4.2. Chilled storage of sperm Ca–F HBSS was the most effective extender in this study to prolong storage time which may relate in part to the similarity in osmolality (301 mOsm/kg) to walking catfish testicular fluid (297 mOsm/kg). Most teleost sperm were quiescent in the testes due to the isotonic osmolality of the testicular fluid but sperm motility occurred when sperm were released to external environment (Morisawa,1985). A hyperpolarization of fish sperm membrane during activation is reported to lead to an increase in intracellular Ca2+ and the initiation of motility (Vines et al., 2002; Krasznai et al., 2003). The suitability of Ca–F HBSS over HBSS for short-term storage of sperm has also been demonstrated in Mekong giant catfish Pangasius gigas (Mongkonpunya et al., 1995). Successful storage of extended semen has been reported in several cultured teleosts, but is subject to individual variation, collection method and storage conditions. Piracanjuba semen diluted (1:10 total volume) in NaCl 200 mM or in Saad solution (NaCl 200 mM, Tris 30 mM) maintained motility above 35% during 7 days of storage at 4 °C, while motility was only 7% from undiluted semen stored for 3 days (Maria et al., 2006) Refrigerated Atlantic cod and haddock sperm diluted 1:3 with modified Mounib's extender retained about 3% motility after chilled storage for 40 and 38 days, respectively, while those of undiluted sperm retained a low motility for only 10–20 days (DeGraaf and Berlinsky, 2004). A gradual decline in motility of extender-preserved sperm during short-term chilled storage has also been reported (Chao

Table 5 Percent fertilization and hatching of walking catfish eggs fertilized with fresh or chilled semen in November 2007. Storage time (days)

Percent fertilization

Percent hatching

Fresh sperm

Chilled sperm

Fresh sperm

Chilled sperm

2 4 6

85.6 ± 2.9a 78.5 ± 3.1a 74.5 ± 4.2a

80.1 ± 3.6a 52.4 ± 3.5b 18.5 ± 4.4c

72.8 ± 3.1a 67.3 ± 2.4a 69.4 ± 3.8a

71.6 ± 3.4a 32.5 ± 3.8b 4.5 ± 2.1c

Fresh sperm refers to freshly collected semen used to fertilize eggs as the control. Extender-preserved sperm after chilled storage for 2, 4 or 6 days at 4 °C was used to fertilize eggs. A number of 18 spermiating males were used in the experiment. Values within a column that were superscripted by the same letter were not significantly different (P N 0.05).

et al., 1992; Bates et al., 1996). Fresh semen in the present study retained their motility at 4 °C for one day before a rapid loss. This was probably related to anaerobic conditions and dehydration effect. Other possibility may be associated with damage of sperm caused by semen preparation from dissected testis (Ciereszko and Dabrowski, 1994). Short-term semen storage may accelerate oxidative damage of fresh semen resulting in a decline in sperm motility (Lahnsteiner et al., 1998). Post-activation motility of undiluted semen from Sarotherodon mossambicus stored at 5 °C declined to zero within 60–120 h (Harvey and Kelley, 1984). Atlantic halibut spermatozoa when stored undiluted, showed significantly lower motility and shorter viability than samples stored under air or oxygen atmosphere (Babiak et al., 2006a). Stored sperm of rainbow trout (Billard,1981) in an oxygen atmosphere showed improved storage time over that under air atmosphere although storage of chinook salmon (Oncorhynchus tshawytscha; Bencic et al., 2001) sperm in air was more effective than oxygen. Biochemical change associated with sperm ageing process that resulted in reduced sperm motility and fertilization capacity during chilled storage has been reported in Atlantic halibut (Billard et al., 1993), rainbow trout (Lahnsteiner et al., 1997) and turbot (Suquet et al., 1998). In the present study, sperm motility of walking catfish after chilled storage did not show significant differences among dilutions of 1:1, 1:2, and 1:4. This indicated that a maximum dilution of 1:4 immediately after dilution would help sustain motility if artificial insemination is delayed several days. Studies on dilution ratio for maintaining motility have been carried out extensively and appear to vary among fish species. In African catfish (C. gariepinus), semen viability was maintained best when it was diluted at a ratio of 1:5 in storage solution (150 mmol/L NaCl, 2.5 mmol/L KCl, 1 mmol/L CaCl2, 1 mmol/L MgSO4, 20 mmol/L Tris (pH 8.5) and 0.5% BSA or 0.5% hen egg yolk) and stored at 4 °C (Mansour et al., 2004). The optimal dilution ratio for Atlantic cod and haddock (DeGraaf and Berlinsky, 2004) and sturgeon (Acipenser oxyrinchus desotoi and A. brevirostrum; Park and Chapman, 2005) was reported to be 1:3. Babiak et al. (2006a) determined factors affecting the success of chilled storage of semen from Atlantic halibut and found that semen diluted in modified Hanks' balanced salt solution 1:5–1:9 retained its viability for an extraordinary long time as compared to other dilution ratios tested. Motility of tropical bagrid catfish Mystus nemurus sperm varied with the type of extender and dilution ratio used from which the highest motility was obtained in Ringer at 1:20 (Muchlisin et al., 2004). Maria et al. (2006) reported dilutions 1:5 and 1:10 were acceptable for piracanjuba spermatozoa. Dilution of semen was reported to reduce the ratio of protective, low-molecular weight seminal components to ram spermatozoa (Ashworth et al., 1994). Diluted semen stored during the middle of spawning season in the present study had the longest successful storage period. Similarly, a higher storage capacity of Atlantic cod sperm was observed during the middle period of the spawning season, compared to the beginning or the end of the spawning season (Rouxel et al., 2008). Billard et al. (1977) reported a decrease in storage capacity of seabass (Dicentrarchus labrax) sperm from 70 h at the beginning of the spawning season to only 30min in the middle or end of the spawning season. Chilled storage of Pikey bream (Acanthopagrus berda) was less successful for semen collected late in the spawning season (Palmer et al., 1994). A decline in the storage time

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of semen collected at the end of the spawning season in the present study was related to changes of sperm characteristics throughout the season. A decrease of sperm motility, sperm density and percentage of spermiating males of walking catfish reflected the change of sperm quality over time. Viability of sperm has been shown to be related with protective effect of seminal plasma proteins (Lahnsteiner et al., 2004). Babiak et al. (2006b) reported that sperm samples of Atlantic halibut from males maintained with natural photoperiod showed significantly longer viability under in vitro storage conditions than sperm from advanced photoperiod males, which had a deterioration of sperm quality.

63

10 days of storage. This method demonstrates a potential application of chilled storage of semen in breeding program of walking catfish with acceptable sperm motility and fertilization rates.

Acknowledgments The authors would like to thank Patinya Ounkwanmurang and Prapan Phanthuchan for technical assistance in rearing and sampling the fish. Financial support for this study was obtained from the Thailand Research Fund (DBG4980006).

4.3. Fertilization A significant decline in fertilization on days 4 and 6 of extended semen was associated with a dramatic decline in sperm motility (44.8% and 13.3%, respectively). At these motilites, sperm may encounter fewer unfertilized eggs per unit time as walking catfish sperm were immediately immotile within b1 min after activation. Shorter period under which limited number of sperm swims to fertilize eggs would provide lesser chance of fertilization success. A higher sperm to egg ratio with low motility sperm during artificial insemination may improve fertilization capacity. Saad et al. (1988) reported that loss of fertilizing ability of carp Cyprinus carpio semen was compensated for by a larger amount of spermatozoa during insemination. As a result, a decline in the proportion of motile sperm in the present study would lower fertilization rate. Chilled storage of diluted semen with extender for a short time prior to fertilization has lengthened fertilization capacity in a number of cultured species i.e. trout (Stoss, 1983), tilapia (Harvey and Kelley, 1984), walleye Stizostedion vitreum (Moore, 1987; Satterfield and Flickinger, 1995), Atlantic sturgeon (DiLauro et al., 1994), paddlefish Polyodon spathula (Brown and Mims, 1995), channel catfish (Christensen and Tiersch, 1996). Sperm to egg ratio, number of sperm, duration of contact between gametes and/or sperm swimming speed have also been reported to influence fertilization success in rainbow trout (Lahnsteiner et al., 1998), walleye (Casselman et al., 2006) and Atlantic cod (Butts et al., 2009). Male walleye with the fastest-swimming sperm had approximately 40% greater fertilization success than males with the slowest-swimming sperm when sperm were used at constant number in fertilization trials (Casselman et al., 2006). Paddlefish sperm diluted 1:1 in a 150-mM NaCl solution and stored at 1 °C exhibited a fertilizing capacity of 73% after 25 days (Brown and Mims, 1995). Fertilization rates in the present study were not likely affected by the number of sperm used to fertilize eggs as an optimal sperm to egg ratio was used. Nevertheless, apparent fertilization rates would have been affected by the level of sperm motility that was significantly declined over time. A significant positive correlation between sperm motility and fertilization percentage has been reported in some teleost species e.g. African catfish C. gariepinus (Mansour et al., 2005), amago salmon Oncorhynchus masou ishikuwue (Ohta et al., 1995). Sperm to egg ratio and sperm motility play an important role for fertilization success. Ohta et al. (1996) obtained 89.6% fertility using 33% motile spermatozoa, which were diluted with artificial seminal plasma 100 times. Additional experiments using different rates of motility and sperm to egg ratios for fertilizing eggs are required to elucidate factors controlling fertilization capacity of walking catfish semen. Further investigation is required to store larger volume of extended semen for production-scale fertilization of commercial walking catfish farming. Hatching percentages in the present study ensured the possibility for insemination of eggs with chilled semen. 5. Conclusion In conclusion, season had a significant influence on semen quality of walking catfish. Highest semen quality was observed during the beginning to middle of the spawning season. Ca–F HBSS was the most suitable extender for chilled storage of walking catfish semen at 4 °C. Semen diluted at a dilution of 1:4 in Ca–F HBSS remained motile for

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