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Motility and fertility of the subtropical freshwater sh streaked prochilod (Prochilodus lineatus) sperm cryopreserved in powdered coconut water
A.T.M. Viveirosa,*, A.F. Nascimentob, L.H. Orfoa, Z.A. Isaa
b a Department of Animal Sciences, Federal University of Lavras, UFLA, P.O. Box 3037, Lavras, MG, 37200-000, Brazil Department of Veterinary Medicine, Federal University of Lavras, UFLA, P.O. Box 3037, Lavras, MG, 37200-000, Brazil

Received 6 October 2009; received in revised form 21 March 2010; accepted 21 March 2010

Abstract Streaked prochilod (Prochilodus lineatus) is a freshwater sh inhabiting many South American rivers. The objective was to determine the effectiveness of coconut water (ACP), combined with methylglycol, as a freezing medium for streaked prochilod sperm. A secondary objective was to compare a computer-assisted sperm analyzer (CASA) system versus subjective microscropic examination as a means of assessing sperm motility. As a control, glucose and methylglycol was used, according to our previous study. Sperm diluted in each medium was loaded into 0.5 mL straws, frozen in liquid nitrogen vapor (in a dry shipper), and stored in liquid nitrogen (-196 C). Half of the samples were evaluated for sperm motility, both subjectively and with CASA; the remainder were evaluated for fertility. There was no difference (P 0.05) between subjective or CASA assessment of post-thaw sperm motility. Although sperm motility was higher in sperm cryopreserved in ACP (85%) than in glucose (75%), cryopreservation in either extender yielded similar fertilization rates (46 48%) and sperm velocities. There were positive correlations (r 0.56 0.8) between all sperm velocities and fertilization rate. In conclusion, streaked prochilod sperm cryopreserved in glucose or ACP and methylglycol was fertile, and thus could be used for research or commercial settings. Furthermore, although the CASA system provided objective data regarding sperm motility, in the present study, subjective evaluation of sperm motility was practical and a good indication of sperm quality; it could readily be done by well-trained personnel under eld or laboratory conditions. 2010 Elsevier Inc. All rights reserved.
Keywords: Prochilodus lineatus; Semen cryopreservation; Sperm motility; Fertility; Fish

1. Introduction The streaked prochilod, Prochilodus lineatus (Valenciennes, 1836), is a migratory Characiformes Brazilian sh species, with a large geographical distribution throughout South America, accounting for 5090% of the total sh biomass in the Paran river basin [1].

* Corresponding author. Tel.: 55 35 38291223; fax: 55 35 38291231. E-mail address: ana.viveiros@dzo.ua.br (A.T.M. Viveiros). 0093-691X/$ see front matter 2010 Elsevier Inc. All rights reserved. doi:10.1016/j.theriogenology.2010.03.018

Their detritivorous habit makes streaked prochilod a dominant element in structuring tropical stream community dynamics by sediment processing activities [2]. Streaked prochilod is one of the freshwater sh species with the greatest importance in the Brazilian aquaculture industry, where it is known as curimba, curimbat, and curimat. Larvae are used as live food for endangered carnivorous sh species, including piracanjuba (Brycon orbignyanus) and ja (Zungaro jahu), whereas adult sh are used for human consumption, mainly in

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northeastern Brazil [3]. Brazilian hydroelectric companies breed streaked prochilod for restocking programs. Finally, because articial reproduction methods are well established and prolicacy is high, the streaked prochilod has been used as a model species for research in sh reproduction. Sperm cryopreservation is an important technique in sh culture, as it facilitates procedures for articial reproduction. With viable sperm stored in liquid nitrogen, it is necessary to induce spawning and collect gametes only from females. Cryopreserved sperm may be kept in germplasm banks for an indenite period, which allows the establishment of breeding programs, eliminates the problem of asynchronous reproductive activity between males and females, and enables maintenance of fewer male broodsh [4]. Prior to freezing, sperm must be diluted in a medium containing an extender and a permeable cryoprotectant agent (CPA). In our previous study, streaked prochilod sperm cryopreserved in a medium containing glucose and methylglycol retained 8695% motility and 4783% fertility [3]. During the past decade, researchers at the State University of Cear (UECE), Brazil, have developed a technology to dehydrate coconut water and produced a stable and standardized powdered coconut water (ACP), which contains minerals, amino acids, vitamins, carbohydrates, growth factors, phytohormones, and saturated fatty acids [5]. The use of ACP as an extender for streaked prochilod sperm extender has undergone preliminarily testing. Although post-thaw sperm quality was limited to a subjective evaluation of sperm motility, results were promising [3]. The objective of this study was to further investigate the effects of ACP as an extender for cryopreservation of streaked prochilod sperm. A secondary objective was to compare a CASA system versus subjective microscropic examination as a means of assessing sperm motility. As a control, sperm cryopreserved in glucose and methylglycol was used, according to our previous study [3]. 2. Materials and methods 2.1. Fish and sperm collection All sh were handled in compliance with published guidelines for animal experimentation [6]. Streaked prochilod (P. lineatus) males were selected from earthen ponds at the Fish Culture Unit of the Hydroelectric Company of Minas Gerais (CEMIG; Itutinga, MG, Brazil) during the spawning season (December and January). Males with detectable running sperm in response to soft abdominal pressure were given a single

dose of carp pituitary extract (Argent Chemical Laboratories, Redmond, WA, USA; 5 mg/kg body weight, IM) and maintained at 26 C. Eight hours after treatment, the urogenital papilla was carefully dried, and sperm was hand-stripped directly into test tubes. Sperm collection was carried out at room temperature (2729 C), and soon after collection, tubes containing sperm were placed in crushed ice ( 5 C). An aliquot (5 L) of each sample was placed on a slide and observed with a light microscope (Model L1000, Bioval, Jiangbei, China) at 400 magnication. Any sperm motility (auto-activation) observed was attributed to urine or water contamination and the sample discarded, as sh sperm in seminal plasma is immotile. In immotile samples (n 8 males), sperm progressive motility was subjectively estimated (in increments of 5%) immediately after the addition of 25 L of 0.29% NaCl as an activating agent [3]. Only samples with at least 80% motility were used in the subsequent analysis. Sperm volume and concentration (hemacytometer/ Neubauer chamber) were also determined. 2.2. Sperm cryopreservation Two freezing media, comprising combinations of two extenders and one CPA (methylglycol: CH3O(CH2)2OH; Vetec Qumica Fina Ltda, Duque de Caxias, RJ, Brazil), were prepared and maintained in crushed ice ( 5 C). Powdered coconut water (ACP, ACP Servios Tecnolgicos Ltda, ACP Biotecnologia, Fortaleza, Cear, Brazil; each 100 mL contained: glucose 4.4 g; proteins: 0.37 mg; phosphorus 6.2 mg; potassium 175 mg; calcium: 17.5 mg; magnesium: 8.5 mg; sodium: 10.5 mg; iron 0.06 mg; among other components [7]; pH 7.8; 300 mOsmol) was tested as extender and a commercial 5% glucose solution (Fresenius-Kabi, Brazil; pH adjusted to 7.6; 277 mOsmol) was used as a control [3]. Sperm samples were diluted in each freezing media (8 males 2 media) at a nal proportion of 10% sperm, 80% extender, and 10% methylglycol. After dilution, sperm samples were immediately aspirated into 0.5 mL straws (n 6 replicate straws) and frozen in a nitrogen vapor vessel (Cryoporter LN2 dry vapor shipper, Cryoport Systems, Brea, CA, USA) at approximately 170 C, which gives a freezing rate of approximately 35.6 C/min between 21 C and 170 C [8]. Thereafter, cryopreserved sperm was transported 50 km by car from CEMIG to the Animal Sciences Department at the Federal University of Lavras (UFLA), Lavras, MG, Brazil, where straws were transferred to liquid nitrogen (M.V.E. Millenium, XC 20, Chart, Minessota, USA) at 196 C within 2024 h for storage.

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In total, 96 straws (6 replicate straws 2 media 8 males) were cryopreserved. Half of the frozen replicate straws were used for a fertilization trial 1 mo after freezing, whereas the remainder was used for sperm motility evaluation 8 mo after freezing, as described below. 2.3. Sperm motility and velocities evaluation Half of the replicate straws (n 3 replicate straws 2 media 8 males) were transferred back to the nitrogen vapor vessel and transported by car ( 350 km) and then by plane ( 3000 km) from UFLA to the Laboratory of Semen Technology at State University of Cear (UECE), Fortaleza, CE, Brazil. After arrival, straws were plunged back into liquid nitrogen. During the following 34 wk, straws were thawed in a 60 C water bath for 8 s [3], and post-thaw sperm motility was immediately estimated, both subjectively as described for fresh sperm and objectively using CASA. Post-thaw sperm was activated in a Makler counting chamber placed on a phase contrast microscope (Nikon H550S, ECLIPSE 50i, Japan), 400 magnication, green lter, and pH1 position. The microscope was connected to a video camera (Basler Vision Technologies A312FC, Ahrensburg, Germany) which generated 25 images/s. Video recording was started at 34 s postactivation, as motility ceased within 93111 s in this species [9]. Each image (n 25) was analyzed using the standard settings for sh by Sperm Class Analyzer software (SCA 2005, Microptics, S.L. Version 3.2.0, Barcelona, Spain). Sperm was considered immotile when velocity was 10 m/s. Although SCA simultaneously assessed more than 15 sperm motility end points, for brevity only curvilinear velocity (VCL), straight line velocity (VSL), and average path velocity (VAP) were considered for further analysis, as similar effects were observed for all end points. To determine these velocities, each individual sperm cell (n at least 500 sperm/straw) was followed throughout the 25 images and a sperm trajectory was calculated. 2.4. Evaluation of fertilization capacity The other half of the straws (n 3 replicate straws 2 media 8 males) were placed in the nitrogen vapor vessel and transported by car ( 50 km) from UFLA back to CEMIG. Straws were thawed as described for motility analysis and used to fertilize fresh oocytes. To harvest oocytes, females (n 3) received two doses (0.5 and 5 mg/kg body weight) of carp pituitary extract at 12 h intervals, and were hand-stripped 5 h after the second dose. All females responded positively to hor-

monal treatment, and all were used for the fertilization trial. An aliquot (100 L of sperm from one replicate straw) was added to 0.2 g of oocytes ( 240 oocytes) of one female (one replicate straw per female). As a control for egg quality, 10 L of pure fresh sperm (fresh control sperm, with 90% motile cells) was added to a similar number of oocytes. Fertilization was initiated by addition of 5 mL tank water, and mixed for 1 min. Subsequently, 10 mL tank water was added and samples mixed for another 2 min. Finally, eggs were transferred to a PVC basket, 10 cm in diameter, with a 0.5 mm mesh bottom [10], and incubated in a ow-through system at 26 C. The number of fertilized eggs, as a percentage of total eggs, was determined 10 h later. 2.5. Statistical analyses Values are reported as means SD. Statistical analyses were conducted with the SISVAR computational program [11]. Sperm motility, velocities, and fertility were tested for normal distribution using the univariate procedure. When data did not have a normal distribution, an arcsin transformation was performed. An ANOVA was used to determine differences between the two extenders. A Spearman test was used to determine the correlation between sperm velocities and fertilization rates. The level of signicance for all statistical tests was set at 0.05. 3. Results Overall, the fresh semen samples had mean volume of 1.9 mL, with 19.2 109 sperm/mL and 92% motility (Table 1). Motility and fertilization rate of frozenthawed sperm are shown (Table 2). For both extenders, post-thaw sperm motility subjectively evaluated using a light microscope was similar (P 0.05) to motility

Table 1 Body weight and fresh sperm characteristics of the subtropical freshwater sh streaked prochilod (n 8 males). Male ID Body weight (kg) 1.4 0.8 0.9 1.3 1.0 1.1 1.2 1.2 1.1 0.2 Volume (mL) 2.5 1.7 1.9 2.1 1.9 1.6 1.7 1.8 1.9 0.3 Sperm concentration ( 109/mL) 20.6 19.4 19.2 20.3 13.2 23.1 16.9 21.2 19.2 3.0 Subjective motility (%) 90 95 85 90 95 90 100 90 92 5

1 2 3 4 5 6 7 8 Mean

SD

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Table 2 Mean SD post-thaw sperm motility, determined both subjectively (light microscope) and by computer-assisted sperm analyzer (CASA) and fertilization rate of streaked prochilod sperm cryopreserved in methylglycol, combined with either powder coconut water (ACP) or 5% glucose, as extenders. Male ID 1 2 3 4 5 6 7 8 Mean
A

Subjective motilityA* (%) ACP 77 70 83 60 77 90 90 80 78 11 0 6 0 6 0 0 0 11a Glucose 64 60 90 60 48 87 83 80 72 6 0 0 0 3 6 5 0 13b

CASA motilityA* (%) ACP 80 74 86 73 82 97 97 87 85 1 7 2 2 3 1 1 0 12a Glucose 63 64 97 64 50 90 90 87 75 1 1 6 1 0 4 2 2 10b

FertilityB (%) ACP 39 39 60 34 42 64 64 43 48 1 2 3 1 1 1 3 3 12 Glucose 38 41 64 40 39 44 58 43 46 1 5 0 5 1 2 3 2 10 0.05).

SD

* Post-thaw sperm motility evaluated by either CASA or subjectively and fertilization between the two freezing media did not differ (P Mean represents (8 males 3 replicate straws/media). B Mean represents (8 males 3 replicate straws 3 females); 1 replicate straw eggs from 1 female. Fresh control sperm yielded 90% of subjective motility and 62% of fertility. a,b Within a row and end point, means without a common superscript differed (P 0.05).

evaluated using CASA. However, CASA data were used to describe and discuss the results. Post-thaw motility was higher (P 0.05) when sperm was cryopreserved in ACP (85%) compared to sperm cryopreserved in glucose (75%). Sperm velocities were similar in sperm cryopreserved in both media. Sperm cryopreserved in ACP had VCL 53.7 25.1 m/s, VSL 23.6 8 m/s, and VAP 36.8 14.8 m/s, whereas the corresponding end points for sperm cryopreserved in glucose were 49.1 19.3 m/s, 24.3 7.4 m/s, and 36.0 13.1 m/s, respectively. Correlations between sperm velocities and the fertilization rates are shown (Fig. 1). The highest correlation (r 0.8) was between fertilization rate and VCL. Sperm cryopreserved in ACP or glucose yielded similar fertilization rates (48 and 46%), respectively. However, fresh control sperm yielded a signicantly higher fertilization rate (62%) than cryopreserved sperm. The estimated mean sperm:egg ratio in this experiment was 8 105 sperm per egg, for both cryopreserved and fresh sperm. 4. Discussion Sperm volume, sperm concentration, and motility rate in the present study were within the range for fresh sperm of this species [4]. This is apparently the rst report on the analysis of streaked prochilod sperm using CASA. When post-thaw sperm motility of streaked prochilod was evaluated, there was no signicant difference between the two methods used, namely a subjective evaluation using a light microscope and an

objective evaluation using CASA. Similarly, in pirapitinga (Piaractus brachypomus), another Characiformes, there was no signicant difference when postthaw sperm motility was analyzed subjectively or using CASA [12]. Subjective scoring of sh sperm motility is used in many laboratories and hatcheries, but produces variable results. The CASA systems, quantify the sperm movement using at least a dozen computercalculated motility characteristics, provides objective methods of assessment and is easy to perform, precise and rapid [13]. For analyses of these indicators of sperm quality, CASA is the most objective and comprehensive quantication currently available [14]. However, the high cost of equipment has restricted the use of CASA to a few laboratories, with even lower use in developing countries. Based on the present study, a well-trained technician and with a light microscope could easily and reliably assess sperm motility, even under eld conditions. However, these results may not be repeatable in all species and with all operators. Another option would be to use a video camera, in conjunction with customized software [14], which provided objective analysis of motility in sh sperm at much lower cost than traditional CASA systems. Motility rate and sperm velocity have been associated with reproductive success in sh. In the present study, there were positive correlations between fertilization rates and all sperm velocities, with the highest correlation (r 0.8) for VCL. Similarly positive correlations between progressive sperm motility velocities (VCL, VSL, and VAP) and fertilization were also reported for African catsh (Clarias gariepinus) [13],

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a wide range of sperm freezing protocols, without involving female sh and therefore, without the variability in egg quality among female sh [18]. The ACP preparation has the same biochemical constituents as the in natura form and yet is standardized and can easily stored and distributed. The use of ACP as sperm extender of sh species was rst tested in streaked prochilod, piapara (Leporinus obtusiden) and piracanjuba (Brycon orbignyanus). In all three species, post-thaw sperm motility was similar between sperm cryopreserved in ACP and in the standard medium [19]. In the present study, streaked prochilod sperm cryopreserved in ACP had higher motility than sperm cryopreserved in glucose. However, neither sperm velocity nor fertilization rate differed between the two media. Therefore, either glucose or ACP, combined with methylglycol, are appropriate extenders for cryopreservation of streaked prochilod sperm. Acknowledgements This research was supported by CNPq (no. 471975/ 2004-4). The authors thank CEMIG for providing the broodsh, the Laboratory of Semen Technology at UECE (Cear, Brazil) for providing ACP and allowing the use of the CASA, and A.N. Maria, C.C.M. Salgueiro, N.O. Pessoa, and M.J.A.F. Vieira for assistance with experiments. References
[1] Bonetto AA. Austral rivers of South America. In: R. Margalef, editor. Limnology now: a paradigm of planetary problems. New York: Elsevier Science. 1994. p. 42572. [2] Flecker AS. Ecosystem engineering by a dominant detritivore in a diverse tropical stream. Ecology 1996;77:184554. [3] Viveiros ATM, Orfo LH, Maria AN, Allaman IB. A simple, inexpensive and successful freezing method for curimba Prochilodus lineatus (Characiformes) semen. Anim Reprod Sci 2009;112:293300. [4] Viveiros ATM, Godinho HP. Sperm quality and cryopreservation of Brazilian freshwater sh species: a review. Fish Phys Bioch 2009;35:13750. [5] Laguna LE, Nunes JF. Physical-chemical evaluation of the coconut water in the beach and dwarf varieties. Revista Brasileira de Reproduo Animal 1997;21:156. [6] Van Zutphen LFM, Baumans V, Beynen AC. Principles of Laboratory Animal ScienceA Contribution to the Humane Use and Care of Animals and to the Quality of Experimental Results. Elsevier B.V., Amsterdam, 1993. [7] Carvalho JM, Maia GA, Sousa PHM, Maia Jr GA. Water of coconut: Nutritional and functional properties and processing. Semina: Cincias Agrrias, Londrina, 2006;43752. [8] Maria AN, Viveiros ATM, Orfo LH, Oliveira AV, Moraes GF. Effects of cooling and freezing on sperm motility of the endan-

Fig. 1. Correlations between sperm velocities parameters and (A) curvilinear (VCL); (B) straight line (VSL); and (C) average path (VAP), and fertilization rates of streaked prochilod eggs fertilized with sperm cryopreserved in () powdered coconut water (ACP)methylglycol (MG) or () 5% glucose (Glu)-MG.

turbot (Psetta maxima) [15], carp (Cyprinus carpio) [16], and rainbow trout (Oncorhynchus mykiss) [17]. The indirect measurement of sperm quality through fertilizing capacity depends on egg quality, which is variable and affects the success of fertilization. In sh farms, it is necessary to assess sperm quality to ensure the success of articial fertilization. Fertilization may be dependent on the number of motile sperm and their velocity. If sperm quality may be related to fertilizing ability, then it is possible to use the CASA routinely in

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A.T.M. Viveiros et al. / Theriogenology 74 (2010) 551556 gered sh piracanjuba Brycon orbignyanus (Characiformes, Characidae). Anim Reprod 2006;3:55 60. Cruz VLB. Criopreservao de smen de curimbat, Prochilodus lineatus scrofa (Characiformes, Prochilodontidae). MSc Thesis. Pontifcia Universidade Catlica de Minas Gerais, Belo Horizonte, Brazil, 2001. Viveiros ATM, So N, Komen J. Sperm cryopreservation of African catsh, Clarias gariepinus: cryoprotectants, freezing rates and sperm:egg dilution ratio. Theriogenology 2000;54:1395 408. Ferreira DF. Analysis of Variance System (SISVAR). Department of Statistics of UFLA, Lavras, Version 4.3 (Build 43) 1999. Nascimento AF, Maria AN, Pessoa NO, Carvalho MAM, Viveiros ATM. Out-of-season sperm cryopreserved in different media of the Amazonian freshwater sh pirapitinga (Piaractus brachypomus). Anim Reprod Sci 2010;118(2 4):324 9. Rurangwa E, Volckaert FAM, Huyskens G, Kime DE, Ollevier F. Quality control of refrigerated and cryopreserved semen using computer-assisted sperm analysis (CASA), viable staining and standardized fertilization in African catsh (Clarias gariepinus). Theriogenology 2001;55:751 69. [14] Wilson-Leedy JG, Ingermann RL. Development of a novel CASA system based on open source software for characterization of zebrash sperm motility parameters. Theriogenology 2007;67:66172. [15] Dreanno C, Cosson J, Suquet M, Seguin F, Dorange G, Billard R. Nucleotides content, oxidative phosphorylation, morphology and fertilizing capacity of turbot (Psetta maxima) spermatozoa during the motility period. Mol Reprod Dev 1999;53:230 43. [16] Linhart O, Rodina M, Cosson J. Cryopreservation of sperm in common carp Cyprinus carpio: sperm motility and hatching success of embryos. Cryobiology 2000;41:24150. [17] Lahnsteiner F. Semen cryopreservation in the Salmonidae and in the Northern pike. Aquacult Res 2000;31:24558. [18] Rurangwa E, Kime DE, Ollevier F, Nasha JP. The measurement of sperm motility and factors affecting sperm quality in cultured sh. Aquaculture 2004;234:128. [19] Viveiros ATM, Maria AN, Orfo LH, Carvalho MAM, Nunes JF. Powder coconut water (ACP) as extender for semen cryopreservation of Brazilian migratory sh species. CybiumInt J Ichthyol 2008;32:215.

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