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Storage of Steindachneridion parahybae oocytes at different temperatures
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Storage of Steindachneridion parahybae oocytes at different temperatures
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Eduardo Antônio Sanches a,∗ , Renan Yoshiharu Okawara b , Danilo Caneppele c , Giovano Neumann d , Robie Allan Bombardelli d , Elizabeth Romagosa b a b c d
State University of São Paulo, UNESP, Registro, SP, Brazil Fishery Institute, APTA, São Paulo, SP, Brazil São Paulo Energy Company, CESP, Paraibuna, SP, Brazil State University of West Paraná, UNIOESTE, Toledo, PR, Brazil
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Article history: Received 27 March 2014 Received in revised form 23 September 2014 Accepted 30 September 2014 Available online xxx
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Keywords: Artificial fertilization Eggs Fish Gamete exposure Larvae Surubim-do-Paraiba
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1. Introduction
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The objective of this study was to assess the influence of temperature and time on the storage of fresh Steindachneridion parahybae oocytes. Two experiments were carried out: (1) the fertilization rates of oocytes exposed to temperatures of 5, 15, 28 (room temperature) and 35 ◦ C were assessed 15 min (control), 115, 235 and 355 min after release; (2) the fertilization and hatching rates, as well as the percentage of normal larvae of oocytes exposed to 14, 17 or 20 ◦ C, 20 min (control) were assessed 50, 80 and 110 min after stripping. In the first experiment, the highest fertilization rates (P < 0.05) were obtained in the control treatment (15 min, 28 ◦ C), with 74.34 ± 5.48% oocytes showing loss of viability over time. In the second experiment, there was a reduction (P < 0.05) in the fertilization rates at the temperatures and times tested. The artificial fertilization of S. parahybae oocytes is recommended immediately after collection, and if storage is necessary, it should be conducted at temperatures between 17 and 20 ◦ C. © 2014 Published by Elsevier B.V.
Artificial propagation is an important technique for intensification of fish production (Romagosa, 2006). Therefore, high quality gametes should be used to ensure maximum fertilization and subsequently, normal development of the embryo (Romagosa, 2008; Bobe and Labbe, 2010). One way to estimate the potential for development is to analyze some parameters such as fertilization, hatching and percentage of normal larvae (Brooks et al., 1997; Coward et al., 2002; Bobe and Labbe, 2010).
∗ Corresponding author. Tel.:+55 13 3828 2900x2930. E-mail addresses: [email protected], [email protected] (E.A. Sanches).
In general, there is little information in the literature on the ex situ viability of gametes from the neotropical fish fauna. The recommended in artificial reproduction procedure is the rapid mixing of gametes (immediately after collection), based on the theory that oocytes and semen lose viability over time (Sanches et al., 2011a, 2013a). Thus, it is important to know the mechanism of viability loss and to develop methods to ensure gamete longevity, to optimize the rational use of broodstocks and the techniques of artificial propagation (Rana, 1995; Babin et al., 2007; Fornari et al., 2011). Temperature is the main factor that affects the quality of oocytes stored in vitro (Sanches et al., 2011a). However, the extent of adverse temperature effects remains unclear for neotropical fish. A simple technique of short-term chilling can be easily applied with positive results in
http://dx.doi.org/10.1016/j.anireprosci.2014.09.022 0378-4320/© 2014 Published by Elsevier B.V.
Please cite this article in press as: Sanches, E.A., et al., Storage of Steindachneridion parahybae oocytes at different temperatures. Anim. Reprod. Sci. (2014), http://dx.doi.org/10.1016/j.anireprosci.2014.09.022
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commercial rearing systems (Sanches et al., 2011a), allowing the maximum utilization of gametes, mainly when there is asynchrony of broodfish spawning following the use of hormonal therapies (Rana, 1995). Asynchrony in broodfish spawning is observed for the surubim-do-Paraíba, Steindachneridion parahybae (Siluriformes: Pimelodidae), a gray catfish endemic to the Paraíba do Sul river basin (Garavello, 2005), which is currently on the red list of Brazilian fauna threatened with extinction (MMA, 2008; IBGE, 2009). This species spawns at different times following hormonal induction (Okawara, 2012) and strategies for its artificial propagation should be adopted, including the management of gametes for in vitro fertilization. The objective of this study was to determine the effect of temperature and time on the storage of fresh S. parahybae oocytes.
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2. Material and methods
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The experiment was conducted at the Hydrobiology and Aquaculture Station of the São Paulo Energy Company–EHA/CESP, in the town of Paraibuna/SP, Brazil (23◦ 24 54 S; 45◦ 35 52 W), using wild S. parahybae broodfish (F0) and other broodfish originating from induced reproduction performed with wild specimens at the same station (F1). The fish were kept in two earthen ponds (200 m2 ) with concrete walls and a sandy bottom, and received extruded commercial feed for carnivorous fish with 40% crude protein at a rate of 5% biomass/week, offered twice-daily, at 08:00 and 16:00, three days per week. To assess the effects of temperature on the oocytes after stripping, two experiments were conducted: (1) four temperatures for up to 355 min; (2) three temperatures for up to 110 min. The second experiment was performed to complement the results of the first experiment. Broodfish that were able to spawn were selected in the pond during the reproductive period (Oct–Feb/2010–2011 and Oct–Feb/2011–2012), as proposed by Caneppele et al. (2009). Semen release was observed for males, and the females were evaluated according to external characteristics, such as a slightly rounded abdomen and the release of a small parcel of oocytes after gentle abdominal pressure. The selected broodfish were transferred to the laboratory, weighed and separated into aquaria (500-L) equipped with aeration, where they remained for hormonal manipulation and stripping (∼24 h). After the females were weighed, they were hormonally induced by injections of crude carp pituitary extract (CCPE) diluted in saline (0.9% NaCl), in two dosages (0.5 and 5.0 mg CCPE/kg), at an interval of 12 h (Caneppele et al., 2009). The males were not hormonally induced (Caneppele, 2011). The oocytes were collected only when they could be easily released after gentle abdominal pressure. The gametes were collected after 240 (9.8 h at 24.56 ± 0.44 ◦ C) and 300 degree-hours (11.9 h at 25.11 ± 3.39 ◦ C), for the first and second experiments, respectively (Caneppele et al., 2009). The gametes were collected after abdominal massage performed from head to tail (by stripping). The semen was collected 15 min before the oocytes, to estimate its quality (Sanches et al., 2011a).
A pool of oocytes collected from three females (two F0 and one F1; mean weight ± SD, 1400 ± 600 g), and semen collected from three F1 males (607 ± 32 g) were used for the first experiment. Another pool of oocytes from two F0 females (2450 ± 212 g) and semen from four F0 males (2133 ± 635 g) were used for the second experiment. To estimate the oocyte production/female, all collected oocytes were weighed. Sperm quality was estimated by computerized sperm analysis using the software IMAGEJ (National Institutes of Health, USA, http://rsbweb.nih.gov/ij/) with the CASA plugin (University of California Howard Hughes Medical Institute, USA, and http://rsbweb.nih.gov/ij/plugins/casa.html), as proposed by Wilson-Leedy and Ingermann (2007) and Sanches et al. (2010, 2013b). Motility rate, curvilinear velocity, straightline velocity and straightness were assessed twice, at the beginning (immediately after collection) and at the end of the period of oocyte exposure (375 and 120 min after collection for the first and second experiments, respectively). In addition to sperm motility, the volume of released semen and sperm concentration were assessed for each experiment. The sperm concentration was assessed using a Neubauer hematimetric counting chamber (Sanches et al., 2011b). A randomized experimental design in a factorial arrangement (4 × 3) was used for the first experiment, considering the exposure of the oocytes to temperatures of 5, 15, 28 (room temperature) and 35 ◦ C, 115, 235 or 355 min after collection. The control treatment consisted of artificial fertilization performed 15 min after stripping at room temperature (28 ◦ C). All the experimental combinations, which represented a total of 39 experimental units, were carried out in triplicate. One hatchery (1.5-L) containing 195 ± 38 oocytes fertilized with 50 L semen and 5 mL water from the hatching system (25.95 ± 1.72 ◦ C; pH 7.32 ± 0.17; 6.30 ± 1.00 mg dissolved oxygen/L) was considered as one experimental unit. The second experiment was conducted using the results from the first experiment. A randomized experimental design in a factorial arrangement (3 × 3) was used, with treatment temperatures of 14, 17 or 20 ◦ C and oocyte exposure times of 50; 80 or 110 min. The control treatment consisted of oocyte fertilization 20 min after stripping at room temperature (23 ◦ C). One hatchery (1.5-L) containing 321 ± 47 oocytes fertilized with 100 L semen and 10 mL water from the hatching system (22.07 ± 0.28 ◦ C; pH 6.96 ± 0.24; 6.89 ± 0.34 mg dissolved oxygen/L) was considered as one experimental unit. The equipment used to conduct each experiment differed. In the first experiment, two refrigerators containing thermostats (±1.0 ◦ C) were used as cooling systems, either at 5 ◦ C or at 15 ◦ C. Room temperature (inside the laboratory) was established at 28 ◦ C. A wooden box heated by an incandescent lamp (240 W) was used as a heating system, with a temperature of 35 ◦ C and a thermostat (±1.0 ◦ C). The temperature was frequently monitored by a mercury thermometer (maximum and minimum). In the second experiment, three water-cooling systems consisting of styrofoam boxes containing water and ice were used. The temperature
Please cite this article in press as: Sanches, E.A., et al., Storage of Steindachneridion parahybae oocytes at different temperatures. Anim. Reprod. Sci. (2014), http://dx.doi.org/10.1016/j.anireprosci.2014.09.022
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Table 1 Sperm and seminal parameters (means ± standard deviation) of Steindachneridion parahybae assessed at the beginning and the end of the two experiments. Parameters
MOT(%) VCL (m/s)* VSL (m/s) STR (%) VOL (mL) CSPZ (spz/mL)
Experiment 01
Experiment 02
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End 01
Beginning
End 02
65.23 ± 13.91 109.78 ± 9.27a 73.18 ± 6.22 89.22 ± 2.25 7.63 ± 3.90 2.28 × 109
72.06 ± 8.58 88.05 ± 6.32b 60.42 ± 11.01 92.28 ± 1.54 10.15 ± 4.49 34.68 × 109
52.23 ± 18.55 62.13 ± 13.41 26.25 ± 6.07 83.71 ± 3.64
45.06 ± 13.95 64.70 ± 6.16 28.49 ± 6.72 82.25 ± 4.39
Beginning = immediately after collection; End 01 = analysis carried out 375 min after collection; End 02 = analysis carried out 120 min after collection. MOT = motility rates, VCL = curvilinear velocity, VSL = straight line velocity, STR = straightness, VOL = Volume of released semen, CSPZ = sperm concentration, SPZ = spermatozoa. * Different letters on the same line indicate P-value = 0.0288 according to a T-test.
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was kept constant and was monitored using a digital thermometer (maximum and minimum) for the three systems. In both experiments, the oocytes were carefully placed in eppendorf tubes (2.0 mL), which were closed and then submitted to the respective temperature control systems. In the first experiment, the tubes were kept in styrofoam racks and in the second, they were directly submerged in water at the requisite temperature. After storage, the oocytes from each eppendorf tube were weighed (0.5 and 1.0 g for the first and second experiment, respectively) and were fertilized by the pool of semen that was kept at room temperature during the experiment. In the first experiment, the effect of the storage temperatures was determined based only on the estimates of oocyte fertilization rates. In the second experiment, the oocyte fertilization rates, egg-hatching rates and percentage of normal larvae were assessed. The percentage of fertilized eggs was estimated based on the number of eggs from each experimental unit 11 h after fertilization, which corresponded to the end of epibolic movement or closure of the blastoporus. Translucent eggs that showed an apparently normal embryonic development (Honji et al., 2012) were considered to be fertilized, and unfertilized eggs were opaque or white. In the second experiment, after the eggs had been counted, they were returned to the incubators for hatching, which allowed the hatching rates to be estimated, based on the total number of eggs in each incubator. For this, all the newly hatched larvae (52.6 ± 4.64 h in water at 22.07 ± 0.28 ◦ C) from each experimental unit were anesthetized with benzocaine (50 mg/L) for 30 s for subsequent classification as normal or abnormal under a stereomicroscope (10×). The fertilization percentages obtained in the first experiment were submitted to a regression analysis of the response surface model. The influence of exposure time (T), and exposure temperature (TE) on the fertilization rates (FR) was assessed using the response surface model: FR = ∂0 + ∂1 T +∂2 TE++∂3 (T × TE) + ∂4 (T )2 + ∂2 (TE)2 + ε where ∂i = constants; ε = error with ∼N(0, ∂2 ). The non-significant higher-order parameters (P > 0.05) were removed progressively by the backward stepwise method. In the case of a significant effect of the response surface model, the partial derivatives of the statistical model were found to obtain a regression line for maximum
values in each exposure time. Apart from the regression analysis, a factorial analysis of variance (factorial ANOVA) at the same level of significance was performed for the fertilization rates obtained in the first experiment, and Duncan’s test for the comparison of means was applied when an effect was observed. The fertilization and hatching rates and the number of normal larvae obtained in the second experiment were submitted to a one-factor analysis of variance (one-way ANOVA) at the 5% significance level. For this, the combination of temperature and exposure time was considered as a factor. In the case of a significant effect, Duncan’s test for the comparison of means was applied at the same level of significance. The seminal parameters obtained at the beginning and end of each experiment were submitted to a t-test at the 5% significance level. The statistical analysis was performed by the software Statistica© . The assumptions were confirmed on the residues as suggested by Myers (1990) and Quinn and Keough (2002). Q2 3. Results In the first experiment, the females released 29.44 ± 13.38 g of oocytes, with 278 oocytes/g, corresponding to 6145 ± 2200 oocytes/kg of female. In the second experiment, the females released 64.21 ± 3.60 g of oocytes, with 294 oocytes/g, corresponding to 7752 ± 1103 oocytes/kg of female. For the sperm parameters, an effect of time after collection (P = 0.0288) was observed only for the curvilinear velocity in the first experiment (Table 1). The effect of exposure time at room temperature was not verified (P > 0.05) for the other sperm parameters (Table 1). The fertilization rates obtained in the first experiment showed a quadratic effect for exposure time (F(1,34) = 13.03, P < 0.01) and temperature (F(1,34) = 31.78, P < 0.01) (Fig. 1). The theoretical temperature that provided the highest results was 17.25 ◦ C from the beginning (time zero) to the end of exposure (355 min) (Fig. 1). In the first experiment, the fertilization rate obtained for the tested periods was different from that in the control treatment (F(6,26) = 29.24, P < 0.01). However, a more pronounced reduction in oocyte quality was evident at extreme temperatures (5 and 35 ◦ C) (Fig. 2). Despite the decrease in fertilization after 115 min at 28 ◦ C, the
Please cite this article in press as: Sanches, E.A., et al., Storage of Steindachneridion parahybae oocytes at different temperatures. Anim. Reprod. Sci. (2014), http://dx.doi.org/10.1016/j.anireprosci.2014.09.022
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Fig. 1. The fertilization rates (Fert) of Steindachneridion parahybae oocytes in relation to the exposure temperature for 355 min. Left—3D graphical representation; right—2D graphical representation.
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temperature of 15 ◦ C provided higher rates at 235 and 355 min (Fig. 2). In the second experiment, the effects of time and temperature on fertilization (F(9,20) = 2.64, P = 0.03) (Fig. 3), hatching (F(9,20) = 2.44, P = 0.05) (Fig. 4) and number of normal larvae (F(9,20) = 2.41, P = 0.05) (Fig. 5) were confirmed. During exposure, variation in the fertilization rate as a function of the different temperatures tested was observed (Fig. 3), with values statistically similar to those in the control treatment only after 80 min of exposure to 14 ◦ C and after 110 min at 17 ◦ C (Fig. 3). With the exception of the hatching rates obtained for oocytes exposed for 110 min at 14 ◦ C, which were lower (P = 0.0467) than in the control treatment, the other hatching rates were similar (P > 0.05) to that in the controls (Fig. 4). A temperature of 14 ◦ C resulted in the highest percentage of defective larvae after 80 min of exposure (P = 0.0489).
Fig. 2. Fertilization rates of Steindachneridion parahybae oocytes in relation to the exposure temperature for 355 min. Different letters in columns indicate P < 0.05 according to Duncan’s test for the comparison of means.
Fig. 3. Fertilization rates of Steindachneridion parahybae oocytes exposed to three temperatures for 110 min. Different letters in columns indicate P < 0.05 according to Duncan’s test for the comparison of means.
Fig. 4. Hatching rates from Steindachneridion parahybae oocytes exposed to three temperatures for 110 min. Different letters in columns indicate P < 0.05 according to Duncan’s test for the comparison of means.
Please cite this article in press as: Sanches, E.A., et al., Storage of Steindachneridion parahybae oocytes at different temperatures. Anim. Reprod. Sci. (2014), http://dx.doi.org/10.1016/j.anireprosci.2014.09.022
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Fig. 5. Rates of normal larvae obtained from Steindachneridion parahybae oocytes exposed to three temperatures for 110 min. Different letters in columns indicate P < 0.05 according to Duncan’s test for the comparison of means.
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However, at 17 and 20 ◦ C, the values were the same for the time periods tested (P > 0.05) (Fig. 5).
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The oocyte production obtained in the present experiment was lower than that found by Caneppele et al. (2009) for S. parahybae of between 9000 and 10,000 oocytes/kg of female. However, the amount of oocytes produced by South American native fish can vary according to the time of year, stage of maturation, nutritional stage and the hormones used in the broodstocks (Romagosa, 2008, 2010). The semen production and sperm concentration were similar to those observed by Caneppele (2011) for non-hormonally induced specimens of S. parahybae, with 9.39 ± 1.19 mL of released semen and a concentration of 9.39 × 109 ± 0.88 × 109 spermatozoa/mL. The sperm parameters evaluated were lower than those found by Sanches et al. (2013b) for S. parahybae with fresh semen, with 89.11 ± 7.41%, 107.23 ± 14.68 m/s, 77.08 ± 22.33 m/s for motility, curvilinear velocity and straight line velocity, respectively. However, these results probably did not influence fertilization, since even with lower values at the beginning of the second experiment, the fertilization rates of the control treatment were higher (85.37 ± 6.16%). Similarly, the reduction in curvilinear velocity in the first experiment 375 min after collection probably did not affect the fertilization rate. The fertilization rate in the first experiment decreased over time, and was highest immediately after stripping (20 min). When oocyte conservation is necessary, it should be performed according to the theoretical line of maximum fertilization. This line obtained in the first experiment suggests a theoretical temperature of 17.25 ◦ C as being optimal for the experimental time tested, which was corroborated by the results of the second experiment, where temperatures of 17 and 20 ◦ C provided better conditions for the exposure of fresh oocytes after 80 min. In the first experiment, 15 ◦ C caused a more rapid reduction in the fertilization rate than 28 ◦ C; however, when compared
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with the other temperatures, exposure to 15 ◦ C produced higher fertilization rates after 235 min of exposure. The temperature of 35 ◦ C caused a rapid fall in the fertilization rate, indicating that a greater degradation of the oocytes occurs at high temperatures, and consequently, a decrease in the fertilization rate. It is notable that gonadal maturation in S. parahybae occurs between November and March (Caneppele et al., 2009) and the mean air temperature in the region during this period can vary from 18 to 35 ◦ C. Thus, these results will be useful in laboratory procedures for the artificial propagation of the species. In other species of fish native to South America, some effects of the relationship between time and temperature on the ex-situ viability of oocytes were observed: For example, Salminus brasiliensis exhibited a reduction in oocyte viability after 30 min of storage at 22.8 ◦ C (Weingartner, 2010). Furthermore, Weingartner (2010) observed that the quality of oocytes obtained from different females influenced the time of ex-situ viability, because higher-quality oocytes were more resistant to the time of exposure. Rizzo et al. (2003) confirmed that a higher viability of Prochilodus marggravii oocytes was promoted at 26 ◦ C than at 18 ◦ C, as well as by ex-situ storage in comparison with that insitu. However, a decrease in the fertilization rate can occur after storage for 60 min at 26 ◦ C and 30 min at 18 ◦ C. Moreover, the authors observed that the decrease in viability at both temperatures was not related to the closure of the micropyle, but to changes in the spatial organization of the cytoskeletal filaments of the oocytes. These changes do not influence fertilization directly, but affect embryonic development and cause death. The different behavior of the ex-situ viability of fresh oocytes from various tropical species might be related to the different cytochemical composition of the oocytes of the different species (Bazzoli and Rizzo, 1990). In comparison with S. parahybae, Okawara (2012) noted that the in-situ storage of oocytes promoted a rapid decrease in their quality, thus, the stripping of females should be performed as soon as ovulation occurs. The results obtained in our study are therefore crucial when the ovulation of different females is not synchronized. In an attempt to maintain oocyte viability, producers often store them at room temperature or in refrigerators at temperatures below 5 ◦ C, leading to a rapid loss of viability. Different results of the short-term exposure of oocytes have also been observed in species of temperate fish. Suquet et al. (1999) found no influence of temperature on the fertilization rates of Psetta maxima oocytes exposed to three temperatures and different conditions with oxygen, atmospheric air and antibiotics,. However, the hatching rates were slightly higher at 13 and 8 ◦ C than at 3 ◦ C. For Cyprinus carpio (koi), Rothbard et al. (1996) observed over 50% of live embryos and larvae when oocytes were exposed to 22–24 ◦ C for 6 h, whereas survival did not exceed 30% at temperatures between 6 and 9 ◦ C. For Acipenser persicus oocytes, a storage temperature of 18 ◦ C was more favorable than 4 ◦ C (Sohrabnezhad et al., 2006). Despite these results, these studies showed some contradictions between the methodologies employed in the storage of oocytes from temperate and tropical species. Therefore, practical and objective investigations are necessary to establish the ideal storage condition for each species.
Please cite this article in press as: Sanches, E.A., et al., Storage of Steindachneridion parahybae oocytes at different temperatures. Anim. Reprod. Sci. (2014), http://dx.doi.org/10.1016/j.anireprosci.2014.09.022
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Ovarian fluid or artificial fluid with the addition of antibiotics have been used with relative success for the short-term preservation of Oncorhynchus mykiss eggs (Goetz and Coffman, 2000; Holcomb et al., 2005; Niksirat et al., 2007). However, the effect of those components on S. parahybae oocytes is still unknown and should be studied to increase ex situ viability. Sanches et al. (2011a) showed that oocytes of Rhamdia quelen (another neotropical catfish) exposed to 35 ◦ C were surrounded by a dense fluid, but became dehydrated when exposed to 5 ◦ C. The authors suggest that during exposure to 35 ◦ C, the oocytes might degenerate, closing the micropyle and preventing the entry of the spermatozoa. This dryness, in turn, might have caused changes in the cytoskeletal filaments and have affected fertilization, as observed by Rizzo et al. (2003). Considering that S. parahybae sperm maintains a satisfactory quality for 8 h of exposure to room temperature between 15 and 25 ◦ C (Sanches et al., 2013c), but oocytes rapidly lose their viability after collection, it is recommended that semen should be collected before the oocytes for the artificial reproduction of S. parahybae. These results are fundamental for artificial reproduction procedures of this species, and allow a better use of the fertilization material, especially in laboratories where the thermal amplitude during the reproductive period is wide. Furthermore, these results can be applied to short-distance transport, in situations where ovulation and spermiation are not synchronized, and in experimental routines with numerous simultaneous replications. Moreover, these results might also provide the basis for other studies with neotropical fish.
5. Conclusion Oocytes of the surubim-do-Paraiba, S. parahybae lose quality over time following collection. Therefore, artificial fertilization should be conducted immediately following collection. It is suggested that semen should collected before the oocytes. When oocyte storage is necessary, it should be conducted between 17 and 20 ◦ C.
Conflict of interest statement None.
Uncited reference Stasoft (2005).
Acknowledgements
The authors would like to thank the National Council for Scientific and Technological Development—CNPq 424 (Grant 478347/2009-0), the São Paulo Research Foun425 dation—FAPESP (Grant 2009/18609-6, 2010/02818-5 and 426 Q5 2013/17426-0) and the São Paulo Energy Company—CESP. 427 Q4 423
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Contents lists available at ScienceDirect
Animal Reproduction Science journal homepage: www.elsevier.com/locate/anireprosci
Storage of Steindachneridion parahybae oocytes at different temperatures
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Eduardo Antônio Sanches a,∗ , Renan Yoshiharu Okawara b , Danilo Caneppele c , Giovano Neumann d , Robie Allan Bombardelli d , Elizabeth Romagosa b a b c d
State University of São Paulo, UNESP, Registro, SP, Brazil Fishery Institute, APTA, São Paulo, SP, Brazil São Paulo Energy Company, CESP, Paraibuna, SP, Brazil State University of West Paraná, UNIOESTE, Toledo, PR, Brazil
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Article history: Received 27 March 2014 Received in revised form 23 September 2014 Accepted 30 September 2014 Available online xxx
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Keywords: Artificial fertilization Eggs Fish Gamete exposure Larvae Surubim-do-Paraiba
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1. Introduction
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The objective of this study was to assess the influence of temperature and time on the storage of fresh Steindachneridion parahybae oocytes. Two experiments were carried out: (1) the fertilization rates of oocytes exposed to temperatures of 5, 15, 28 (room temperature) and 35 ◦ C were assessed 15 min (control), 115, 235 and 355 min after release; (2) the fertilization and hatching rates, as well as the percentage of normal larvae of oocytes exposed to 14, 17 or 20 ◦ C, 20 min (control) were assessed 50, 80 and 110 min after stripping. In the first experiment, the highest fertilization rates (P < 0.05) were obtained in the control treatment (15 min, 28 ◦ C), with 74.34 ± 5.48% oocytes showing loss of viability over time. In the second experiment, there was a reduction (P < 0.05) in the fertilization rates at the temperatures and times tested. The artificial fertilization of S. parahybae oocytes is recommended immediately after collection, and if storage is necessary, it should be conducted at temperatures between 17 and 20 ◦ C. © 2014 Published by Elsevier B.V.
Artificial propagation is an important technique for intensification of fish production (Romagosa, 2006). Therefore, high quality gametes should be used to ensure maximum fertilization and subsequently, normal development of the embryo (Romagosa, 2008; Bobe and Labbe, 2010). One way to estimate the potential for development is to analyze some parameters such as fertilization, hatching and percentage of normal larvae (Brooks et al., 1997; Coward et al., 2002; Bobe and Labbe, 2010).
∗ Corresponding author. Tel.:+55 13 3828 2900x2930. E-mail addresses: [email protected], [email protected] (E.A. Sanches).
In general, there is little information in the literature on the ex situ viability of gametes from the neotropical fish fauna. The recommended in artificial reproduction procedure is the rapid mixing of gametes (immediately after collection), based on the theory that oocytes and semen lose viability over time (Sanches et al., 2011a, 2013a). Thus, it is important to know the mechanism of viability loss and to develop methods to ensure gamete longevity, to optimize the rational use of broodstocks and the techniques of artificial propagation (Rana, 1995; Babin et al., 2007; Fornari et al., 2011). Temperature is the main factor that affects the quality of oocytes stored in vitro (Sanches et al., 2011a). However, the extent of adverse temperature effects remains unclear for neotropical fish. A simple technique of short-term chilling can be easily applied with positive results in
http://dx.doi.org/10.1016/j.anireprosci.2014.09.022 0378-4320/© 2014 Published by Elsevier B.V.
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commercial rearing systems (Sanches et al., 2011a), allowing the maximum utilization of gametes, mainly when there is asynchrony of broodfish spawning following the use of hormonal therapies (Rana, 1995). Asynchrony in broodfish spawning is observed for the surubim-do-Paraíba, Steindachneridion parahybae (Siluriformes: Pimelodidae), a gray catfish endemic to the Paraíba do Sul river basin (Garavello, 2005), which is currently on the red list of Brazilian fauna threatened with extinction (MMA, 2008; IBGE, 2009). This species spawns at different times following hormonal induction (Okawara, 2012) and strategies for its artificial propagation should be adopted, including the management of gametes for in vitro fertilization. The objective of this study was to determine the effect of temperature and time on the storage of fresh S. parahybae oocytes.
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2. Material and methods
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The experiment was conducted at the Hydrobiology and Aquaculture Station of the São Paulo Energy Company–EHA/CESP, in the town of Paraibuna/SP, Brazil (23◦ 24 54 S; 45◦ 35 52 W), using wild S. parahybae broodfish (F0) and other broodfish originating from induced reproduction performed with wild specimens at the same station (F1). The fish were kept in two earthen ponds (200 m2 ) with concrete walls and a sandy bottom, and received extruded commercial feed for carnivorous fish with 40% crude protein at a rate of 5% biomass/week, offered twice-daily, at 08:00 and 16:00, three days per week. To assess the effects of temperature on the oocytes after stripping, two experiments were conducted: (1) four temperatures for up to 355 min; (2) three temperatures for up to 110 min. The second experiment was performed to complement the results of the first experiment. Broodfish that were able to spawn were selected in the pond during the reproductive period (Oct–Feb/2010–2011 and Oct–Feb/2011–2012), as proposed by Caneppele et al. (2009). Semen release was observed for males, and the females were evaluated according to external characteristics, such as a slightly rounded abdomen and the release of a small parcel of oocytes after gentle abdominal pressure. The selected broodfish were transferred to the laboratory, weighed and separated into aquaria (500-L) equipped with aeration, where they remained for hormonal manipulation and stripping (∼24 h). After the females were weighed, they were hormonally induced by injections of crude carp pituitary extract (CCPE) diluted in saline (0.9% NaCl), in two dosages (0.5 and 5.0 mg CCPE/kg), at an interval of 12 h (Caneppele et al., 2009). The males were not hormonally induced (Caneppele, 2011). The oocytes were collected only when they could be easily released after gentle abdominal pressure. The gametes were collected after 240 (9.8 h at 24.56 ± 0.44 ◦ C) and 300 degree-hours (11.9 h at 25.11 ± 3.39 ◦ C), for the first and second experiments, respectively (Caneppele et al., 2009). The gametes were collected after abdominal massage performed from head to tail (by stripping). The semen was collected 15 min before the oocytes, to estimate its quality (Sanches et al., 2011a).
A pool of oocytes collected from three females (two F0 and one F1; mean weight ± SD, 1400 ± 600 g), and semen collected from three F1 males (607 ± 32 g) were used for the first experiment. Another pool of oocytes from two F0 females (2450 ± 212 g) and semen from four F0 males (2133 ± 635 g) were used for the second experiment. To estimate the oocyte production/female, all collected oocytes were weighed. Sperm quality was estimated by computerized sperm analysis using the software IMAGEJ (National Institutes of Health, USA, http://rsbweb.nih.gov/ij/) with the CASA plugin (University of California Howard Hughes Medical Institute, USA, and http://rsbweb.nih.gov/ij/plugins/casa.html), as proposed by Wilson-Leedy and Ingermann (2007) and Sanches et al. (2010, 2013b). Motility rate, curvilinear velocity, straightline velocity and straightness were assessed twice, at the beginning (immediately after collection) and at the end of the period of oocyte exposure (375 and 120 min after collection for the first and second experiments, respectively). In addition to sperm motility, the volume of released semen and sperm concentration were assessed for each experiment. The sperm concentration was assessed using a Neubauer hematimetric counting chamber (Sanches et al., 2011b). A randomized experimental design in a factorial arrangement (4 × 3) was used for the first experiment, considering the exposure of the oocytes to temperatures of 5, 15, 28 (room temperature) and 35 ◦ C, 115, 235 or 355 min after collection. The control treatment consisted of artificial fertilization performed 15 min after stripping at room temperature (28 ◦ C). All the experimental combinations, which represented a total of 39 experimental units, were carried out in triplicate. One hatchery (1.5-L) containing 195 ± 38 oocytes fertilized with 50 L semen and 5 mL water from the hatching system (25.95 ± 1.72 ◦ C; pH 7.32 ± 0.17; 6.30 ± 1.00 mg dissolved oxygen/L) was considered as one experimental unit. The second experiment was conducted using the results from the first experiment. A randomized experimental design in a factorial arrangement (3 × 3) was used, with treatment temperatures of 14, 17 or 20 ◦ C and oocyte exposure times of 50; 80 or 110 min. The control treatment consisted of oocyte fertilization 20 min after stripping at room temperature (23 ◦ C). One hatchery (1.5-L) containing 321 ± 47 oocytes fertilized with 100 L semen and 10 mL water from the hatching system (22.07 ± 0.28 ◦ C; pH 6.96 ± 0.24; 6.89 ± 0.34 mg dissolved oxygen/L) was considered as one experimental unit. The equipment used to conduct each experiment differed. In the first experiment, two refrigerators containing thermostats (±1.0 ◦ C) were used as cooling systems, either at 5 ◦ C or at 15 ◦ C. Room temperature (inside the laboratory) was established at 28 ◦ C. A wooden box heated by an incandescent lamp (240 W) was used as a heating system, with a temperature of 35 ◦ C and a thermostat (±1.0 ◦ C). The temperature was frequently monitored by a mercury thermometer (maximum and minimum). In the second experiment, three water-cooling systems consisting of styrofoam boxes containing water and ice were used. The temperature
Please cite this article in press as: Sanches, E.A., et al., Storage of Steindachneridion parahybae oocytes at different temperatures. Anim. Reprod. Sci. (2014), http://dx.doi.org/10.1016/j.anireprosci.2014.09.022
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Table 1 Sperm and seminal parameters (means ± standard deviation) of Steindachneridion parahybae assessed at the beginning and the end of the two experiments. Parameters
MOT(%) VCL (m/s)* VSL (m/s) STR (%) VOL (mL) CSPZ (spz/mL)
Experiment 01
Experiment 02
Beginning
End 01
Beginning
End 02
65.23 ± 13.91 109.78 ± 9.27a 73.18 ± 6.22 89.22 ± 2.25 7.63 ± 3.90 2.28 × 109
72.06 ± 8.58 88.05 ± 6.32b 60.42 ± 11.01 92.28 ± 1.54 10.15 ± 4.49 34.68 × 109
52.23 ± 18.55 62.13 ± 13.41 26.25 ± 6.07 83.71 ± 3.64
45.06 ± 13.95 64.70 ± 6.16 28.49 ± 6.72 82.25 ± 4.39
Beginning = immediately after collection; End 01 = analysis carried out 375 min after collection; End 02 = analysis carried out 120 min after collection. MOT = motility rates, VCL = curvilinear velocity, VSL = straight line velocity, STR = straightness, VOL = Volume of released semen, CSPZ = sperm concentration, SPZ = spermatozoa. * Different letters on the same line indicate P-value = 0.0288 according to a T-test.
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was kept constant and was monitored using a digital thermometer (maximum and minimum) for the three systems. In both experiments, the oocytes were carefully placed in eppendorf tubes (2.0 mL), which were closed and then submitted to the respective temperature control systems. In the first experiment, the tubes were kept in styrofoam racks and in the second, they were directly submerged in water at the requisite temperature. After storage, the oocytes from each eppendorf tube were weighed (0.5 and 1.0 g for the first and second experiment, respectively) and were fertilized by the pool of semen that was kept at room temperature during the experiment. In the first experiment, the effect of the storage temperatures was determined based only on the estimates of oocyte fertilization rates. In the second experiment, the oocyte fertilization rates, egg-hatching rates and percentage of normal larvae were assessed. The percentage of fertilized eggs was estimated based on the number of eggs from each experimental unit 11 h after fertilization, which corresponded to the end of epibolic movement or closure of the blastoporus. Translucent eggs that showed an apparently normal embryonic development (Honji et al., 2012) were considered to be fertilized, and unfertilized eggs were opaque or white. In the second experiment, after the eggs had been counted, they were returned to the incubators for hatching, which allowed the hatching rates to be estimated, based on the total number of eggs in each incubator. For this, all the newly hatched larvae (52.6 ± 4.64 h in water at 22.07 ± 0.28 ◦ C) from each experimental unit were anesthetized with benzocaine (50 mg/L) for 30 s for subsequent classification as normal or abnormal under a stereomicroscope (10×). The fertilization percentages obtained in the first experiment were submitted to a regression analysis of the response surface model. The influence of exposure time (T), and exposure temperature (TE) on the fertilization rates (FR) was assessed using the response surface model: FR = ∂0 + ∂1 T +∂2 TE++∂3 (T × TE) + ∂4 (T )2 + ∂2 (TE)2 + ε where ∂i = constants; ε = error with ∼N(0, ∂2 ). The non-significant higher-order parameters (P > 0.05) were removed progressively by the backward stepwise method. In the case of a significant effect of the response surface model, the partial derivatives of the statistical model were found to obtain a regression line for maximum
values in each exposure time. Apart from the regression analysis, a factorial analysis of variance (factorial ANOVA) at the same level of significance was performed for the fertilization rates obtained in the first experiment, and Duncan’s test for the comparison of means was applied when an effect was observed. The fertilization and hatching rates and the number of normal larvae obtained in the second experiment were submitted to a one-factor analysis of variance (one-way ANOVA) at the 5% significance level. For this, the combination of temperature and exposure time was considered as a factor. In the case of a significant effect, Duncan’s test for the comparison of means was applied at the same level of significance. The seminal parameters obtained at the beginning and end of each experiment were submitted to a t-test at the 5% significance level. The statistical analysis was performed by the software Statistica© . The assumptions were confirmed on the residues as suggested by Myers (1990) and Quinn and Keough (2002). Q2 3. Results In the first experiment, the females released 29.44 ± 13.38 g of oocytes, with 278 oocytes/g, corresponding to 6145 ± 2200 oocytes/kg of female. In the second experiment, the females released 64.21 ± 3.60 g of oocytes, with 294 oocytes/g, corresponding to 7752 ± 1103 oocytes/kg of female. For the sperm parameters, an effect of time after collection (P = 0.0288) was observed only for the curvilinear velocity in the first experiment (Table 1). The effect of exposure time at room temperature was not verified (P > 0.05) for the other sperm parameters (Table 1). The fertilization rates obtained in the first experiment showed a quadratic effect for exposure time (F(1,34) = 13.03, P < 0.01) and temperature (F(1,34) = 31.78, P < 0.01) (Fig. 1). The theoretical temperature that provided the highest results was 17.25 ◦ C from the beginning (time zero) to the end of exposure (355 min) (Fig. 1). In the first experiment, the fertilization rate obtained for the tested periods was different from that in the control treatment (F(6,26) = 29.24, P < 0.01). However, a more pronounced reduction in oocyte quality was evident at extreme temperatures (5 and 35 ◦ C) (Fig. 2). Despite the decrease in fertilization after 115 min at 28 ◦ C, the
Please cite this article in press as: Sanches, E.A., et al., Storage of Steindachneridion parahybae oocytes at different temperatures. Anim. Reprod. Sci. (2014), http://dx.doi.org/10.1016/j.anireprosci.2014.09.022
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Fig. 1. The fertilization rates (Fert) of Steindachneridion parahybae oocytes in relation to the exposure temperature for 355 min. Left—3D graphical representation; right—2D graphical representation.
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temperature of 15 ◦ C provided higher rates at 235 and 355 min (Fig. 2). In the second experiment, the effects of time and temperature on fertilization (F(9,20) = 2.64, P = 0.03) (Fig. 3), hatching (F(9,20) = 2.44, P = 0.05) (Fig. 4) and number of normal larvae (F(9,20) = 2.41, P = 0.05) (Fig. 5) were confirmed. During exposure, variation in the fertilization rate as a function of the different temperatures tested was observed (Fig. 3), with values statistically similar to those in the control treatment only after 80 min of exposure to 14 ◦ C and after 110 min at 17 ◦ C (Fig. 3). With the exception of the hatching rates obtained for oocytes exposed for 110 min at 14 ◦ C, which were lower (P = 0.0467) than in the control treatment, the other hatching rates were similar (P > 0.05) to that in the controls (Fig. 4). A temperature of 14 ◦ C resulted in the highest percentage of defective larvae after 80 min of exposure (P = 0.0489).
Fig. 2. Fertilization rates of Steindachneridion parahybae oocytes in relation to the exposure temperature for 355 min. Different letters in columns indicate P < 0.05 according to Duncan’s test for the comparison of means.
Fig. 3. Fertilization rates of Steindachneridion parahybae oocytes exposed to three temperatures for 110 min. Different letters in columns indicate P < 0.05 according to Duncan’s test for the comparison of means.
Fig. 4. Hatching rates from Steindachneridion parahybae oocytes exposed to three temperatures for 110 min. Different letters in columns indicate P < 0.05 according to Duncan’s test for the comparison of means.
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Fig. 5. Rates of normal larvae obtained from Steindachneridion parahybae oocytes exposed to three temperatures for 110 min. Different letters in columns indicate P < 0.05 according to Duncan’s test for the comparison of means.
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However, at 17 and 20 ◦ C, the values were the same for the time periods tested (P > 0.05) (Fig. 5).
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The oocyte production obtained in the present experiment was lower than that found by Caneppele et al. (2009) for S. parahybae of between 9000 and 10,000 oocytes/kg of female. However, the amount of oocytes produced by South American native fish can vary according to the time of year, stage of maturation, nutritional stage and the hormones used in the broodstocks (Romagosa, 2008, 2010). The semen production and sperm concentration were similar to those observed by Caneppele (2011) for non-hormonally induced specimens of S. parahybae, with 9.39 ± 1.19 mL of released semen and a concentration of 9.39 × 109 ± 0.88 × 109 spermatozoa/mL. The sperm parameters evaluated were lower than those found by Sanches et al. (2013b) for S. parahybae with fresh semen, with 89.11 ± 7.41%, 107.23 ± 14.68 m/s, 77.08 ± 22.33 m/s for motility, curvilinear velocity and straight line velocity, respectively. However, these results probably did not influence fertilization, since even with lower values at the beginning of the second experiment, the fertilization rates of the control treatment were higher (85.37 ± 6.16%). Similarly, the reduction in curvilinear velocity in the first experiment 375 min after collection probably did not affect the fertilization rate. The fertilization rate in the first experiment decreased over time, and was highest immediately after stripping (20 min). When oocyte conservation is necessary, it should be performed according to the theoretical line of maximum fertilization. This line obtained in the first experiment suggests a theoretical temperature of 17.25 ◦ C as being optimal for the experimental time tested, which was corroborated by the results of the second experiment, where temperatures of 17 and 20 ◦ C provided better conditions for the exposure of fresh oocytes after 80 min. In the first experiment, 15 ◦ C caused a more rapid reduction in the fertilization rate than 28 ◦ C; however, when compared
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with the other temperatures, exposure to 15 ◦ C produced higher fertilization rates after 235 min of exposure. The temperature of 35 ◦ C caused a rapid fall in the fertilization rate, indicating that a greater degradation of the oocytes occurs at high temperatures, and consequently, a decrease in the fertilization rate. It is notable that gonadal maturation in S. parahybae occurs between November and March (Caneppele et al., 2009) and the mean air temperature in the region during this period can vary from 18 to 35 ◦ C. Thus, these results will be useful in laboratory procedures for the artificial propagation of the species. In other species of fish native to South America, some effects of the relationship between time and temperature on the ex-situ viability of oocytes were observed: For example, Salminus brasiliensis exhibited a reduction in oocyte viability after 30 min of storage at 22.8 ◦ C (Weingartner, 2010). Furthermore, Weingartner (2010) observed that the quality of oocytes obtained from different females influenced the time of ex-situ viability, because higher-quality oocytes were more resistant to the time of exposure. Rizzo et al. (2003) confirmed that a higher viability of Prochilodus marggravii oocytes was promoted at 26 ◦ C than at 18 ◦ C, as well as by ex-situ storage in comparison with that insitu. However, a decrease in the fertilization rate can occur after storage for 60 min at 26 ◦ C and 30 min at 18 ◦ C. Moreover, the authors observed that the decrease in viability at both temperatures was not related to the closure of the micropyle, but to changes in the spatial organization of the cytoskeletal filaments of the oocytes. These changes do not influence fertilization directly, but affect embryonic development and cause death. The different behavior of the ex-situ viability of fresh oocytes from various tropical species might be related to the different cytochemical composition of the oocytes of the different species (Bazzoli and Rizzo, 1990). In comparison with S. parahybae, Okawara (2012) noted that the in-situ storage of oocytes promoted a rapid decrease in their quality, thus, the stripping of females should be performed as soon as ovulation occurs. The results obtained in our study are therefore crucial when the ovulation of different females is not synchronized. In an attempt to maintain oocyte viability, producers often store them at room temperature or in refrigerators at temperatures below 5 ◦ C, leading to a rapid loss of viability. Different results of the short-term exposure of oocytes have also been observed in species of temperate fish. Suquet et al. (1999) found no influence of temperature on the fertilization rates of Psetta maxima oocytes exposed to three temperatures and different conditions with oxygen, atmospheric air and antibiotics,. However, the hatching rates were slightly higher at 13 and 8 ◦ C than at 3 ◦ C. For Cyprinus carpio (koi), Rothbard et al. (1996) observed over 50% of live embryos and larvae when oocytes were exposed to 22–24 ◦ C for 6 h, whereas survival did not exceed 30% at temperatures between 6 and 9 ◦ C. For Acipenser persicus oocytes, a storage temperature of 18 ◦ C was more favorable than 4 ◦ C (Sohrabnezhad et al., 2006). Despite these results, these studies showed some contradictions between the methodologies employed in the storage of oocytes from temperate and tropical species. Therefore, practical and objective investigations are necessary to establish the ideal storage condition for each species.
Please cite this article in press as: Sanches, E.A., et al., Storage of Steindachneridion parahybae oocytes at different temperatures. Anim. Reprod. Sci. (2014), http://dx.doi.org/10.1016/j.anireprosci.2014.09.022
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Ovarian fluid or artificial fluid with the addition of antibiotics have been used with relative success for the short-term preservation of Oncorhynchus mykiss eggs (Goetz and Coffman, 2000; Holcomb et al., 2005; Niksirat et al., 2007). However, the effect of those components on S. parahybae oocytes is still unknown and should be studied to increase ex situ viability. Sanches et al. (2011a) showed that oocytes of Rhamdia quelen (another neotropical catfish) exposed to 35 ◦ C were surrounded by a dense fluid, but became dehydrated when exposed to 5 ◦ C. The authors suggest that during exposure to 35 ◦ C, the oocytes might degenerate, closing the micropyle and preventing the entry of the spermatozoa. This dryness, in turn, might have caused changes in the cytoskeletal filaments and have affected fertilization, as observed by Rizzo et al. (2003). Considering that S. parahybae sperm maintains a satisfactory quality for 8 h of exposure to room temperature between 15 and 25 ◦ C (Sanches et al., 2013c), but oocytes rapidly lose their viability after collection, it is recommended that semen should be collected before the oocytes for the artificial reproduction of S. parahybae. These results are fundamental for artificial reproduction procedures of this species, and allow a better use of the fertilization material, especially in laboratories where the thermal amplitude during the reproductive period is wide. Furthermore, these results can be applied to short-distance transport, in situations where ovulation and spermiation are not synchronized, and in experimental routines with numerous simultaneous replications. Moreover, these results might also provide the basis for other studies with neotropical fish.
5. Conclusion Oocytes of the surubim-do-Paraiba, S. parahybae lose quality over time following collection. Therefore, artificial fertilization should be conducted immediately following collection. It is suggested that semen should collected before the oocytes. When oocyte storage is necessary, it should be conducted between 17 and 20 ◦ C.
Conflict of interest statement None.
Uncited reference Stasoft (2005).
Acknowledgements
The authors would like to thank the National Council for Scientific and Technological Development—CNPq 424 (Grant 478347/2009-0), the São Paulo Research Foun425 dation—FAPESP (Grant 2009/18609-6, 2010/02818-5 and 426 Q5 2013/17426-0) and the São Paulo Energy Company—CESP. 427 Q4 423
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