Growth and reproductive characteristics of Rhamdia quelen males fed on different digestible energy levels in the reproductive phase

Aquaculture 326-329 (2012) 74–80

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Growth and reproductive characteristics of Rhamdia quelen males fed on different digestible energy levels in the reproductive phase Lucélia Tessaro a,⁎, Cesar Pereira Rebechi Toledo a, Giovano Neumann a, Ricardo Andrei Krause a, Fábio Meurer b, Maria Raquel Marçal Natali c, Robie Allan Bombardelli a a b c

Universidade Estadual do Oeste do Paraná, Rua da faculdade, 645, Jardim Santa Maria, Toledo, Paraná, CEP: 85903-000, Brazil Universidade Federal do Paraná, Rua Pioneiro, 2153, Palotina, Paraná, CEP: 85950-000, Brazil Universidade Estadual de Maringá, Av. Colombo, 5.790, Jd. Universitário, bloco H79, Maringá, Paraná, CEP 87020-900, Brazil

a r t i c l e

i n f o

Article history: Received 5 July 2011 Received in revised form 9 November 2011 Accepted 10 November 2011 Available online 19 November 2011 Keywords: CASA Histology Liver Nutrition Reproduction Semen

a b s t r a c t The growth, hepatocyte morphology, gonadal maturation and semen and sperm characteristics of silver catfish (Rhamdia quelen) were assessed. Males were fed for 210 days with food pellets containing 30% digestible protein (DP) and digestible energy (DE) corresponding to 2850, 3100, 3350, 3600, and 3850 kcal DE kg diet− 1. The length, weight, weight gain, and condition factor were evaluated. Between October and February hormones were administrated (carp pituitary extract) and semen was collected to evaluate the semen volume, semen pH, sperm motility, sperm velocity, sperm survival, sperm concentration and sperm normality. In February, six animals from each treatment were euthanized and dissected to measure the viscerosomatic, hepatosomatic and gonadosomatic indices, the histological maturation stage and the average hepatocyte area. Monthly changes were observed only (pb 0.05) in seminal parameters, indicating the occurrence of a reproductive peak in November and December. Food pellets containing 30% digestible protein and 2850 kcal DE kg diet− 1 may be used as a male silver catfish diet without a loss of growth or reproductive performance. © 2011 Elsevier B.V. All rights reserved.

1. Introduction The silver catfish (Rhamdia quelen) is a siluriform fish widely distributed throughout South America (Gomes et al., 2000). This fish is a potential species to fish farming by presenting fast growth and accepting artificial diets (Parra et al., 2008). Furthermore, it has a high carcass yield and its meat is appreciated, for its taste and texture (Baldisseroto, 2009). Despite presenting these characteristics, this species is generally produced below their potential due to a lack of knowledge of their biological responses to rearing techniques (Parra et al., 2008). Nutrition and reproduction are highly related (Bombardelli et al., 2010; Schneider, 2004), and the quality of offspring is directly associated with broodstock nutrition (Bombardelli et al., 2009; Izquierdo et al., 2001). Investigations of gamete quality are usually related to the oocytes and fewer studies are carried out to the male gametes (Bombardelli et al., 2010). However, semen and sperm quality also deserve attention because they have a great influence on the production of healthy larvae (Rurangwa et al., 2004).

⁎ Corresponding author at: Rua 25 de julho 565, Catanduvas, PR, Brazil. Tel.: + 55 45 88044691. E-mail addresses: [email protected] (L. Tessaro), [email protected] (C.P.R. Toledo), [email protected] (G. Neumann), [email protected] (R.A. Krause), [email protected] (F. Meurer), [email protected] (M.R.M. Natali), [email protected] (R.A. Bombardelli). 0044-8486/$ – see front matter © 2011 Elsevier B.V. All rights reserved. doi:10.1016/j.aquaculture.2011.11.012

Studies related to the digestible energy requirements of fish have been performed for different species and rearing phases (Boscolo et al., 2005; Navarro et al., 2006, 2007; Pezzato et al., 2004). In studies with silver catfish broodstocks, Reidel et al. (2010) suggest that better protein value to females is 35% of crude protein associated to lower energy levels. However, precise information regarding the digestible energy requirements to silver broodstock is scarce. The diet of the animal influences both sperm (Bombardelli et al., 2010; Vassalo-Agius et al., 2001) and semen (Ferrell, 1991; Watanabe and Vassalo-Agius, 2003) production and quality. The diet energy levels have also been shown to promote changes in the sperm concentration and normality of Nile tilapia (Bombardelli et al., 2010). For silver catfish, the ration energy levels have been reported to influence the duration of sperm motility after activation (Sanches et al., 2006a). Despite knowledge about the important relationship between nutrition and reproduction in fish (Izquierdo et al., 2001), there is little information available to date regarding Neotropical species. The goal of this study was to evaluate the effect of digestible energy levels on animal performance, reproductive parameters, and the histological aspects of liver and testicular tissue in male silver catfish (R. quelen) during their reproductive period. 2. Materials and methods The experiment was conducted at the Laboratory of Reproductive Technology for Farmed Aquatic Animal, at the Institute for

L. Tessaro et al. / Aquaculture 326-329 (2012) 74–80

Environmental Aquaculture Research at the State University of west Paraná (Toledo, Paraná, Brazil), and the Laboratory of Animal Histotechnology at the State University of Maringá (Maringá, Paraná, Brazil). The study was conducted over 210 days, from September 2009 to April 2010. To simulate the business models employed in the farming of this species, males or females were reared together. A total of 150 males (35.37 ± 10.42 g; 16.5 ± 1.70 cm) and 210 females (33.26 ± 9.62 g; 15.92± 1.49 cm) of silver catfish (R. quelen) were included. The fish were stocked in 15 tanks (area of 16 m2), under natural temperature and photoperiod conditions. The water temperature in the experimental tanks was measured daily using a mercury thermometer (±0.1 °C). Every two weeks the pH (Tecnal® Tec 5) and dissolved oxygen content (YSI® 550A) were measured (Reidel et al., 2010) at 6:00 and 16:00 h (Bombardelli et al., 2010). The experiment was performed as a randomized complete design with five treatments and three replications. Each tank corresponded to an experimental unit. The animals were fed for 210 days with isoproteic diets containing 30% digestible protein (DP) and five different digestible energy (DE) levels, corresponding to 2850, 3100, 3350, 3600, and 3850 kcal DE kg − 1 diet (Table 1). Prior to the diet formulation, the ingredients were evaluated for nutrient composition. The digestible protein and energy values were calculated according to Oliveira Filho and Fracalossi (2006). Food rations were ground, mixed and pelleted (3 mm in diameter) according to Meurer et al. (2005) and Bombardelli et al. (2010). The fish were fed twice a day (10:00 h and 17:00 h) based on a feeding rate of 2% biomass each day, adjusted every two weeks. At the beginning and the end of the experimental period, the weight and length of the animal were individually measured and further used to calculate the standard length, body weight, weight gain and allometric condition factor (K = Wt / Ls b, where Wt = weight total, Ls = length standard, and b = the slope of the regression between Wt / Ls) (Vazzoler, 1996). The semen and sperm parameters were evaluated monthly from October 2009 to February 2010. Males were randomly selected from each experimental unit which released semen under slight abdominal pressure (Bombardelli et al., 2006).

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These fishes were individually weighed and placed in 1000-L tanks in water recirculation system, equipped with aeration and water temperature control (24 ± 1.0 °C). Reproductive manipulation consisted of hormonal induction with carp pituitary extract (CPE) (Woynarovich and Horvath, 1983), which was applied intramuscularly to the dorsal region as a single dose containing 2.5 mg CPE per kg of the animal (Bombardelli et al., 2006). After a period of 240 degree–hours (10 h, water at 24 °C) from the beginning of the hormonal application (Sanches et al., 2010a), sperm was collected from each individual, by cephalo-caudal abdominal massage (Sanches et al., 2011). The volume of semen released was measured using graduated test tubes with a precision of 0.1 mL. The semen pH was measured immediately after collection using litmus paper (Merck®). For these evaluations, 30-μL semen samples were used, and the color was compared to the colorimetric standard (Bombardelli et al., 2010) on a scale of 1–14. The semen was then refrigerated (±12 °C) during the time period required to perform other semen and sperm analyses (adapted from Asturiano et al., 2001). Sperm motility and swimming velocity (straight-line velocity, curvilinear velocity, and average path velocity) parameters were evaluated using computer-assisted sperm analysis (CASA), by open source software, adapted to the species by Sanches et al. (2010a). The images were captured in light microscope with 100× magnification. The sperm survival index was measured in light microscope by counting approximately 300 sperm cells stained by the eosin–nigrosin staining technique (magnification of 400×) (Fig. 1A) (Bombardelli et al., 2006, 2010; Kavamoto and Fogli da Silveira, 1986; Sanches et al., 2009). To measure the sperm concentration, the sperm cell count was performed in a Neubauer hematimetric chamber, in light microscope with 400× magnification (Wirtz and Steinmann, 2006).

Table 1 Ingredients and proximate composition of the experimental diets. Ingredients (%)

Energy level (kcal of DE kg ration− 1) 2.850

3.100

3.350

3.600

3.850

Fish meal Soybean meala Corna Soybean oila Inert Mineral and vitamin mixb Salt (NaCl) Antioxidant (BHT)

30.53 31.37 32.94 0.00 2.64 2.00 0.50 0.01

30.57 31.39 32.47 3.05 0.00 2.00 0.50 0.01

30.99 31.53 27.93 7.02 0.00 2.00 0.50 0.01

31.41 31.68 23.40 10.99 0.00 2.00 0.50 0.01

31.82 31.83 18.86 14.97 0.00 2.00 0.50 0.01

Proximate composition Crude energy (kcal kg) Crude fibre (%) Crude protein (%) Digestible protein (%) Mineral matter (%) Dry matter (%) Fat (%) Calcium (%) Linoleic acid Total lysine

4054.89 2.37 36.75 30.00 6.18 89.43 4.47 1.48 0.87 2.25

4326.91 2.34 36.75 30.00 6.18 89.51 7.48 1.48 2.51 2.25

4547.72 2.12 36.72 30.00 6.20 89.97 11.31 1.50 4.57 2.26

4768.52 1.89 36.69 30.00 6.23 90.43 15.13 1.52 6.63 2.27

4989.33 1.66 36.66 30.00 6.24 90.89 18.96 1.54 8.69 2.28

a

a

Digestible values to silver catfish according to Oliveira Filho and Fracalossi, 2006 Basic composition: folic acid: 200 mg, pantotenic acid: 4000 mg; biotin: 40 mg; Cu: 2000 mg; Fe: 12,500 mg; I: 200 mg; Mn: 7500 mg; niacin: 5000 mg; Se: 70 mg; vitamin A: 1,000,000 UI; vitamin B1: 1900 mg; vitamin B12: 3500 mg; vitamin B2: 2000 mg; vitamin B6: 2400 mg; vitamin C: 50,000 mg; vitamin D3: 500,000 UI; vitamin E: 20,000 UI; vitamin K3: 500 mg; Zn: 25,000 mg. b

Fig. 1. A) Survival index evaluation. L = live spermatozoa and →Dead spermatozoa. Gray scale, 400×. B) Silver catfish's morphology spermatozoa. N = normal spermatozoa; Nn = no normal spermatozoa. Gray scale, 400×.

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The sperm normality index (Rurangwa et al., 2004; Streit et al., 2005) was obtained by evaluating the semen samples fixed in a saline-buffered formalin solution. Spermatozoa were marked by Bengal rose staining technique (Hafez and Hafez, 2004; Streit et al., 2004) and were evaluated from samples placed on microscope slides and covered with coverslips, observed in 400× magnification (Sanches et al., 2010b). Approximately 300 spermatozoa were counted and classified as either “normal” or “no normal” (Fig. 1B) (CBRA, 1998; Chenoweth, 2005; Kavamoto et al., 1999; Streit et al., 2006). In February 2010, six males from each treatment were randomly selected and euthanized by dislocation of the cervical spine (CFMV, 2008), conforming to the experimental protocol #79/09 approved by the Ethics Committee for Animal Experimentation and Practical Classes (State University of west Paraná). Euthanized animals were dissected and the individual body, viscera, liver, and gonads were weighed immediately after dissection. From these data, the viscerosomatic (VSI), hepatosomatic (HSI) (Bombardelli et al., 2009, 2010), and gonadosomatic indices were calculated (GSI) (Vazzoler, 1996). The testes and livers of two males from each experimental unit (n= 6 for each treatment) were then separated for histological evaluation. The collected organs were fixed with Bouin's solution for 24 h and then transferred to a 70% alcohol solution. For processing, the material was dehydrated through increasing alcohol concentrations, cleared in xylene, and embedded in paraffin to obtain semi-serial transverse sections with a thickness of 5 μm and 7 μm for the testis and liver, respectively. The sections were stained with hematoxylin–eosin (HE). Approximately five sections of each testis were analyzed using an optical microscope and classified according to the gonadal maturation stage (adapted from Ghiraldelli et al., 2007; Reidel et al., 2010), and the percentage of mature males were calculated. Images of random fields were captured from the liver sections in regions adjacent to blood vessels, where the individual areas of 200 hepatocytes were measured, with Image Pro plus® software. With the obtained values, the average hepatocyte area for each animal was calculated. In the data evaluation, the results for the average weight gain, average length, average final weight, and condition factor were evaluated by one-way ANOVA. As for the male reproductive parameters, only the pH was assessed by bi-factorial variance analysis (month and DE level in the diet) because variability was not observed in all of the months. The other variables (semen volume, survival, concentration, morphology, and sperm normality) were subjected to repeated measures ANOVA, considering the energy levels and months evaluated. Index data, such as motility, survival, and sperm normality, were arcsine square root transformed. The velocity data (velocity average path, velocity

straight-line and curvilinear velocity) were submitted to principal component analysis (PCA) and correlated. The verified correlation, generated a common factor that was subjected to repeated measures ANOVA, as with the other parameters. The percentage of mature males and the average hepatocyte area were subjected to one-way ANOVA. Differences between means were assessed by Tukey's multiple comparison procedure with 5% significance (Gotelli and Ellison, 2004). Data were processed in the Statistica 7.0® software. 3. Results The physical and chemical parameters of the water were the same between treatments (p > 0.05). The temperature during the experimental period was 22.4 ± 2.1 °C, and the amount of dissolved oxygen was 4.31 ± 2.1 mg/L and the pH of the water was 6.8 ± 0.4. There was no significant effect (p > 0.05) between treatments for the standard length, body weight or weight gain parameters (Table 2). The same was observed for the final condition factor, the somatic indices and the average hepatocyte area that was not affected (p > 0.05) by the diets energy levels (Table 2). In relation to the reproductive aspects, the milt samples had a white and slimy appearance. The variance analysis showed that the semen volume and pH, along with the sperm parameters of motility, velocity, concentration, survival, and normality, were not influenced by the energy levels in diets (p> 0.05) (Table 3). However, except for the semen pH and sperm concentration (p> 0.05), other semen and sperm variables were changed (pb 0.05) throughout the reproductive period (Table 4). The highest semen productions (volume) were observed in December and January (Table 4). The highest normality values were observed during November and February (p b 0.05) (Table 4). The sperm survival index that varied among the different months showed better average in December (p b 0.05). The motility observed was the lowest (pb 0.05) in October, followed by higher values in November and December (Table 4). In January, a decrease (pb 0.05) in the percentage of motile cells was observed. The lowest rates were recorded in February (pb 0.05) (Table 4). Among the sperm motility parameters, three types of velocity were evaluated, the curvilinear velocity (VCL), straight-line velocity (VSL), and average path velocity (VAP), which appeared to be highly correlated (r = 78, r = 94, and r = 94) and, therefore, were grouped and analyzed by principal component analysis (PCA). The resulting factor was a denominated linear combination of sperm velocity given by the following equation: SV ¼ −ð0:566ÞVCL–0:598ðVAPÞ–0:566ðVSLÞ;

Table 2 Growth performance, somatic indices and average hepatocyte area of male silver catfish (Rhamdia quelen) fed on diets with different digestible energy levels. Variable

IW (g) IL (cm) ICF FW (g) FL (cm) FCF WG (g) VSI (%) HSI (%) GSI (%) 2

Hp (μm )

Digestible energy level (kcal DE kg diet− 1) 2.850

3.100

3.350

3.600

3.850

p

35.54 ± 0.86 12.95 ± 0.13 6.98 ± 3.27 191.86 ± 21.3 21.21 ± 0.52 1.49 ± 0.44 156.0 ± 11.41 10.85 ± 1.28 (n = 6) 0.95 ± 0.06 (n = 6) 7.32 ± 1.28 (n = 6) 65.07 ± 3.56

36.64 ± 0.54 13.06 ± 0.10 5.02 ± 2.06 196.29 ± 20.72 21.05 ± 0.32 2.68 ± 0.9 159.66 ± 12.41 9.89 ± 1.22 (n = 6) 0.86 ± 0.05 (n = 6) 5.29 ± 1.2 (n = 6) 62.81 ± 3.13

35.06 ± 0.73 13.03 ± 0.09 10.02 ± 6.74 151.58 ± 44.21 19.63 ± 0.84 1.69 ± 0.19 116.51 ± 24.99 9.81 ± 0.66 (n = 6) 0.86 ± 0.05 (n = 6) 4.74 ± 0.68 (n = 6) 58.82 ± 2.47

35.01 ± 0.49 12.99 ± 0.24 6.53 ± 0.14 166.57 ± 14.41 20.11 ± 0.15 1.28 ± 0.53 131.56 ± 8.79 9.72 ± 0.95 (n = 6) 0.79 ± 0.05 (n = 6) 4.73 ± 1.16 (n = 6) 55.32 ± 1.89

35.14 ± 0.52 12.96 ± 0.19 9.19 ± 1.53 149.89 ± 32.51 19.44 ± 0.58 2.43 ± 1.9 114.74 ± 19.63 9.24 ± 0.67 (n = 6) 0.77 ± 0.03 (n = 6) 4.87 ± 0.58 (n = 6) 53.34 ± 4.43

0.49 0.99 0.85 0.21 0.13 0.82 0.24 0.84 0.08 0.35 0.13

Mean ± standard error. IW = initial weight; IL = initial standard length; ICF = initial condition factor; FW = final weight; FL = final standard length; FCF = final condition factor; WG = weight gain; VSI = viscerosomatic index; HSI = hepatosomatic index; GSI = gonadosomatic index; Hp = average hepatocyte area.

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Table 3 Semen and sperm parameters and percentage of mature males of silver catfish (Rhamdia quelen) fed on diets with different digestible energy levels. Variable

Mat (%) pH SV (mL kg− 1) Nor (% Sur (%) Mot (%) Vel (μm s− 1) Conc (spz mL− 1)

Digestible energy in diet (kcal DE kg diet− 1) 2.850

3.100

3.350

3.600

3.850

p

50 ± 0.00 8.02 ± 0.02 58.77 ± 7.37 70.4 ± 4.66 95.9 ± 1.5 73.43 ± 6.41 53.72 ± 2.21 3.35 × 1010 ± 2.45 × 1009

66.67 ± 16.67 8.0 ± 0.00 56.80 ± 8.34 72.56 ± 2.37 98.05 ± 0.39 84.96 ± 3.56 57.96 ± 1.99 4.27 × 1010 ± 3.48 × 1009

66.67 ± 16.67 7.98 ± 0.02 55.34 ± 6.74 72.62 ± 3.03 97.75 ± 0.36 83.4 ± 3 58.28 ± 2.03 3.75 × 1010 ± 3.69 × 1009

66.67 ± 33.33 8.06 ± 0.04 45.55 ± 7.81 70.48 ± 2.69 97.82 ± 0.79 80.0 ± 4.5 46.73 ± 1.7 3.93 × 1010 ± 4.04 × 1009

66.67 ± 16.67 8.02 ± 0.02 50.13 ± 6.53 71.41 ± 2.82 98.51 ± 0.3 76.9 ± 4.88 45.57 ± 1.34 3.38 × 1010 ± 3.591009

0.96 0.26 0.47 0.89 0.08 0.11 0.27 0.41

Mean ± standard error. Mat = mature males; SV = relative semen volume; Nor = sperm normality; Sur = sperm survival index; Mot = sperm motility; Vel = sperm velocity; Conc = sperm concentration; spz = spermatozoa.

where SV = the sperm velocity; VCL = the curvilinear velocity; VAP = the average path velocity; and VSL = the straight-line velocity. The diet energy levels had no effect on the sperm velocity (p > 0.05) (Table 3); however, the monthly variation was significant (p b 0.05) and January and February showed the lowest values (Table 4). The last evaluated aspect was the gonadal development stage. Macroscopically, the testis occurred as paired organs that were large and whitish and that contained many projections (Fig. 2). Cysts were observed to differentiate at various stages of maturation (spermatogonia, spermatocyte, spermatid, and spermatozoa) and to be randomly distributed throughout the testis (Fig. 2). Most of the evaluated animals were classified as “mature,” and the maturation stage was not influenced (p > 0.05) by the diet energy levels (Table 3). The predominance of animals at this stage (mature) (Table 3) explains the abundance of spermatozoa and the loosening of the tissue around the cysts (Fig. 2). Due to the period of the collection (February), “immature” animals or a gonadal “regression” stage and the resorption of cysts or residual sperm were not observed. 4. Discussion During the experimental period, the physical and chemical parameters on the water remained within the recommended to silver catfish (Baldisseroto and Radünz Neto, 2004). The energy levels tested did not affect the animal performance to silver catfish males. In another species and other rearing phases, Bomfim et al. (2005) found no effect of their tested energy levels on animal performance in curimbatá (Prochilodus affins) fingerlings. In contrast, Navarro et al. (2010) reported a quadratic effect of the energy level on the body weight (average values from 9.45 to 17.45 g) in females of “piavuçu” (Leporinus macrocephalus), but their final length was unaffected. In investigations with broodstock, Bombardelli et al. (2009, 2010) found no effect of different energy levels on the animal performance of Nile tilapia (Oreochomis niloticus) and Sanches et al. (2006b),

using increasing levels of digestible energy, in isoproteic diets, found no effect on the animal or reproductive performance of silver catfish. About the somatic indices, there was no observed effect on the diet on these parameters. The gonadosomatic index and condition factor are commonly used as parameters that are indicative of reproductive activity (Andrade et al., 2010; Vazzoler, 1996). For silver catfish, Barcellos et al. (2002) observed that an increase in the gonadosomatic index was related to the peak gonadal maturation in the animals, in which an increase in the testicular mass coincided with increased testosterone production. This increase in the testicular mass has been attributed to the proliferation of the germinal epithelium (Schulz et al., 2010). Also, hepatosomatic index and hepatocyte area were not affected by the energy levels, however, it has been described that liver changes in farmed fish are promoted by the diet. For example, increases in the cell diameter of hepatocytes (Caballero et al., 1999), the presence of intracellular vacuoles and the occurrence of steatosis (Bolla et al., 2011; Caballero et al., 2004) have been reported. Furthermore, Bombardelli et al. (2010), in testing energy levels for broodstock, found a direct proportional relationship between an increase in the diet energy levels and lipid deposition in the hepatocytes of male Nile tilapia (O. niloticus). In this manner, other studies must be led to clarify the relation of the diet on the hepatic metabolism in males of this species. The milt characteristics observed, after collection, were also observed by Borges et al. (2005) in the same species. In relation to the semen volume, the values observed in this experiment were higher than those obtained by Carneiro and Mikos (2008), who tested several hormonal inducers and obtained values ranging from 4.0 to 38.8 mL semen kg − 1 of fish. Under the same hormonal induction protocol used in the present study, Bombardelli et al. (2006) observed a volume of 36.0 ± 8.0 mL of semen kg − 1 in males. The variation in sperm production may be related to factors such as hormonal induction protocol (Godinho, 2007; Viveiros et al., 2002), season, age and reproductive maturation of the individual.

Table 4 Values of the semen and sperm parameters of silver catfish (Rhamdia quelen) in the reproductive phase. Parameters

Month

p

October

November

December

January

February

n pH SV (mL kg− 1) Conc (spz.mL− 1) Nor (%) Sur (%) Mot (%) Vel (μm s− 1)

15 nc 15.94 ± 2.61c 3.83 × 1010 ± 5.35 × 109 56.25 ± 4.16d 98.5 ± 0.24ab 74.47 ± 6.23c 50.32 ± 2.09b

15 8±0 40.76 ± 4.40b 3.96 × 1010 ± 4.36 × 109 79.99 ± 1.06ab 95.53 ± 1.45b 93.52 ± 0.85a 53.43 ± 1.89a

30 8.04 ± 0.03 76.94 ± 5.44a 4.33 × 1010 ± 2.35 × 109 68.8 ± 1.28c 99.03 ± 0.29a 90.24 ± 2.34ab 53.39 ± 2.09a

60 8.03 ± 0.03 75.4 ± 4.06a 3.17 × 1010 ± 1.89 × 109 71.72 ± 1.19bc 97.96 ± 0.34ab 77.9 ± 3.22bc 53.16 ± 2.15b

60 8±0 57.57 ± 4.70b 3.4 × 1010 ± 1.61 × 109 80.71 ± 1.47a 97.02 ± 0.79ab 62.57 ± 3.62c 51.97 ± 1.94b

– 0.58 0.00 0.08 0.00 0.00 0.00 0.00

Mean ± standard error. The same letters represent similar means according to Tukey's test (p ≤ 0.05). nc = no collected data. SV = relative semen volume; Conc = sperm concentration; spz = spermatozoa; Nor = sperm normality; Sur = sperm survival index; Mot = sperm motility; Vel = sperm velocity.

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Fig. 2. Silver catfish testis. H.E. A: projections of testes; B: cysts with spermatozoa (SPZ) and rupture to envoltory (Δ) 400×; C: differentiation of cysts: spermatocytes (SPC), spermatids (SPT) and spermatozoa (SPZ), 400×; D: spermatogonia (SPG) 1000×.

Other semen parameter evaluated was the seminal pH, and this did not show effect on the diet or on the period. The seminal pH is directly involved with the onset of sperm activation (Alavi and Cosson, 2005). In rainbow trout (Oncorhynchus mykiss), the extracellular pH (seminal plasma) controls, in part, the sperm activation by interfering with the ATP hydrolytic activity (Woolsey and Ingermann, 2003). Thus, it is important that formulated diets do not modify the specific pattern of the semen pH. The sperm parameters were affected by the experimental period. The average sperm concentration in the treatments ranged from 3.35 × 1010 ± 2.45 × 109 to 4.27 × 1010 ± 3.48 × 109 spermatozoa mL− 1 (Table 3). Normally, the sperm concentration is reduced by hormonal induction due to the increase in semen volume (Godinho, 2007). Indeed, Borges et al. (2005) observed that wild silver catfish (without hormonal induction) have sperm concentrations of 5.0 ± 1.2 × 10 10 and 6.6 ± 3.6 × 10 10 spermatozoa mL− 1 of semen during winter and spring, respectively. For animals bred in captivity and hormonally induced, the values reported are below 2 × 10 10 spermatozoa mL− 1 of semen (Bombardelli et al., 2006; Sanches et al., 2006a) and up to 4.5 ± 1.57 × 1010 spermatozoa mL− 1 of semen (Sanches et al., 2010a). These results show a high intraspecific variation of this parameter in populations, which may be associated with the hormonal induction protocol, the method and frequency of collections, and especially, the period of the year in which samples were collected. The sperm concentration is generally used for milt characterization (Billard et al., 1995). This is an important aspect in the processes related to fish artificial propagation to optimization of the sperm:oocyte ratio in the artificial fertilization procedures (Bombardelli et al., 2006; Chereguini et al., 1999; Sanches et al., 2009). The values of normality found in the present experiment, were similar to that found in Bombardelli et al. (2006), in the same species, which observed indices of 66.9% of normal spermatozoa. But, the sperm morphology parameter is still poorly explored, and its influence on the process of artificial reproduction in Neotropical fish is

not well understood. However, morphology has been shown to influence sperm velocity in Atlantic cod (Gadus morhua) (Tuset et al., 2008a), and the presence of sperm deformities also appears to influence both motility parameters and fertilizing capacity (Rurangwa et al., 2004). Motility is the first parameter measured to evaluate the sperm quality (Alavi and Cosson, 2005; Billard et al., 1995). The high values observed in this experiment (Table 3) revealed that the diets used did not have deleterious effects on the sperm quality. The average sperm motility values in the silver catfish semen ranged from 62.57 ± 3.62% to 93.52 ± 0.85% throughout the experimental period. Other studies of the same species have shown the percentages of sperm motility to be from 60 to 80% (Fogli da Silveira et al., 1985) and 72.5 to 88.3% (Ferreira et al., 2001). Using computer analyses for this species and other confined animals, Sanches et al. (2010a) reported values of 65.37% motility. A comparison between previously reported values, with those of the present study, suggests a pattern of motility for this species between 70 and 80% of motile cells. Although the sperm velocity suggests an important relationship with sperm fertility, as observed by Tuset et al. (2008b) in rainbow trout (O. mykiss), more studies are needed to elucidate their influence. The morphological characteristics observed in testis, as projections, are commonly found in Siluriformes (Andrade et al., 2010; Santos et al., 2001). Microscopically, there was an unrestricted germinal epithelium, in which there was no observable directional pattern of maturation or migration of the cysts (Schulz et al., 2010). The stages of testicular maturation observed at the end of the reproductive period (February), in particular, the absence of animals in a regression stage, indicated a continuous process of cell differentiation that occurred in a satisfactory manner under captivity conditions. Semen quality is defined as the ability of sperm to fertilize oocytes efficiently, and many seminal parameters are used as indicators of this capacity. The indicators most commonly used are the density, the semen osmolarity, the semen pH, the chemical composition, the

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enzyme activity, and the sperm concentration, morphology, and motility (Rurangwa et al., 2004). In the experimental animals, a high production of semen (volume) and sperm cells (sperm concentration) observed, still showed a few alterations and high motility rates, demonstrating the semen quality produced by the males of this species. In general, the ration energy levels did not affect the growth performance of the fish, and even less of the effect was found for the reproductive performance. These results indicate that the use of diets with lower levels of digestible energy (2850 kcal DE kg − 1 diet) promotes satisfactory productive and reproductive indices with a lower production cost. 5. Conclusion The male reproductive peak occurs in late spring. Within the reproductive period and pellet diets, it is recommended to use diet containing 30% DP and 2850 kcal DE kg − 1 diet. References Alavi, S.M.H., Cosson, J., 2005. Sperm motility in fishes. I. Effects of temperature and pH: a review. 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