Use of biofloc technology during the pre-maturation period of Litopenaeus vannamei males: effect of feeds with different protein levels on the spermatophore and sperm quality
Abstract
The objectives of this study were: (1) Compare two systems for pre-maturation of Litopenaeus vannamei in terms of spermatophore and sperm quality, (2) Compare the effect of feeds with different protein levels on reproductive quality of males reared in a biofloc-dominated system. Animals (36.40 ± 3.13 g) reared under biofloc technology (BFT) were used in the 30-day experiment, which involved four treatments: one in a clear water system (CW) and other three in a BFT system. The BFT treatments were differentiated by feed: mix of fish, squid and crab (BFT+FF) composed of 68.48% dietary protein (DP); broodstock feed (BFT+BF) composed of 52.51% DP; and juvenile feed (BFT+JF) composed of 39.91% DP. Feed in the CW was also the mix of fresh food. Spermatophore and sperm quality were analyzed at the beginning and end of the experiment. Higher normal sperm rate was recorded in the CW compared with the BFT+FF. Among the BFT treatments, the BFT+FF had the lowest normal sperm rate. Thus, the use of BFT for pre-maturation of L. vannamei allowed the reduction in dietary protein levels from 68.48% (BFT+FF) to 39.91% (BFT+JF) and the maintenance of spermatophore and sperm quality compared to the system based on high daily exchange rate.
Introduction
Among dietary organic compounds, it has been suggested that protein is more associated with the quality of spermatophores and sperm than are lipids or carbohydrates because protein is related to the formation of spermatozoal spike (Sánchez, Pascual, Sánchez, Vargas-Albores, Moullac & Rosas 2001; Goimier, Pascual, Sánchez, Gaxiola, Sánchez & Rosas 2006). The spermatozoal spike is normally a straight structure that is involved in the initial contact between the spermatozoa and the egg coat at the beginning of the dendrobranchiate fertilization process (Braga, Nakayama, Poersch & Wasielesky 2013). Thus, when the spike is absent, spermatozoa are classified as being abnormal (Alfaro 1993) because they are generally unable to attach to the egg (Wang, Misamore, Jiang & Browdy 1995). Concerning protein levels in male diets, Goimier et al. (2006) evaluated the effect of three dietary protein levels (35%, 45% and 55% DPL) on the reproductive capacity of Litopenaeus setiferus (Linnaeus, 1767) males in a clear water system that had a 200% daily exchange rate. They reported that a lower normal sperm rate was observed in males fed 35% and 55% DPL feeds. Thus, males should be fed at 45% DPL to maintain their reproductive quality.
Feed management for shrimp maturation is not generally based on dietary protein levels, instead it is based on diversifying foods to increase the chance of reaching the broodstock nutritional requirements (Wouters, Zambrano, Espin, Calderon, Lavens & Sorgeloos 2002). Such feed management includes fresh food, e.g. squid, polychaete worms, bivalves, crabs and fish, with or without the addition of commercial feeds (Browdy 1992, 1998; Harrison 1997). Braga, Nakayama, Martins, Colares and Wasielesky (2010) reported the need to include fresh food in the diets of Farfantepenaeus paulensis (Pérez Farfante, 1967) males to avoid any decrease in spermatophore quality in a clear water system that had a 90% daily exchange rate. Likewise, Perez-Velazquez, Lawrence, Gatlin, González-Félix and Bray (2002) reported lower survival rates and changes in the sperm counts of Litopenaeus vannamei (Boone, 1931) males that were fed only dry feed in a recirculating system. The authors concluded that including fresh food in the male shrimps' diet enhanced the reproductive quality. These examples corroborate the importance of diets that contain fresh food on the reproductive quality of male shrimps in traditional systems.
Although the typical combination of fresh food is important for reproductive quality, it is not nutritionally optimal for domesticated L. vannamei males (Perez-Velazquez, González-Félix, Lawrence, Bray & Gatlin 2003). When shrimp are fed with unbalanced or incomplete diets, they have impaired reproductive performance or which can completely inhibit the reproduction process (Bray & Lawrence 1992). Fertility problems of penaeid male shrimps broodstocks may compromise larval production (Alfaro 1993). Thus, the effects of food on reproductive quality of males still require further investigations.
Associated with the nutritional problems in domesticated broodstock that remain unsolved (Wouters, Lavens, Nieto & Sorgeloos 2001), the production of close-life broodstock is primarily stimulated for factors such as increases in the demand for postlarvae as a consequence of the intensification of aquaculture, the transmission of diseases from wild broodstock, and the sustainability of the shrimp farming industry through more ecological and economical production methods compared with the traditional systems (Preston, Brennan & Crocos 1999; Regunathan 2008). Thus, the use of biofloc technology (BFT) in the pre-maturation of broodstocks has been investigated (Emerenciano, Gaxiola & Cuzon 2012).
Pre-maturation phase of domesticated broodstocks has been mentioned as the period between the grow-out and maturation phases, when shrimp are stocked at lower density in comparison to the grow-out phase and fresh food is usually added to feed management. Thus, this phase is considered as a phase of preparation of the broodstocks, aiming to enhance the reproductive quality before the maturation phase (Emerenciano, Cuzon, Arévalo & Gaxiola 2014; Emerenciano, Cuzon, Arévalo, Miquelajauregui & Gaxiola 2013).
Biofloc technology has been widely used in nursery and grow-out systems and shows several advantages, including waste retention, biosecurity, and productivity intensification (Cohen, Samocha, Fox, Gandy & Lawrence 2005; Avnimelech 2006; De Schryver, Crab, Defoirdt, Boon & Verstraete 2008). Furthermore, the microbial flocs may serve as a food source for the reared animals and improve the physiological status compared with organisms fed only formulated feeds (Becerra-Dorame, Martinez-Cordova, Martínez-Porchas, Hernández-López, López-Elías & Mendoza-Cano 2013). In addition, some authors have reported that when the reared animals have bioflocs, available as food source, the dietary protein levels of feed may be reduced (Burford, Thompson, McIntosh, Bauman & Pearson 2004; Wasielesky, Atwood, Stokes & Browdy 2006). For example, the optimal dietary protein level for F. paulensis juveniles reared in a clear water system is 45% crude protein (CP) (Fróes, Abe, Wasielesky, Prentice & Cavalli 2006); however, Ballester, Abreu, Cavalli, Emerenciano, Abreu and de & Wasielesky W. (2010) demonstrated that when this species is reared in a suspended microbial flocs system, the dietary protein level can be reduced to 35% CP. Similarly, Xu, Pan, Zhao and Huang (2012) reported that the protein levels in the diet of L. vannamei juveniles reared in zero-water exchange biofloc-based systems can be reduced to 25% CP without affecting shrimp growth.
The contribution of microbial flocs as the food source to broodstock is poorly understood. Among the scarce investigations available on this contribution, Emerenciano, Cuzon, Arévalo & Gaxiola (2014) compared the reproductive performance of Farfantepenaeus duorarum (Burkenroad, 1939) females previously maintained in clear water and BFT with and without fresh food supplementation systems during a 120-day pre-maturation period. The authors reported better results of female shrimps maintained in BFT during the pre-maturation than those reared in the clear water system; however, there were no differences in performance among female shrimps raised in biofloc with or without fresh food supplementation. In contrast, L. vannamei females that were fed fresh food for 20 days in biofloc conditions prior to ablation had better reproductive performance than other raised in the same conditions, but without fresh food supplementation (Emerenciano, Cuzon, Arévalo, Miquelajauregui et al. 2013).
For domesticated L. vannamei males, the effects of the use of BFT systems and the feed offered during the pre-maturation phase on spermatophore and sperm quality is still unknown. Thus, this study aimed to compare: (1) two systems for pre-maturation of L. vannamei in terms of sperm and spermatophore quality, and (2) the effect of practical feeds on reproductive quality of L. vannamei males reared in a zero-water exchange biofloc-dominated system.
Materials and methods
Animals and experimental design
Litopenaeus vannamei postlarvae (PL) were obtained from a commercial hatchery (Aquatec, Canguaretama, RN, Brazil) and were reared in the Marine Station of Aquaculture (FURG). The grow-out was divided into two phases (grow-out I and grow-out II), which were differentiated primarily by the stocking density and culture system. In grow-out I, shrimp (0.08 ± 0.02 g) were stocked at 180 animals m−2 in nine 600-m2 ponds that were lined with 1.5-mm-thick high-density polyethylene (HDPE) and filled with 594 m3 of groundwater and 6 m3 of biofloc-rich water obtained from a L. vannamei nursery study in 35-m2 tanks. Shrimp were harvested after 120 days and had a final mean weight of 10 g (±2 g). During the harvest, shrimp were selected according to their apparent health condition. Their size, the absence of melanization on the body, the presence of all appendages, and having full hepatopancreas and guts were used as parameters to evaluate the condition of the animals. The selected shrimp were stocked at 40 animals m−2 in two 35-m2 rectangular tanks that were lined with HPDE and sheltered in an enclosed greenhouse for 120 days (grow-out II). Fifteen cubic metres (15 m3) of biofloc-rich water from a preceding L. vannamei grow-out study was mixed with 20 m3 of natural seawater to fill each tank. During both phases, shrimp were reared in BFT and were fed twice daily with a commercial 38% CP feed (Potimar Active 38, Guabi, Campinas, Brazil). Furthermore, the water quality parameters were monitored and maintained within the adequate range for the development of penaeids, as recommended by Van Wyk and Scarpa (1999).
At the end of the grow-out II phase, 60 10-month-old adults males (36.40 ± 3.13 g) were randomly selected from the 35-m2 tanks and were stocked in 20 150-L indoor tanks (0.49 m2). In these units, an experiment was conducted in two types of pre-maturation systems. The first system consisted of clear water (CW), in which five 150-L tanks were filled with 100% of seawater. In these tanks, the daily exchange rate was 90% and feed was composed of mixed blue crab Callinectes sapidus Rathbun, 1896, fish Menticirrhus americanus Linnaeus, 1758 and squid Illex argentinus (Castellanos, 1960). The second system was based on biofloc without water exchange and was composed of 15 150-L tanks filled with 100% of the biofloc used in the grow-out II phase. The 15 150-L tanks were divided into three treatments differentiated by feed. The feed treatments used in the BFT system were: fresh food consisting of the same mix of fresh food offered in the CW (BFT+FF); a broodstock feed consisting of a commercial feed for maturation (Breed S INVE Aquaculture, Baasrode, Belgium) (BFT+BF); and a juvenile feed consisting of a commercial feed for grow-out (Potimar Active 38) (BFT+JF). The experiment lasted for 30 days, and the four treatments (one in clear water and three in the BFT system) had five replicates each. Feed was offered three times daily (at 09:00, 12:00 and 17:00 hours) ad libitum in all treatments. The apparent consumption of feed was checked daily via feeding trays that were installed in all tanks in order to adjust the amount of offered feed. The artificial photoperiod was 13:11 light:dark.
Proximate composition of the feeds and biofloc
The proximate compositions of the fresh food mix and broodstock and juvenile feeds used in the distinct treatments were analyzed. Likewise, the proximate composition of the biofloc of each treatment was analyzed at the beginning and at the end of the trial. The method described in AOAC (2000) was used for the proximate analysis of the feeds and the biofloc. The percentages of proteins, lipids, carbohydrates, fibre and ash were provided by the dry weights of the feeds and the biofloc.
Water quality
Temperature, dissolved oxygen (DO), pH and salinity were measured once daily using an YSI 556 multiparameter probe (YSI Yellow Springs, USA). The floc volume, alkalinity, total ammonium nitrogen (TA-N), dissolved inorganic nitrite (NO2-N), dissolved inorganic nitrate (NO3-N) and phosphate (PO4-P) were monitored weekly. The floc volume was measured based on the sedimentation of the flocs contained in a 1-L water sample after 15–20 min in an Imhoff cone (Avnimelech 2012). The alkalinity was estimated following the APHA (1998) methodology. TA-N was determined following the UNESCO (1983) methodology, whereas NO2-N, NO3-N and PO4-P were measured using the methodologies described by Aminot and Chaussepied (1983).
Spermatophore and sperm quality
Spermatophore and sperm quality were evaluated at the beginning and the end of the experimental period. In both the initial and final samplings, spermatophore quality was measured using spermatophore weight, sperm count and the presence of melanization, whereas sperm quality was measured using the normal and dead sperm rates. At the beginning of the experimental period, both spermatophores of all male shrimp were extruded manually in order to have a unique condition. However, one spermatophore was selected at random, weighed to the nearest 0.001 g and homogenized in a 2 mL calcium-free saline solution and 0.1 mL trypan blue. Sperm count, abnormal cells (i.e. malformations of the main body or absence of the spike) and dead cells (blue coloration) were estimated by counting the cells present in the resulting homogenization of the sperm, saline solution and trypan blue using a haemocytometer under a light microscope following the method described by Leung-Trujillo and Lawrence (1987b). Spermatophore melanization was checked by visually examining the coxae on the fifth pereopod pair and the extruded spermatophore.
Statistical analyses
Prior to the analyses, the data on percentages (i.e. survival, melanization and normal and dead sperm rates) were arcsine transformed (only the untransformed values are presented), and the statistical assumptions were evaluated when required. For the water quality parameters, the Kruskal–Wallis nonparametric analysis was employed to identify significant differences among treatments (Sokal & Rohlf 1995). One-way analyses of variances (anova) were used to identify significant differences among the mean values of survival and spermatophore and sperm quality parameters. An anova was followed by a Tukey's post-hoc comparison test when significant differences were found. All statistical analyses were examined at P < 0.05.
Results
Proximate composition of the feeds and the biofloc
The mix of squid, crab and fish had a higher protein level than the commercial feeds, whereas the broodstock feed had a higher protein level than the juvenile feed. The ash and fibre levels were low in the broodstock feed, though it had a higher lipids level than the other feeds. The carbohydrate level was much lower in the fresh food mix than in the commercial feeds (Table 1).
| Mix of fresh food | Commercial diets | ||
|---|---|---|---|
| Broodstock feed | Juvenile feed | ||
| Protein | 68.48 | 52.51 | 39.91 |
| Lipids | 9.40 | 15.90 | 9.29 |
| Carbohydrate | 1.93 | 20.50 | 35.59 |
| Fib | 4.32 | 1.10 | 3.09 |
| Ash | 15.87 | 9.99 | 12.12 |
All organic compounds in the biofloc used at the beginning of the study, except for ash, trend to decrease in all treatments after the experimental period. The protein levels were similar among treatments at the end of the experimental period (Table 2).
| Initial | BFT + FF | BFT + BF | BFT + JF | |
|---|---|---|---|---|
| Protein | 29.27 | 18.64 | 23.17 | 22.96 |
| Lipids | 2.03 | 0.64 | 1.35 | 0.95 |
| Carbohydrate | 28.11 | 24.97 | 25.68 | 24.78 |
| Fibre | 7.36 | 1.46 | 1.88 | 2.72 |
| Ash | 33.23 | 54.29 | 47.92 | 48.59 |
Water quality
The mean temperature, dissolved oxygen and pH were 29.5 °C, 6.5 mg L−1 and 7.7, respectively, and significant differences were not observed among treatments. Salinity was significantly lower in the CW than in the other treatments (P < 0.05) (Table 3). The mean values of the weekly water quality parameters for each treatment are shown in Table 4. Among the selected parameters, significant differences were only observed for NO3-N and PO4-P between the CW and the BFT treatments during the experimental period (P < 0.05).
| CW | BFT + FF | BFT + BF | BFT + JF | |
|---|---|---|---|---|
| Temperature (°C) | 29.50 ± 1.28 | 29.79 ± 1.36 | 29.49 ± 1.72 | 29.59 ± 1.52 |
| Dissolved oxygen (mg L−1) | 6.56 ± 0.42 | 6.48 ± 0.34 | 6.48 ± 0.37 | 6.59 ± 0.37 |
| pH | 7.76 ± 0.09 | 7.57 ± 0.53 | 7.65 ± 0.13 | 7.71 ± 0.09 |
| Salinity (g L−1) | 27.79 ± 0.83a | 29.51 ± 1.24b | 29.51 ± 1.24b | 29.59 ± 1.44b |
| CW | BFT + FF | BFT + BF | BFT + JF | |
|---|---|---|---|---|
| Floc volume (mL L−1) | – | 22.18 ± 19.23 | 21.37 ± 22.16 | 17.65 ± 16.68 |
| Alkalinity (mg L−1) | 144.37 ± 12.50 | 122.06 ± 20.85 | 129.41 ± 12.31 | 130.83 ± 7.72 |
| TA-N (mg L−1) | 0.11 ± 0.08 | 0.15 ± 0.22 | 0.12 ± 0.21 | 0.13 ± 0.20 |
| NO2-N (mg L−1) | 0.05 ± 0.01 | 0.04 ± 0.07 | 0.06 ± 0.08 | 0.06 ± 0.08 |
| NO3-N (mg L−1) | 1.36 ± 0.31a | 30.46 ± 8.03b | 28.04 ± 6.60b | 28.18 ± 6.12b |
| PO4-P (mg L−1) | 0.15 ± 0.04a | 3.45 ± 1.53b | 3.93 ± 1.72b | 3.63 ± 1.61b |
Survival and spermatophore and sperm quality
Survival, spermatophore weight, melanization rate and sperm count did not show significant differences among treatments. However, significant differences were found for the sperm quality parameters between the initial and final samplings. Normal sperm rates significantly increased (P < 0.05) in the CW and in the BFT treatments at the end of the experimental period, except for BFT+FF. The mean values of dead sperm rates trended to decrease in all treatments; however, significant differences were only found for the BFT+BF and BFT+JF (P < 0.05) when they were compared with the initial sampling (Table 5).
| Initial | CW | BFT + FF | BFT + BF | BFT + JF | |
|---|---|---|---|---|---|
| Survival (%) | – | 53.33 ± 38.01 | 40 ± 27.89 | 60 ± 27.89 | 66.67 ± 23.57 |
| Spermatophore weight (mg) | 32.04 ± 16.36 | 24 ± 12.38 | 25.80 ± 9.18 | 25 ± 7.76 | 34.89 ± 17.64 |
| Melanization (%) | 42.23 ± 11.93 | 40 ± 54.77 | 62.50 ± 47.87 | 33.33 ± 47.14 | 26.67 ± 43.46 |
| Sperm count (×106) | 25.58 ± 16.49 | 26.97 ± 13.88 | 24.72 ± 20.58 | 25.67 ± 12.17 | 33.63 ± 13.18 |
| Normal sperm rate (%) | 54.89 ± 13.90a | 87.04 ± 8.18b | 65.97 ± 8.53a | 89.29 ± 7.79b | 86.08 ± 9.26b |
| Dead sperm rate (%) | 5.84 ± 3.11a | 1.85 ± 2.01a,b | 1.08 ± 1.86a,b | 0.34 ± 0.62b | 0.40 ± 0.94b |
Discussion
Significant differences in salinity, NO3-N and PO4-P between CW and BFT treatments observed in this study are associated with principles of BFT widely described in the literature, such as zero-exchange water, evaporation and accumulation of those inorganic compounds throughout the culture (Ebeling, Timmons & Bisogni 2006; Avnimelech 2012). Despite these differences, all of the selected daily and weekly water quality parameters were also within the range recommended for penaeid culture and/or reproduction (Bray & Lawrence 1992; Cavalli, Peixoto & Wasielesky 1998; Van Wyk & Scarpa 1999) and, hence, did not affect the spermatophore and sperm quality.
Some authors have shown that the spermatophore quality can be affected by factors such as stress, degeneration of the reproductive tract, long time in captivity and nutrition (Leung-Trujillo & Lawrence 1987a; Alfaro 1993). In this study, spermatophore weights and sperm counts were not different among treatments. A wide range of mean values of spermatophore quality parameters has been reported in studies for L. vannamei under different culture conditions (Alfaro-Montoya 2010). The mean values of the spermatophore weights reported in this study (24–34 mg) were similar to those reported for 10-month-old L. vannamei that weighed 30 g and were reared in tidal ponds (Ceballos-Vázquez, Rosas & Racotta 2003). Likewise, similar values to the sperm counts found in this study (24–33 × 106 cells spermatophore−1) were previously reported for both wild and captive male shrimp (Heitzmann, Diter & Aquacop, 1993; Wouters et al. 2002; Ceballos-Vázquez, Aparicio-Simón, Palacios & Racotta 2004).
Another spermatophore quality parameter considered in this study was the melanization, which is an immune process that promotes the production of melanin, a toxic molecule, by prophenoloxidase system. The melanization was initially described as a process triggered by infectious syndrome or stress associated with the captivity conditions (Alfaro, Lawrence & Lewis 1993; Braga et al. 2010). According to Sánchez et al. (2001), when melanization is associated with stress, the increase in melanin produces cell degeneration and finally male sterilization. However, it is currently known that the melanization is also triggered as a natural process for spermatophore regeneration (Alfaro-Montoya 2010). In this study, it is not possible to ensure which factor triggered the melanization observed in all treatments. However, the rates did not also show significant differences among treatments and, consequently, it seems that the type of culture system and/or feed did not affect the melanization process.
Concerning the sperm quality, a possible effect of type of system and dietary protein level was recorded in this study. In a traditional maturation system with clear water, Goimier et al. (2006) reported that L. setiferus males fed 35% and 55% DP had lower sperm quality than those that were fed 45% DP. The authors suggested that the 35% feed did not have enough protein, whereas the 55% DP had too much dietary protein, which affected the sperm quality. In this study, the protein level in the fresh food mix (68.37%) was higher than the level that Goimier et al. (2006) suggested to be an excess of dietary protein for L. setiferus.
In the BFT treatments, L. vannamei males had access to the protein supplied in the feeds in addition to the protein contained in the microbial flocs. Thus, in the BFT+FF, the protein levels of the fresh food (68.37%) and of the microbial floc (18.64%) could have represented an excess of dietary protein. On the other hand, the dietary protein level of the mix of fresh food seemed to meet the nutritional requirement of the L. vannamei males raised in the clear water system, enhancing the sperm quality in terms of normal sperm rate in comparison to the BFT+FF. Among BFT treatments, the increase in normal sperm rate was also recorded in that treatments composed of the commercial feeds with lower dietary protein level than the fresh food (see Table 1). In addition, the dead sperm rate significantly decreased in those treatments in comparison to the beginning of the experimental period.
Those results suggest that the pre-maturation phase of domesticated L. vannamei males could be conducted in clear water systems using fresh food, ensuring an improvement of sperm quality compared with the end of the grow-out II phase. However, the pre-maturation phase could be also successfully conducted in BFT system using commercial feeds without affecting the spermatophore and sperm quality in comparison to the traditional system. Thus, the use of this system in pre-maturation phase, besides decreasing the water use and environmental impacts and increasing biosecurity (De Schryver et al. 2008), could allow the use of commercial feed and, consequently, the reduction in dietary protein level, as previously observed for juveniles (Ballester et al. 2010; Xu et al. 2012). The use of commercial feeds is advantageous because they are easy to manage and stock, do not require preparation, and present less danger of contamination (Wouters, Nieto & Sorgeloos 2000).
Differences in the nutritional requirements of female shrimp and male shrimp are apparent because the reproductive performance of L. vannamei females improved when they were fed fresh food for 20 days under biofloc conditions prior to ablation (Emerenciano, Cuzon, Arévalo, Miquelajauregui et al. 2013). Thus, the pre-maturation of female shrimp and male shrimp reared in a pre-maturation BFT system could be conducted separately, which would reduce the dependence on diets with high protein levels, such as those containing fresh food. However, some questions should be evaluated further, such as the useful reproductive time of male shrimp that have been fed diets with low protein levels without the supplementation of fresh food.
Conclusion
The pre-maturation phase improves the sperm quality of domesticated L. vannamei males compared to the end of the grow-out phase. The pre-maturation could be conducted in clear water systems under high daily exchange rates and using fresh food. However, the use of biofloc-dominated, zero-exchange system allows the reduction in dietary protein levels and the maintenance of spermatophore and sperm quality compared with the conventional system.
Acknowledgements
The authors are grateful for the financial support provided by the National Council for Scientific and Technological Development (CNPq), the Ministry of Fishery and Aquaculture (MPA) and the Coordination for the Improvement of Higher Level Personnel (CAPES). A special thanks goes to Centro Oeste Rações S.A. (GUABI) for donating the feed for the grow-out phases. W.J. Wasielesky and L.H. Poersch are research fellows of CNPq.
